SLAS2017 Scientific Podium Program Abstract Compendium

Podium presentations are organized into seven educational tracks. Podium abstracts and speaker information are organized first by track and then by session below.

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To view a complete schedule of podium presentations and schedule of events for SLAS2017 and to view speaker bios and photos, please visit the SLAS2017 Event Scheduler.

Advances in Bioanalytics, Biomarkers and Diagnostics Track

Track Chairs: Dieter Drexler, Bristol-Myers Squibb and Melanie Leveridge, GlaxoSmithKline

Label Free Bioanalytical Techniques for Hit Identification and Optimisation

Session Chair: Melanie Leveridge, GlaxoSmithKline

• High-throughput MALDI TOF mass spectrometry for drug discovery in the ubiquitin system
  Matthias Trost, MRC PPU, University of Dundee
  High-throughput MALDI TOF mass spectrometry has become an exciting new method for drug discovery. It allows ultra-fast, label-free screening and has with its flexibility the potential to become the tool of choice for many drug discovery efforts. Here, we present the current state of MALDI TOF mass spectrometry and demonstrate a novel screening method to assay E1-E2-E3 ubiquitin ligase activity and specificity using high-throughput MALDI-TOF mass spectrometry. The ubiquitin system consists of the ligating enzymes E1, E2, E3 while deubiquitylases (DUBs) reverse the reaction. E3 ligases recognize substrates with high specificity and are thus of key interest as drug targets. Deregulation of the ubiquitin network is correlated with a variety of human diseases, particularly neurodegenerative disease and cancer. In vitro, active E2/E3 ligase complexes produce free or attached poly-ubiquitin chains, thus free ubiquitin rapidly disappears from the assay solution. It is therefore possible to determine the activity of E2/E3 ligases by measuring the disappearance of monoubiquitin in the assay. We have developed a fast and sensitive assay to analyse in vitro activity and specificity of E2-E3s by MALDI-TOF mass spectrometry. In this assay, we quantitate the decrease in the amount of free monoubiquitin in an in vitro E2/E3 ligase ubiquitylation reaction. Quantification is facilitated by using 15N-labelled ubiquitin as an internal standard for light ubiquitin quantitation. As proof of concept we used three well studied E3 ligases: MDM2 (a RING-type E3 ligases relevant for the stability of p53 oncosuppressor), ITCH (a HECT domain-containing E3 ligase) and HOIP (an E3 ligase part of the LUBAC complex). Firstly, we measured the kinetics of our three E3 ligases paired with E2 reported in literature to be functional. Secondly, we can show that the assay can be used to identify the specificity of the E3s to form active pairs with any of 32 recombinantly expressed E2 ligases which is currently undertaken laboriously and non-quantitatively using SDS-PAGE. Finally, we evaluated whether the MALDI-TOF E2/E3 assay had potential to be deployed to assess potency and selectivity of E2/E3 inhibitors. To undertake this, we tested a ~1,600 compounds against these three E3 ligases and further determined of the hits inhibition kinetics and IC50s. We identified a number of hits which we are currently further validating in cell-based assays.
• High throughput acoustic mass spectrometry: Development and delivery of a biochemical screen
  Jonathan Wingfield, AstraZeneca, Discovery Sciences
  This presentation will be recorded at SLAS2017 and made available free-of-charge to SLAS dues-paying members and full conference attendees post-event.
  
  In the two years since receiving the 2015 SLAS Innovation Award, our acoustic mass spectrometry project has made significant progress towards a commercial instrument. To deliver high throughput screening capability, we use a modified Echo acoustic dispenser to eject and electrically charge a droplet mist directly from an assay plate into a Waters mass spectrometer via a custom transfer interface. This Echo-MS system can run at sampling speeds of 3 samples per second and allows for label-free, high throughput screening (HTS) of biochemical assays.
  
  Elimination of the liquid chromatographic (LC) separation step allows our system to run at true high throughput speeds. Optimization and redesign of sample buffers improves sensitivity and minimizes ion suppression while maintaining biological activity. We have demonstrated this by modifying several traditional labelled assays to run on the Echo-MS and will share two examples where kinase assays have been successfully converted to an Echo-MS end point.
  
  We demonstrate the ability to deliver true HTS by using the Echo-MS to develop and screen compounds to identify inhibitors of glutathione reductase. Both substrate and product readily ionise and therefore we were able to run the Echo-MS in kinetic mode to rapidly optimize the assay conditions, reducing the time required to days. For HTS, the assay was run in batch mode using a formic acid stop reagent. The Echo-MS system was able to screen around 300,000 samples from our collection. Despite manually processing plates through the detector, we were still able to achieve a cycle time for each plate around 6 minutes. Testing at scale not only enabled us to demonstrate the robustness of the hardware but also integrate the output with corporate data analysis tools. Typical Z' values of 0.6 were achieved and less than 0.2% of wells failed to generate data. All actives were confirmed using traditional LCMS.
  
  In summary, we have demonstrated the robustness and the utility of the Echo-MS by screening a pharmaceutically relevant target at sampling rates that support HTS. In addition we utilised the system to reduce assay development timelines and, following the screen, confirm the mechanism of action (MOA) of the hits. The ability to operate the system unattended is clearly necessary to support HTS long term so we have integrated the Echo-MS with simple robotic automation, giving us up to 12 hours of walk-away operation.
• The Use of Backscattering Interferometry to Study Compound Binding to GPCRs
  Jonathan Ellery, Takeda Cambridge Ltd
  G-protein coupled receptors (GPCRs) are an important class of drug target. Many successful drug molecules act primarily through targeting a specific member of this family and historically were described pharmacologically as either an agonist or antagonist. More recent detailed in vitro studies have revealed that some of these drug molecules do not act directly at the orthosteric binding site of the cognate ligand, but at an allosteric site. The realisation that drug molecules do not need to target the orthosteric site has opened up a whole range of novel GPCR pharmacology. This in turn has the potential promise of realising better drug molecules through greater specificity and the possibility to alter the overall output of target GPCRs to enhance beneficial functional responses. Whilst the ability to measure, and understand, the functional consequence of allosteric compounds in vitro has progressed rapidly the ability to directly measure the binding affinity of these compounds and their effects on the binding of cognate ligands has lagged behind. A relatively new technique to help address this gap in our capabilities is Backscattering Interferometry (BSI). BSI is a homogeneous, label free, biophysical technique that can be used to primarily measure the interaction between molecules. Conformational changes that occur when one molecule binds to another leads to a measurable change in refractive index in a concentration dependent manner. The utility of this technology for the study of compound interaction with GPCRs will be discussed.
• High Throughput Screening and Label-Free Characterization of Molecular Interactions Using a 32-spot Array
  Klaus Wiehler, Sierra Sensors GmbH
  High-throughput analysis and characterization of molecular interactions using label-free detection methods requires the precise choreography of continuous flow sample delivery and detection sensitivity. The utility of Hydrodynamic Isolation™ (HI), a novel microfluidic sample delivery technology and highly sensitive SPR+ detection, is evaluated for array-based, high-throughput kinetic and affinity analysis of large and small molecule interactions.

Targeted Biomarker Analysis

Session Chair: Dieter Drexler, BMS

• Effect of Controlled Diet on Biomarker Measurements in the Clinic
  Petia Shipkova, Bristol-Myers Squibb
  The human metabolome, defined as a combination of all small molecules found in the body, including those introduced and modified by diet or medication, holds important information about an individual's phenotype and includes many biomarkers linked to various diseases. In pre-clinical studies, inter-subject variability is minimized by using genetically-similar animals kept on identical diet under identical housing conditions. In clinical trials, differences in diet and lifestyle add complexity to inherent human genetic heterogeneity and may contribute to inter-subject variability when quantitating biomarkers. However, additional hospitalization solely for diet standardization is costly and therefore not typically performed. Here we present a metabolomics study designed to evaluate the effect of standardized diets on the small molecule metabolome of healthy volunteers enrolled in a phase 1 clinical trial.
• The SOMAscan® assay and SOMAmer® reagents: Translatable tools from high-throughput biomarker discovery to targeted assays
  Sheri Wilcox, SomaLogic, Inc.
  SOMAmer reagents are novel affinity binders made from single-stranded DNA engineered with amino-acid like side chains. These reagents combine the best properties of antibodies and aptamers — high affinity to thousands of proteins, reproducibly made synthetically. The hydrophobic nature of these binding interactions results in exquisite shape complementarity between the reagents and their protein targets. SOMAmer reagents have been proven effective in biomarker discovery, diagnostic products, and research tools. SomaLogic has developed a proteomic assay called the SOMAscan assay for biomarker discovery that transforms protein concentrations in a biological sample into a corresponding DNA signature that can be measured using any DNA quantification technology. The commercially available SOMAscan assay measures over 1,300 human proteins simultaneously in biological samples, and has been used to find candidate biomarkers in applications from cardiovascular disease to rare genetic disorders to oncology. Recent SOMAscan data have shown that certain SOMAmer reagents are even able to distinguish between proteins resulting from single-nucleotide polymorphisms (SNPs) and wild-type proteins in human plasma. Applications of the SOMAscan assay range from broad proteomic profiling of thousands of proteins to exquisite specificity measurements for certain analytes, including targeted enrichment coupled to mass spectrometry.
• 2017 SLAS Innovation Award Top 10 Finalist — Inkjet printing technology for facilitated at will antimicrobial susceptibility testing (FAST) in under 5 hours: addressing the needs of a so-called 'post-antibiotic era'.
  James Kirby, Beth Israel Deaconess Medical Center and Harvard Medical School
  The time between when a clinician starts empiric antibacterial therapy and adjusts therapy based on availability of antimicrobial susceptibility testing (AST) data is called the antimicrobial testing gap. A prolonged gap and mismatch between initial therapy and organism susceptibility — a much more likely occurrence for emerging multidrug-resistant (MDR) pathogens - leads to adverse outcome. Furthermore, AST for agents used to treat MDR pathogens such as colistin often must be sent to reference laboratories for broth or agar microdilution testing, manual methods too complex for hospital-based clinical laboratories to perform, thus leading to further unacceptable delay. We therefore developed a novel prototype AST platform, FAST, to close the AST gap. It makes use of inkjet printer-based, digital dispensing technology to enable precise microdilution testing of any antimicrobial at will in 384-well microplate format. The million-fold range of possible inkjet droplet sizes allows the exact amount of antimicrobial needed to be dispensed into each well from a single antibacterial stock solution. Notably, the microdilution assay was found to achieve better precision and equal accuracy to the gold standard reference comparator used for FDA clearance studies. This technology further enables sub-doubling dilution AST near susceptibility breakpoint cutoffs, affording the ability to precisely tailor antimicrobial dosing for borderline susceptible pathogens, where reference methods in current use do not provide sufficient granularity. Using this technology, we also can facilely perform two-dimensional synergy testing. We thus comprehensively examined 56 pairwise combinations of FDA-approved antimicrobials for activity against multiple carbapenem-resistant Enterobacteriaceae (CRE) strains, and identified 4 heretofore undescribed combinations where inhibitory concentrations of each antibacterial when used together were brought into the susceptible range (minocycline:rifampin, fosfomycin:rifampin, minocycline:colistin, and cefepime:fosfomycin). This flexible format was further enabled by establishing the novel use of inkjet printing for automated introduction of bacterial pathogens in appropriate concentrations into microplates. R-squared for bacterial dispensing of E. coli was 0.99 over a wide dynamic range, and a wide variety of major Gram-negative and -positive pathogens tested printed appropriately, including those with chained and clustered forms. We further combined this inkjet assay set up with real-time fluorescent tests for bacterial growth and viability (through use of permeant and impermeant DNA-binding dyes). These parameters were read continuously on a highly sensitive multi-mode plate reader with onboard incubator to provide susceptibility calls within five hours. We expect the entire system to form the basis of a prototype diagnostic platform that will allow highly accurate AST for any antimicrobial at will within a few hours and with single pipetting steps to load organisms and antimicrobial stock solutions. We envision that a fully developed, automated platform will significantly close the antimicrobial testing gap, eliminate need for reference laboratory testing, and provide early direction for treating MDR infections.
• Earwax: A Neglected Body Secretion or a Step Ahead in Clinical Diagnosis!
  Engy Shokry, Laboratory of Methods of Extraction and Separation, Institute of Chemistry, Federal University of Goias (LAMES-IQ-UFG)
  Cerumen, commonly known as earwax, is a secretion of the ceruminous gland "a modified apocrine" sweat gland together with sebaceous gland in humans and other mammals. Until recently, earwax was looked upon as a neglected fluid or body secretion and its potential as a biological matrix for bio-monitoring and clinical diagnosis is still to be explored. In this work, a trial to combine the advantages of earwax as a non invasive sampling technique with volatolomics as a growing research field for detection and monitoring of biomarkers for diabetes mellitus (types I & II) has been achieved with success. These biomarkers can be used to determine with high accuracy diabetes risk at very early point as well as for monitoring success of treatment. Several biological fluids have been utilized for this purpose as plasma, serum, urine even sweat but earwax represents a major advantage of not only being a non-invasive sampling technique but being more protected from the external environment making the sample less liable to contamination, without time consuming extraction procedure, with low preparation and analysis cost when compared with other biological fluids. In our method, earwax samples were collected from diabetic patients of both types (I & II), analyzed directly by headspace gas chromatography/mass spectrometry (HS/GC-MS) without any extraction or pretreatment procedures for analysis of volatiles and confronted to the volatile earwax composition of healthy individuals using an internal standard. Data acquisition was performed in the full scan mode. The identification of all the volatile compounds was conducted using NIST Mass Spectral Library (NIST 05) and by analysis of reference standards, comparing retention times and mass spectral data. Autoscaling of the data matrix was performed before being subjected to a chemometric evaluation for classification and discrimination between the three types of groups. A significant difference in the volatiles composition and quantitative profiles was observed especially in alcohols and ketones, and it was useful to achieve a complete discrimination between the three groups. It means that the developed method is cheap, fast, highly accepted by patients and involves no tedious sample extraction or preparation procedures. Thus the earwax analysis is an extremely promising and valuable approach to determine the health state of individuals with regard to diabetes mellitus.

Target Identification After Phenotypic Screening

Session Chair: Shaun McLoughlin, AbbVie Laboratories

• Strategies and technologies to identify targets from phenotypic screens by quantitative proteomics
  Dirk Ullmann, Evotec AG
  Using phenotypic screens as a strategy to identify potential drug candidates has gained tremenduous attention in recent years. The aim is to identify chemical matter that corrects disease-relevant cellular phenotypes with the potential to be truly disease-modifying. To this end, the industry has established innovative and novel disease-relevant screening paradigms along with target identification methods. Understanding the mechanism and binding partner(s) of any hit compounds identified from phenotypic screening has posed a challenge for many years. Today a number of technologies are available each with its advantages and caveats in covering the target space, the number and methods of hit compounds to be introduced and the compound binding affinity. The presentation will discuss and differentiate these technologies for its drug discovery potential. Particular focus will be given to technologies which combines quantitative mass spectrometry based chemical proteomics to identify the target(s) of small molecule hit compounds. The technologies use cellular background to reveal a compound's cellular binding partners including binding affinities thus allowing deciphering the network of intracellular targets and enabling lead optimization.
• Deconvolution of Targets for Small Molecules Using a Palladium-Cleavable Capture Tag
  Rachel Friedman Ohana, Promega Corporation
  The benefits posed by phenotypic screening are often encumbered by difficulties in identifying the underlying cellular targets. We have developed a new approach utilizing a cleavable chloroalkane capture tag, which can be chemically attached to bioactive compounds to isolate their respective targets for subsequent identification by mass spectrometry. The tag was shown to minimally affect compound potency and membrane permeability, allowing target engagement to occur within intact cells under appropriate physiological conditions. Effective enrichment of these targets was achieved through selectivity in both their capture and elution. Selective capture relied on rapid binding of the tagged compound with its bound targets onto immobilized HaloTag. Selective elution was achieved through palladium catalyzed cleavage of an allyl-carbamate linkage incorporated into the tag. Selective tag cleavage provides a robust elution of bound targets unbiased by their binding interactions with the bioactive compound, particularly interactions that are covalent or have a prolonged residence time. The putative targets identified with mass spectrometry can be readily verified using resonance energy transfer. By exchanging the chloroalkane tag for a fluorophore, direct binding to the targets can be revealed through proximity of the fluorophore to a small luciferase (NanoLuc, 19 kD) genetically fused to the putative target. Using two kinase inhibitors ibrutinib and BIRB796, as models compounds, we demonstrate the capabilities of this approach to identify intracellular targets exhibiting a range of binding characteristics (including both long residence time and covalent interactions).
• Unravelling the target and novel MOA of a tuberculosis phenotypic hit
  Chun-Wa Chung, GSK
  Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. Whole genome sequencing of spontaneous resistance mutants generated against an indazole sulfonamide (GSK3011724A) found by phenotypic screening has identified several specific single nucleotide polymorphisms in the essential Mycobacterium tuberculosis beta-ketoacyl synthase (kas) A gene. This genomic-based target assignment was confirmed by biochemical and biophysical assays, chemoproteomics and finally a high resolution X-ray structure of the KasA-GSK3011724A complex. This structure reveals a surprising and novel mode of inhibition that explains both the profile of resistance mutations and the selectivity of GSK3011724A for KasA over related beta-ketoacyl synthases involved in bacterial fatty acid biosynthesis.
• Discovery of the NLRP3 inflammasome inhibitor CP-456,773 (CRID3) through phenotypic screening
  Fabien Vincent, Pfizer
  The pro-inflammatory cytokine IL-1 is associated with a range of diseases, including COPD, CAPS and Gout. Several biological agents targeting this cytokine or its receptor have been approved for therapeutic use. The recent discovery of multiple inflammasomes, multimeric proteins containing pattern recognition danger signals, has revealed key signaling pathways leading to IL-1b production and secretion by macrophages. As CAPS is caused by mutations in the NLRP3 gene, we sought to identify inhibitors of the NLRP3 inflammasome as potential anti-inflammatory agents. A phenotypic screening approach was employed, which involved the stimulation of primary human cells (PBMCs) by a dual LPS and Nigericin stimulus coupled with an IL-1b secretion readout. Hits from our Chemogenomics library were triaged using counterscreens monitoring IL-6 secretion as well as cytotoxicity. Further hit validation was conducted using a high content imaging assay monitoring inflammasome speck formation in primary human monocytes. As hypothesized prior to the screen, clinical candidate CP-456,773 (aka CRID3) developed in the 1990's as an inhibitor of ATP-induced IL1b secretion emerged as a valid inhibitor of the NLRP3 inflammasome pathway. CP-456,773 was itself the product of a phenotypic drug discovery approach and the story of its original discovery will be shared, along with new mechanistic information related to the (unsuccessful) identification of its precise molecular target and its pharmacology. In summary, CP-456,773 represents a novel, orally bioavailable inhibitor of the NLRP3 Inflammasome pathway displaying efficacy in preclinical models of disease and inflammation.

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Assay Development and Screening Track

Track Chairs: Cathy Tralau-Stewart, University of California, San Francisco and Edward Ainscow, Carrick Therapeutics

Biochemical and Biophysical Screening

Session Chair: Razvan Nutiu, NIBR

• Use of Biophysical Technologies to Identify and Characterize Hit Compounds for NR3A Subunit of NMDA Receptors
  Chen-Ting Ma, Sanford Burnham Prebys Medical Discovery Institute
  High-throughput screening (HTS) assays are an essential part of the drug discovery process. Many lead identification projects rely on functional assays, such as catalytic activity or native ligand displacement assays, and involve direct compound binding assays only at later stages during hit validation and characterization. In this study, we describe the use of direct compound binding assays as primary HTS approach for the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) 3A (NR3A) subunits followed up with functional receptor signaling assays in the hit confirmation stage. NMDAR is essential for normal function of the central nervous system, including memory and learning. However, excessive activation of NMDARs mediates neuronal or synaptic damage in many neurological disorders including Alzheimer's disease (AD). Therefore, blockade of overactivation of NMDARs in the pathological condition of AD may be an effective treatment to prevent disease progression. One approach is to utilize the inhibitory effect of a novel family of NMDAR subunits, the NR3A and NR3B. It is expected that NR3A-specific modulators should interact with NMDARs consisting of NR3 and NR1/NR2 and offer neuroprotection, without disrupting normal NR1/NR2 functions.
• Analysis of bispecific interactions with the switchSENSE biosensor
  Ulrich Rant, Dynamic Biosensors GmbH
  High-affinity and bispecific antibody formats are challenging analytes for interaction analysis systems, because the apparent binding kinetics crucially depend on how the target molecules are presented on the sensor surface. In order to emulate the presentation of heterogeneous antigens on a cell surface with a biosensor platform it is necessary to functionalize the sensor with different antigens at a defined stoichiometry and to control the spatial arrangement of these antigens relative to each other, which has not been feasible up to now. Here, we introduce the DNA-templated assembly of different ligands on a switchSENSE sensor and demonstrate the quantitative measurement of binding cooperativity, i.e., avidity effects. The influence of different ligand arrangements on the binding kinetics, in particular the off-rate, is discussed for antibody formats and bifunctional small proteins. We show how the ligand-to-ligand distance can be controlled with sub-nanometer precision using bifunctional DNA scaffolds, i.e. nanolevers with adjustable arm-lengths. Measurements with these templated ligand arrangements are compared to the conventional case of random ligand adsorption at different densities and steric influences are exemplified. We investigate bispecific interactions with similar (koff,1~koff,2) and dissimilar (koff,1>10 x koff,2) dissociation rates and describe the resulting avidities. Moreover, a comparison of the obtained results to the predictions of a theoretical dissociation model based on monovalent off-rates reveals substantial differences, which underlines the importance of experimental studies for the understanding of binding cooperativity. We believe the introduced workflows will be highly instrumental in the discovery and selection of bispecific biotherapeutics.
• Targeting Structurally and Functionally Diverse Nucleic Acids with Druglike Small Molecules
  John Schneekloth, Chemical Biology Laboratory, National Cancer Institute
  The past twenty years have seen an explosion of knowledge about the structure and function of nucleic acids in roles distinct from coding for protein sequence. However, examples of small molecules that target pharmacologically important nucleic acid structures are exceedingly rare in comparison to proteins. Technologies to identify and study nucleic acid binding molecules are similarly lacking. In this seminar, I will discuss my laboratory's approach to identify and study DNA and RNA-binding small molecules. We use a small molecule microarray (SMM) screening approach, which enables us to screen a library of ~25,000 druglike small molecules against an RNA target in a single day, or to profile small molecule selectivity against a variety of targets. Several case studies of successful SMM screens will be discussed, including selective targeting of the MYC G-quadruplex, microRNAs, and riboswitches with druglike small molecules.
• Unlocking the RNA target space: In search for selective small molecule RNA binders
  Razvan Nutiu, NIBR
  Aberrant RNA expression, regulation, structure and function leads to human disease. Despite the progress made towards understanding RNA biology, the efforts towards finding small molecules against RNA and RNA / protein targets have been generally timid. Recently, several academic and industrial groups reported small molecules that selectively bind RNA and modulate its function, highlighting the potential and triggering an increased interest in RNA as a therapeutic target. The presentation will describe our strategy for biochemical screening and validation of small molecules against RNA targets, with an emphasis on toxic RNA repeats.

Phenotypic and High Content Screening Assays

Session Chair: Susanne Heynen-Genel, Sanford Burnham Prebys Medical Discovery Institute

• Kinetic Imaging of Calcium and Voltage Activity in hiPSC-Derived Dopaminergic Neurons Relevant to Parkinson's
  Jeffrey Price, Vala Sciences
  Parkinson's Disease (PD) occurs due to loss of dopaminergic neurons in the Substantia Nigra, an area of the brain involved in motor control. Certain environmental toxins (e.g., paraquat, rotenone) increase the risk of PD. Additionally, evidence links the protein alpha-synuclein (α-Syn) to PD, including the presence of α-Syn in Lewy bodies, the neurotoxicity of "prion-like" α-Syn aggregates, and the predisposition to early-onset PD in carriers of the α-Syn-A53T mutation. We present progress on a Kinetic Image Cytometry (dynamic HCS) assay to test chemicals for PD-relevant neurotoxicity. Accordingly, we utilized an isogenic pair of human induced pluripotent stem cell (hiPSC) lines, featuring wild-type cells and cells with the A53T mutation of α-Syn, respectively, which were differentiated to dopaminergic neurons (from Cellular Dynamics International). The activity of the neurons was investigated in 96-well plates using fluorescent indicators for intracellular calcium (Fluo-4) and membrane voltage (FluoVolt) by recording digital movies on living neurons in each well, and performing single cell (cell-by-cell) analyses of characteristics of the Ca++ transients and action potentials. The synchronization of spontaneous calcium transients and action potentials between multiple neurons varies with conditions and cell type. For example, rotenone diminishes the synchronized activity in a time (e.g., effect at 2 days› over night› 2 hr) and dose-dependent (effect at 1 µM › 0.1 µM) fashion, particularly in the A53T-α-Syn neurons, suggesting this mutation increases the sensitivity of the neurons to rotenone toxicity. The assay enables high-throughput testing of compounds for toxicity and efficacy relevant to PD.
• High-content imaging and single cell analysis for the systematic discovery and molecular dissection of novel pathways regulating nuclear architecture
  Gianluca Pegoraro, High-Throughput Imaging Facility/Center for Cancer Research/NCI/NIH
  The spatial organization of the genome has an important role in maintaining gene expression programs and in the DNA damage and repair pathway. Furthermore, alterations in the proteins that maintain the proper 3D structure of the cell nucleus can lead to pathologies such as cancer and aging. To study these processes in a systematic fashion, we implemented advanced single-cell image processing and data analysis pipelines to design and execute several high-content imaging (HCI) assays aimed at the discovery of novel cellular factors regulating nuclear architecture. These HCI assays were used in RNAi screens to identify DNA replication as major pathway regulating gene positioning, and to uncover a novel function for the NRF2 transcription factor as an important component of a pathway protecting against cellular aging, respectively. Furthermore, we also developed hiBA-FISH (High-throughput Break-Apart FISH), a novel HCI approach, to precisely measure extremely rare chromosomal translocation events amongst tens of thousands of interphase nuclei, and to quantify the impact of epigenetics modifiers on chromosomal DNA breakage susceptibility. Altogether, the results of these studies demonstrate that the combined use of HCI and single cell analysis is a powerful approach to gain novel insights in the molecular mechanisms controlling nuclear architecture in both physiological and pathological settings.
• Parsing cellular homogeneity from heterogeneous stem cell cultures
  Daniel Hoeppner, Lieber Institute
  The origin of cell type diversity in stem cell culture remains a key question in basic cell biology. However, in disease modeling and drug discovery, cell type diversity is more frequently considered a liability than an asset. The most commonly used pluripotent stem cells are induced pluripotent stem cells (iPSCs). These are now readily derived from a patient or control biopsy and can be converted into most functional cell types of the adult. iPSCs reflect the epiblast stage in early development where immediately prior to gastrulation, cell fates are spatially segregated by migration during an epithelial to mesenchymal transition in response to the organizing factor nodal. Similarly, cultured pluripotent cells self-organize into discrete spatial domains creating inherent cellular heterogeneity. To bring benefit to CNS disease modeling and drug discovery, we applied this dynamic developmental context to identify domains of cellular homogeneity, within heterogeneous colonies, that are suitable for pharmacological manipulation. In these studies, we (1) developed a high-throughput image processing methodology that enables the automated identification of homogeneous cellular domains in spatially-unconstrained iPSC cultures, (2) demonstrate differential responsiveness in each domain to pharmacological modulators of the AKT pathway, (3) establish that cell lines differ in their kinetics of domain formation and (4) use gene expression modules to demonstrate that stem cells share unique properties with donor brains from which they were derived. Together these results support the expanded application of iPSC technology in disease modeling and drug discovery.
• Light sheet microscopy for high content 3-D imaging of 3-D tissue cultures in a 96-well plate format
  Vincent Maioli, Imperial College London
  Light sheet fluorescence microscopy is a high-speed 3-D imaging technique providing low out-of-plane photobleaching and phototoxcity. However, conventional light sheet fluorescence microscopy uses two microscope objective lenses orientated at an angle of 90° relative to one another in order to image a sample and so is not compatible with imaging 96-well plates. Oblique plane microscopy (OPM) is an alternative approach to light sheet microscopy that uses a single high numerical aperture microscope objective to provide both fluorescence excitation and detection. OPM can be implemented on a commercially available fluorescent microscope frame and has previously been applied to calcium imaging of isolated heart muscle cells in 2-D at up to 950 frames per second and in 3-D at up to 25 volumes per second. We present the development of a stage scanning OPM (ssOPM) approach for light sheet fluorescence imaging in 96-well plates. The plate is translated at a constant velocity through the tilted light sheet of the OPM system and image acquisition is electrically synchronised to the motion of the stage. This approach enables rapid 3-D imaging across each well of a 96-well plate with sub-cellular resolution. We demonstrate the application of ssOPM to 3-D imaging of WM-266-4 melanoma cells seeded onto the bottom a 96-well plate in collagen I gel. The plate was subsequently incubated for 48 hours allowing cells to invade up into the gel prior to fixation. Image volumes of 1.6 x 0.28 x 0.14 mm³ were acquired for each well of a plate for a range of siRNA knockdowns. The total volume of raw image data for the plate was 0.4 TB. We demonstrate the use of automatic 3-D morphological quantification of the image data to establish different morphological phenotypes between knockdowns. In conclusion this work will present the development of a stage-scanning OPM plate-reader system for automatic 3-D fluorescence imaging with sub-cellular resolution imaging in 96-well plates.

Assay Platforms for Biologics

Session Chair: Rob Howes, AstraZeneca

• Discovering Antibodies to a Moving Target
  Caroline Colley, MedImmune
  In response to infections and irritants, the respiratory epithelium releases the alarmin interleukin (IL)-33 to elicit a rapid immune response. This makes IL-33 an attractive target for drug discovery. Work performed at MedImmune (Cohen et al, Nature Communications 2015) has recently shown for the first time that IL-33 can form intra-chain disulphide bonds resulting in large conformational changes within the IL-33 protein that disrupt the binding of IL-33 to its receptor, ST2. These conformational changes occur rapidly in in vivo and in vitro, leading to challenges in identifying antibodies that can bind to and inhibit the action of IL-33.
• Developing an Assay Platform for Screening and Characterisation of Antibody Drug Conjugates
  Rachel Forfar, MRC Technology
  Antibody-drug conjugates (ADCs) are increasingly being designed and used as highly targeted cancer therapies — a modern "magic bullet" approach. They consist of an antibody conjugated to a cytotoxic drug via a chemical linker, thereby enabling delivery of the toxic payload to cancer cells expressing a surface antigen of interest. This approach relies on low level or no expression on healthy cells, high expression on the cancer cells and internalisation of the antigen-ADC complex to allow efficient delivery of the small molecule and a reduction in side effects. MRC Technology has a growing portfolio of ADC programs and has built an assay platform for the screening and characterisation of novel ADCs. One of our most advanced programs is on ALK-ADC. Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase expressed on neuroblastoma cancer cells, is associated with poor prognosis in young patients. Current therapies target the intracellular signalling cascades, but these kinase inhibitors can have reduced sensitivity if there are mutations in ALK. Instead, a novel approach is being investigated whereby cytotoxic agents are conjugated to ALK-specific antibodies as a mechanism of directly targeting the ALK-expressing neuroblastoma cells. Antibody:target characterisation and validation of internalisation is crucial early in the process, with cell based assays an essential part. This presentation will provide an overview of the cell-based assays set up at MRC Technology for this purpose, using both ALK and Her2 (Trastuzumab) as examples. The suite of assays includes flow cytometry on the Intellicyt iQue screener, imaging on high content platforms, and assessment of cell cytotoxicity using secondary antibodies conjugated to potent toxins. The challenges of screening, from the initial stages with hybridoma supernatants through to candidate antibodies, towards effectively identifying antibodies with the desired properties of an ADC will be highlighted.
• 2017 SLAS Innovation Award Top 10 Finalist — Automated 3D Cell Culture Platform for Investigating Chemoresistance and Efficacy of Antibody-based Therapeutics
  Christopher Millan, CellSpring AG
  Taxol is a routinely employed first-line small molecule drug for treating ovarian and breast cancer. However, the majority of patients that show an initial positive response to Taxol treatment eventually develop resistance to the drug. A number of phenotypic alterations have been identified in comparisons between sensitive and resistant cell populations and in vitro models are lacking to investigate methods for circumvention. One promising alternative to small molecule drugs is to employ antibodies that specifically bind targets that are overexpressed on the surface of cancer cells. Here, we present an in vitro 3D culture model, called 3D Bloom, for inducing Taxol resistance in cancer cells. Cells cultured in 3D Bloom exhibit hallmark chemoresistance-associated changes in expression of. Common cancer cell lines (i.e. SKOV3 and SKBR3) were cultured either in standard 2D well plates or in 3D Bloom for four days and proteome changes were analyzed via liquid chromotagraphy-mass spectrometry. Strikingly, after just 4 days in culture more than 10% of the proteins identified exhibited significantly altered expression in 3D including chemoresistance-associated proteins related to stress response, metabolism, and protein biosynthesis. Chemoresistance was confirmed by comparing growth inhibition of cells cultured either in 2D or 3D and viability was assessed via the CellTiter Glo assay. Seeding of cells, media changes, drug dosing, and the final viability assay were all performed in automated fashion on a Tecan Freedom EVO liquid handler. Resistance indices (calculated by dividing IC50 values of cells cultured in 3D by those in 2D) ranged from ˜10 in ovarian cancer cells to over 5,000 for breast cancer cells. In stark contrast, antibodies developed against targets known to be overexpressed in a number of cancer lines were shown to be highly effective in suspending growth of cancer cells in the 3D model. The same cells cultured in 2D did not respond to antibody-based treatment. In situ immunostaining was used to confirm antibody-target binding in 3D and increased expression of targets, including HER2 and Integrin α5β1, by cells cultured in 3D compared to 2D. Western blots for these targets corroborated the heightened expression and explained increased sensitivity of the cells to trastuzumab or volociximab (resp.) when cultured in 3D. These results support the current paradigm shift in biology towards using 3D cell culture systems to address more complicated biological questions, especially as they relate to cancer biology and response of cancer cells to therapeutic treatment. The model presented here, 3D Bloom, is the first chemically defined scaffold-based system to combine critical biological relevance, wide ranging versatility, and, most importantly, reproducibility and scalability via full automation.
• Evaluation of protein adsorption from peptides to antibodies to laboratory consumabales
  Peter Rezk, Wheaton Industries
  Protein adsorption to laboratory consumables such as microplates during sample prep and storage has been a challenge for bioanalytical researchers. This challenge often results in analytical imprecision as well as loss of valuable sample over time. Currently, there are different treatments employed in overcoming this nonspecific protein binding issues that involve siliconization for glass articles, BSA coating, and commercially available low protein binding plates. This study investigates the adsorption profile of different classes of proteins ranging from polypeptides, small proteins

Rational Screen Design

Session Chair: Edward Ainscow, GNF

• Phenotypic personalized medicine as a powerful discovery platform for the optimization of multidrug treatments for cancer
  Patrycja Nowak-Sliwinska, University of Geneva
  Current response rates to clinically used chemotherapeutics or targeted agents used as single compounds remain relatively low, mainly due to acquired resistance and tumor / patient heterogeneity. Therefore, the development of a personalized combinatory treatment approach has become a major goal in the field of experimental cancer therapy. Effective optimization of a drug combination requires the consideration of numerous parameters, e.g. the type of drugs or treatment modalities included, the drug dose/dose ratio applied, the application route and treatment schedule. Moreover, currently used approaches are still far from optimal for these applications. We propose to challenge these difficulties by using a phenotype-based screening platform, based on the design of experiment approach and response surface methodology called the streamlined feedback system control method (s-FSC). This platform enables the analysis of drug-drug interactions, the rapid elimination of antagonistic drugs and the identification of solely synergistic drug combinations. This rapid technique could enable the application of a patient tailored optimal drug / dose combination throughout the entire course of the disease, i.e. based on adapting the optimal regimen as a function of the patient's response over time. We tested an initial set of ten targeted and clinically used drugs that target parallel pathways in an in vitro assay for cell viability inhibition in five human renal cell carcinoma cell lines characterized by different origins and mutations, representing samples of five different patients. In only four iterative in vitro experiments, i.e. after testing of only around 0.2% of the full search space, the optimal, three-drug combinations were identified for each cell line. By analyzing a cell viability in response to the administered drug mixtures we developed models that included both individual and drug-drug interaction terms that enabled rapid elimination of antagonistic drugs. The optimized drug mixtures were cell line specific and were significantly more potent than monotherapies of all individual drugs (p < 0.001, combinatory index < 0.3). Moreover, the optimal drug combinations contained significantly reduced drug doses for each compound, as compared to optimal doses for each drug as a monotherapy. These results confirm the necessity of a personalized approach for the successful design of cancer treatments. This study provides a proof-of-concept that phenotype-based screening is a rapid and cost-effective way to identify powerful, low-dose drug combinations that can directed as a precision medicine towards a selected cancer (sub)type.
• Designing workflows and compound sets for combination screens
  Frederick King, The Genomics Institute of the Novartis Research Foundation
  The trend toward the use of more complex assay systems in compound screens can necessitate more thoughtful decisions on the screen design and workflow. Complexity can arise even with relatively simplistic cell viability assays, for example, when combinations of compounds are assessed. We have investigated different approaches to the design of screens of up to four compounds per cell. The results suggest ways to balance the need to keep screen sizes reasonable while maintaining confidence that the scheme is able to identify synergistic combinations. Another common aspect of designing complex screen is the selection of the compound set. The molecules interrogated should have well-characterized and diverse mechanism of actions in order to maximize the biological space interrogated during the screen. We developed an approach that surveyed an extensive collection of screening data and was used to identify such tool compounds. Furthermore, the model was tested by profiling the compounds through a large collection of reporter gene assays that evaluated their activity and specificity.
• Fragment-Based Assisted HTS: a Novel Approach to Traditional Drug Discovery
  Louis Scampavia, Scripps Florida; Department of Molecular Therapeutics
  High Throughput Screening (HTS) has become a well-established strategy for hit identification that requires a sizable investment in compound libraries and careful selection of "target-focused" and "diversity" sets to span biologically relevant chemical-space. Recent shifts in drug screening toward nontraditional and/or orphan-ligand targets have proved challenging, often resulting in large expenditures and poor prospects from traditional HTS approach. Fragment Based Drug Discovery (FBDD) has grown in popularity as an alternative to HTS and as a means of salvaging drug development efforts for these demanding targets. Despite the exponential growth of interest in this field, FBDD represents a significant paradigm shift in terms of methodology and work-flow; requiring a significant investment and cost in medicinal chemistry efforts as well as protracted timelines for development. The Scripps Research Institute had served as one of four principal HTS screening centers during the NIH-funded Molecular Libraries Production Centers Network program; having performed well over 200 HTS campaign on a large variety of traditional and nontraditional targets. Cheminformatic analysis of the NIH MLPCN library indicates that it was serendipitously composed of over 8,000 fragment-like compounds that are compliant with the "Rule of Three"; ideal fragments for FBDD. Further analysis found many of these fragments to be representative scaffolds for hierarchical related compounds also within the MLPCN library. Data mining HTS screening results of various campaigns has revealed that the fragment sub-library portion produced similar hit rates to the entire library deck of over 300K compounds; but their hierarchical related compounds had proportionately enhanced hit rates (up to~50X); hence fragments serving as a hit predictor and guide for compound selection. Presented are the informatics and case scenarios that support a novel methodology to HTS screening referred to as "Fragment-Based Assisted HTS". Leveraging this approach one can mitigate risk when screening difficult targets by only requiring a small fraction of a traditional uHTS library with the advantage of also gaining early structure-activity relationship (SAR) pedigree feedback.
• Using Data to Decide Which Compounds to Screen: Considering Cytotoxicity in Compound Selection, and Utilizing 'Informer Compound Sets' and Iterative Screening to Decrease Experimental Effort and Cost
  Andreas Bender, University of Cambridge
  While compound screens are still a source of hits in drug discovery programs, it has by now been realized that both the cost and time it takes for running such screens can be immense, and that the return in terms of quality hits can often be rather low. In this presentation, we will hence illustrate how chemical and biological data can be used to improve the odds of identifying bioactive matter with the desired properties in a screening campaign. Firstly, we will discuss the impact of cytotoxicity on the quality of screening collections[1], which has been found to be of significant importance, given that even low level cytotox is significantly associated with adverse reactions observed in the clinic. However, given information obtained from a large-scale cytotoxicity screen of 300,000 compounds at AstraZeneca we can conclude that we can both suggest mechanisms of cytotoxicity for individual compounds based on the available data, as well as implement a model which allows for the selection of suitable screening deck compounds, considering also their likely cytotoxicity. Secondly, we will illustrate how even a rather small-scale initial screen of only a few thousand compounds, a so-called 'Informer Set'[2], can be used to identify compounds that are more likely to be hits in a particular assay than those chosen randomly, while at the same time preserving scaffold diversity. Furthermore, this information gathered on a rather small compound set can be used to perform 'Iterative Screening'[3], where over a series of screens only the most promising subsets are screened subsequently. On the 34 in-house datasets of Novartis which the method was validated on consistently a significant fraction of active compounds could be retrieved when screening only a minority of the full deck. Hence we can conclude that, while compound screening will continue to require significant resources in the future, we apparently can improve the odds of finding a compound with the desired properties by taking chemical and biological data into account. [1] Lewis Mervin et al. Understanding Cytotoxicity and Cytostaticity in a High-Throughput Screening Collection. ACS Chem. Biol. 2016 (in press). [2] Shardul Paricharak et al. Data-Driven Derivation of an "Informer Compound Set" for Improved Selection of Active Compounds in High-Throughput Screening. J. Chem. Inf. Model. 2016 (in press, DOI: 10.1021/acs.jcim.6b00244). [3] Shardul Paricharak et al. Analysis of Iterative Screening with Stepwise Compound Selection Based on Novartis In-house HTS Data. ACS Chem. Biol. 2016 (11) 1255 - 1264.

Cellular Manipulation and Genome Editing in Screening Assay Design

Session Chair: Ralph Garippa, Memorial Sloan-Kettering Cancer Center

• Developing CRISPR/Cas9 tools to functionalize the coding and noncoding genome.
  Joana Vidigal, Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center
  CRISPR-based genome editing tools have revolutionized the way we query gene function, by facilitating the generation of site-specific DNA double stranded breaks. In our lab, we are taking advantage of these tools to generate somatic mutations in adult mice in order to dissect their contribution to tumorigenesis. In addition, we are actively developing approaches to design and construct CRISPR libraries against the noncoding genome. Disruption of noncoding RNA or DNA elements often requires the engineering of genomic deletions through the concomitant expression of two gRNAs. To facilitate the generation of loss-of-function screens against these elements we have developed an experimental approach to construct paired-gRNA libraries. In addition, we have developed GuideScan, a novel computational tool that facilitates the construction of comprehensive and fully customizable gRNA databases for any genome and CRISPR endonuclease of choice. GuideScan outperforms other tools in the identification of potential off-targets, generates high-density gRNA databases, and enables batch design of single- and paired-gRNA vectors for genome-wide screens.
• Droplet microfluidic- based 3D tumor models for cancer therapeutic screening
  Pooja Sabhachandani, Northeastern University
  This presentation will be recorded at SLAS2017 and made available free-of-charge to SLAS dues-paying members and full conference attendees post-event.
  
  One of the widely used cancer models today to evaluate the efficacy of drugs at preclinical stage, 2D monolayers, fail to mimic the in-vivo human tumor biology and do not consider the effect of tumor microenvironment when testing therapeutic efficacy of new drug candidates. As a result, 90% of the promising preclinical drugs fail to transform into efficacious treatment options. Also, the 3D spheroids systems cited in literature, are loose aggregates of cells without an extracellular matrix. Here we describe a robust, Microfluidic technique to generate 3D tumors, which resembles tumor microenvironment and can be used as a more effective preclinical drug testing and screening model. Monodisperse cell-laden alginate droplets were generated in Polydimethylsiloxane (PDMS) microfluidic devices that combine T-junction droplet generation and external gelation for spheroid formation. The proposed approach has the capability to incorporate multiple cell types. For the purposes of the preliminary studies, we generated spheroids with breast cancer cell lines (MCF-7 drug sensitive and resistant) and co-culture spheroids of MCF-7 together with a fibroblast cell line (HS-5). The device has the capability to house 1000 spheroids on chip for drug screening and other functional analysis. Cellular viability of spheroids in the array part of the device was maintained for two weeks by continuous perfusion of complete media into the device. The functional performance of our 3D tumor models and a dose dependent response of standard chemotherapeutic drug, Doxorubicin (Dox) and standard drug combination Dox and Paclitaxel (PCT) was analyzed on our chip-based platform. Currently, we are developing spheroid models to monitor pathophysiological gradients which are generally seen in larger tumors. Additionally, we have generated immunogenic tumor models which are currently being used to dynamically evaluate the effect of the immune cells in the tumor microenvironment during chemotherapy administration. Altogether, our work provides a simple, novel and high-throughput in vitro platform to generate, image and analyze 3D monodisperse Alginate hydrogel tumors for various Omic studies and therapeutic efficiency screening, an important translational step before in vivo studies.
• Genome-wide siRNA high-content screen identifies novel muscle differentiation regulators as therapeutic targets for rhabdomyosarcoma
  Yueming Wang, High-Throughput Bioscience Center, Chemical Biology and Therapeutics, St. Jude Children's Research Hospital
  Chemotherapy is an important modality of cancer therapy. The conventional chemotherapy is a standard treatment for rhabdomyosarcoma (RMS), the most common type of sarcoma believed to arise from cells of muscle origin. However, pediatric RMS patients usually face significant treatment-related side effects, such as toxicity on vital organs, neuropathy, and development of second cancers. A new therapeutic approach to induce RMS cell differentiation is especially appealing because it is potentially less toxic and more selective. Development of such therapeutic approach requires a comprehensive understanding of the molecular mechanism underlying regulation and dysregulation of muscle cell differentiation. Here, we performed a functional genome-wide siRNA screen on LHCN-M2 cells, which are immortalized normal human skeletal myoblasts and can undergo myogenic differentiation in differentiation condition, to identify novel muscle differentiation regulators (MDRs) whose dysregulation can lead to failed terminal muscle differentiation and the development of RMS-like phenotype. Using this model, we first developed a high-content microscopic assay allowing quantitative evaluation of the differentiation stages of the cells in the growth or differentiation condition by multivariate analysis including the expression levels of multiple differentiation markers, the cell morphology, the numbers of multinucleated myotubes, and cell fusion index etc. The primary siRNA screen of 16,383 human genes showed a high degree of assay sensitivity and reproducibility with the absolute strictly standardized mean difference (SSMD) value of the controls at 3.17 ± 0.57 (n = 68) across all assay plates. In a confirmation siRNA screen of top 319 hits, we identified 63 genes as the putative negative MDRs and 175 genes as the positive MDRs whose knockdown promoted or suppressed LHCN-M2 differentiation, respectively. In a parallel screen of the 319 genes in the RD cells (a human RMS cell line), we successfully validated 27 genes as negative MDRs and 58 genes as positive MDRs for their myogenic differentiation. Further, we determined the gene expression levels of the 319 genes in LHCN-M2 cells, RD and RH30 RMS cancer cells using a high-throughput real-time PCR approach and found 11 negative MDR genes were exclusively overexpressed in RD cells but not in LHCN-M2 cells, suggesting targeting them for RMS treatment can be selective. Functional studies on the selected candidate genes indicated that knockdown of some of them significantly inhibited RMS cells' growth and induced their differentiation, and enhanced their sensitivity to conventional chemotherapeutic drugs such as doxorubicin and etoposide by ~20 and ~60 folds, respectively. Overall, the novel MDR genes identified in this study provide insights into the molecular mechanism of muscle differentiation and RMS development and serve as putative targets for novel therapy development. The current assay and fully automated high-content screen method can be employed in drug discovery for other muscle-related diseases using the large chemical library.
• A Genome-wide pooled CRISPR knockout screen identifies necrosome regulatory mechanisms.
  Marinella Callow, Genentech Inc.
  Necroptosis, a caspase-independent form of programmed cell death, relies on the activity of kinases RIPK1 and RIPK3, core components of the necrosome, which coordinately phosphorylate the pseudokinase MLKL to drive necroptosis. RIPK1 also participates in competing complexes that alternatively induce apoptosis, cell survival and inflammation, and the full complexity of regulatory and downstream signaling pathways remains incompletely characterized. Pharmacological attenuation of necroptosis presents an opportunity to ameliorate a variety of pathological conditions associated with acute organ injury such as myocardial infarction, stroke, drug induced kidney damage, or diseases associated with inflammation such as inflammatory bowel disease. We performed a whole-genome, pooled CRISPR screen to identify novel loci that, upon disruption, confer resistance to a necroptotic death-inducing cocktail of TNF and pan-caspase inhibitor zVAD-fmk. We used a custom CRISPR library of approximately 155,000 sgRNA divided into 9 sub-libraries with 8 sgRNAs per gene, preferentially targeting the most 5' shared exon between splice variants. Cas9 was stably expressed in L929 fibroblast cells, and then sgRNA expression vectors were introduced by lentiviral infection separately for each sub-library. We used gene-level ranking statistics to identify a total of 76 hits. MLKL, RIPK1, RIPK3 and TNFRSF1A scored as the top 4 hits for sgRNA enrichment, confirming the quality of our screening platform. Known RIPK1 regulators CYLD and SPATA2, as well as new candidate necrosome regulators, showed lower but statistically significant sgRNA enrichment. We categorized hit genes further by examining sgRNA depletion in the absence of TNF/zVAD selection, indicating reduced cell survival or proliferation. Necrosome regulator 'hit' genes were validated using synthetic CRISPR reagents in arrayed assay format, and one of these was determined to act by altering RIPK1 alternative splicing.

Screening the Undruggable

Session Chair: John Lazo, University of Virginia

• Interrogating Protein-Protein Interactions in Cancer
  Haian Fu, Emory Chemical Biology Discovery Center, Emory University School of Medicine
  Protein-protein interactions (PPI) dictate intricate intra- and inter-cellular signaling networks, which are essential for diverse physiological processes. Because of their critical roles in transmitting physiological and pathological signals, the once "undruggable" PPIs have emerged as a viable class of molecular targets for therapeutic interventions. Our work focuses on cancer-genomics-based PPI target discovery and interrogation with the development of informative screening platforms. Case studies will be presented.
• Hot spotting with thermal scanning: a ligand- and structure-independent assessment of target drugability/ligandability
  Stefan Geschwindner, Discovery Sciences, AstraZeneca R&D; Gothenburg
  The technical feasibility to find and develop novel chemical starting points towards successful drug discovery activities has seen a tremendous improvement during the last decades. This is contrasted by the increasing challenge to generate differentiated chemical equity for novel drug targets, as those display very frequently a low drugability, i.e. a low feasibility to develop a small molecule drug against a given target. Some strategies like encoded library technologies (ELT), fragment-based drug discovery (FBDD) or new modalities like cyclic peptides or RNA-based therapeutics are nowadays applied to unlock those undruggable targets.
• 2017 SLAS Innovation Award Top 10 Finalist — Identifying Allosteric Modifiers of K-Ras using Second Harmonic Generation
  Frank McCormick, University California San Francisco
  The K-Ras gene is mutated in 20-30% of all human cancers, accounting for about one million cancer deaths per year worldwide. Mutated K-Ras proteins have been deemed un-druggable because they do not contain pockets or active sites that can be exploited by traditional medicinal chemistry. However, recent advances in chemical biology have enabled new approaches to targeting K-Ras. One of these approaches harnesses an optical phenomenon known as Second Harmonic Generation (SHG) in a technique that is extremely sensitive to small changes in orientation of a protein relative to a membrane surface and can measure conformational change in real-time. We have built and optimized a robust SHG assay to screen for compounds that cause allosteric changes in K-Ras. In a screen of an ~2,800 fragment library we have identified compounds that bind to K-Ras in a GTP-dependent or GDP-dependent manner and promote distinct conformational changes in the protein. We hope that these fragments will; i) reveal novel allosteric binding pockets for drug targeting and, ii) prove to be useful tools for studying the relationship between K-Ras conformation and its biological functions.
• Drugging Protein Phosphatases for the Treatment of Cancers
  John Lazo, University of Virginia
  Protein phosphatases counterbalance the enzymatic activity of protein kinases and, consequently, these two superfamilies have a central role in determining protein phosphorylation status, which can alter stability, macromolecular interactions, enzyme activity, subcellular localization and, ultimately, protein function controlling normal homeostasis and disease processes including cancer. Mathematical modeling suggests the activity of the ~500 kinases encoded in the human genome primarily controls the amplitude of a given signal while the ~100 phosphatases, which dephosphorylate serine, threonine or tyrosine on protein substrates, appear to regulate the signal rate and duration, thus providing an orthogonal mode by which cellular processes can be managed. No two enzyme superfamilies better illustrate drug surfeit and dearth. Currently there are >30 FDA approved protein kinase inhibitors but an almost complete absence of FDA approved protein phosphatase agonists or antagonists. Protein tyrosine phosphatases historically were ignored as therapeutic targets, because they were considered unregulated housekeeping genes. There is now a sizable literature indicating that phosphatases are highly regulated and genetic studies provide incontrovertible evidence that phosphatases have a central role in many disease processes. Nonetheless, phosphatases have become one of the premier members of the undruggable caste. We will review some of the previous attempts to screen for drug-like molecules that alter phosphatase function and explore the recent successes targeting the protein phosphatases with small molecules and biologics. Particular emphasis will be placed on attempts to drug PTP4A3, PTP1B, and SHP2 for the treatment of cancer.

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Automation and High-Throughput Technologies

Track Chairs: Craig Schulz, Amgen and Taosheng Chen, St. Jude Children's Research Hospital

High Content and High Throughput Automation

Session Chair: Louis Scampavia, Scripps Florida; Department of Molecular Therapeutics

• Past, present & future — AstraZeneca's experience, learning and vision in the delivery of automated High Content Screening
  Paul Harper, AstraZeneca
  As High Throughput screening approaches evolve, the ability to generate value adding, multi-parametric assay analysis is expanding in the fields of High Content screening, transcriptomics, mass spectroscopy and flow cytometry. In this presentation we will focus on the practical delivery of phenotypic assays for High Content analysis. We will review the basic steps of the assay, its staining, image acquisition and analysis, in alignment to equipment and automation to successfully prosecute the screening campaign. We will present how the screening infrastructure has evolved and expanded over the past decade, in combination with the developing imaging capability; to deliver successful high throughout HCS; reflecting on our key learnings for robust assays, equipment task prosecution, automated protocol scheduling and management of the image storage & analysis. Looking forward we will share the development of our new generation of HCS automation to facilitate ultra HTS imaging capabilities through the deployment of next generation imagers and scheduling robotics. Finally we will review how, at this step changing scale, we propose to manage the daunting task of dealing with image storage, retrieval and analysis to deliver maximum benefit from these systems.
• Multiplexed Live Cell Phenotypic Screening: Cell level vs Multi-variant Well Level Analysis
  James LaRocque, AMRI
  With the improved resolution and signal collection specifications of high-content instruments, there is need to develop unbiased, predictive and cost-efficient data analyses routines. In order to develop these routines, we have conducted a live cell 1536-well plate based high content screen (HCS) utilizing three stains to help visualize their morphological characteristics to monitor cytoskeletal structure, induction of apoptosis and cell death, as a single well multiplexed assay. Applying multi-parametric analysis with machine learning and non-biased statistical analysis to images, numerical data sets and combinations of both, we selected phenotype characteristics which accurately identify the effect on cells, based on operator positive cell selection. This technique was applied to each of the three positive control compounds vs the negative controls to establish minimum thresholds on a per cell basis for each morphological class. The features from this cellular positive vs negative measurement was aggregated for each phenotype and applied to the 105,000 compound test well population to identify hits based on a higher percentage of cells per well for each positive morphological class. Separately, a multi-parametric analysis was used to identify the top ten morphological features that distinguished each of the three positive control phenotypes on a per well basis to identify 3 distinct classes of hits without operator bias to identify positive wells. The hits for each phenotypic class identified by both of these methods were tested to confirm the results and make comparisons between the two hit identification methods in regards to the false positive / false negative rates and the overlap in phenotypic classes along with the ability to identify structural classes or mechanisms of action of lead compounds.
• The European Lead Factory: an efficient open-innovation platform with unique screening capabilities
  Steven van Helden, Pivot Park Screening Centre
  The European Lead Factory has been designed to create unrivalled opportunities for the discovery of new drug lead molecules by giving academics and SMEs access to an 'industry-like' discovery platform. Seven large pharmaceutical companies have joined forces and contributed compounds from their proprietary collections to the core Joint European Compound Library (JECL). This unique library will grow from 300,000 compounds in 2013 to 500,000 compounds in 2017 and is screened against a wide variety of targets. Scientists with innovative biology target and chemistry scaffold owners are welcome to participate in the European Lead Factory.
  
  All High Throughput Screening campaigns for the European Lead Factory are performed at the Pivot Park Screening Centre in Oss, The Netherlands. As of June 2016, 47 full-deck HTS campaigns have been completed. These cover a wide range of target classes, including a high proportion of more demanding classes like ion channels, transcription factors and protein-protein interactions. Almost all HTS assays have been successfully miniaturized to 1536 format in order to perform them in a cost-efficient manner. The high productivity has been possible thanks to the implementation of industry-standard workflows and efficient interaction with target-owners across Europe. A robustness set containing "clean" and "nuisance" compounds has been implemented to guide assay design. Particular emphasis has been put on the triaging capabilities in the European Lead Factory. The large number of projects, many involving challenging innovative targets, has led to unique experience in the design of deselection and orthogonal assays for following up the HTS campaigns. As a result, high quality hits are delivered to the target owners.
  
  The research leading to these results has received support from IMI under grant agreement n° 115489 and EFPIA companies' in-kind contribution.
• App Store and High Performance Computing for High Content Screening
  Seungtaek Lee, PerkinElmer
  There's a trend in drug discovery to move from classical target-based screening towards phenotypic screening aiming at greater physiological relevance to reduce failure rates and more predictive results; leading to increase in cellular and live cell assays, use of complex disease models, and characterization across multiple and orthogonal assays. The dimension and type of screening experiments is growing, and flexibility and computing time are becoming limiting factors. Additionally, the move from 2D to 3D is challenging the capacity of computing and storage systems even more. Using today's data analysis systems, researchers conducting phenotypic screening campaigns at pharmaceutical companies processing approximately 500,000 compounds estimate image and data analysis time of at least three months. Furthermore, different software systems are used at various stages of the workflow including image analysis, cell level data analysis, well level data analysis, hit stratification, multivariate/machine learning data analysis and visualization, reporting, collaboration, and persistence. PerkinElmer Signals for Screening is an instrument agnostic, comprehensive screening software solution that combines the Signals App Store for Screening with modern Big Data storage and computing technologies in an open, end-to-end screening data analysis and management platform designed to support the complete data flow of a wide range of screening workflows and applications with unmatched performance and scalability. The platform easily integrates screening assay data and phenotypic data and enables scientists to integrate, search, and retrieve relevant data from across internal and external sources. The Signals App Store for Screening is an analytics and workflow management platform that allows users to browse, download, and build screening applications to TIBCO Spotfire. Apps can be combined to build protocols for distribution. Also, custom Apps can be provisioned by Spotfire users providing ultimate flexibility in the types of analytics and applications it can support. Intrinsic domain model awareness enables Apps to configure themselves and guide user through input parameter selection. Furthermore, protocols can be used interactively during assay development or can be fully automated for the analysis of screening campaigns. PerkinElmer Signals for Screening leverages high-performance computing (HPC) to parallelize all applications to provide ultimate performance in image and data analysis. Batch re-analysis jobs that took months can be completed in days. Clustering and other machine learning methods that took hours can be completed in minutes. It provides a perfect balance of flexibility, automation, and scalability for large and small organizations. We will provide few use cases where these Big Data storage, analysis, and computing technologies have been leveraged in a high content screening context.

Screening Automation: Modular vs. Highly integrated systems

Session Chair: Paul Anderson, GNF

• Development of Small Modular Systems to Enable New Scientific Applications
  Jason Matzen, The Genomics Institute of the Novartis Research Foundation
  GNF has been developing and manufacturing high throughput drug discovery platforms since being founded in 1999. These HTS and Automated Cellular Profiling systems feature high capacity, excellent performance and extreme reliability. This approach has enabled routine screening of millions of compounds in complex cell-based assays as well as massive profiling of leads from HTS as well as biologics. These highly integrated systems are complemented by smaller modular systems. GNF's modular systems are lower throughput but are rapidly reconfigurable for new projects. The use of collaborative robots with modular systems reduces the amount of system guarding and access limitations, while still ensuring the necessary safe work environment. GNF has leveraged its expertise with highly integrated systems to now build small scale modular systems. The same scheduling software is used to drive both types of platform, thus leveraging 15 years of development and improvements in the user interface. Specific details comparing the two types of systems will be discussed including case studies where modular systems are more appropriate than highly integrated systems.
• Connecting Hits to Leads through Fit-for-Purpose Automation Solutions
  Jonathan Lippy, Bristol Myers Squibb
  In the mid 1990's, the availability of commercial laboratory automation to support High-Throughput Screening (HTS) was minimal. As a result, pharmaceutical companies turned to in-house engineering teams to build and support automated screening systems for simple and linear assay processes. Here we describe a transformational approach to evolve from in-house to commercial automation and enable multi-modality capabilities from Hits-to-Leads. Over the past 15 years, the marketplace has grown significantly and in-house solutions have become obsolete. The movement towards non-typical reagents such as, whole blood, primary cells and human matrices has driven our requirement to establish flexible automation. Furthermore, complex assay formats such as high content imaging, flow cytometry and kinetic readouts have pushed demands beyond traditional single mode biochemical and reporter based readouts. We have implemented a fit-for-purpose approach that provides high-fidelity integrated automation to drive HTS while providing connectivity with Lead Optimization through modular flexible systems. We have delivered a fleet based and standardized solution to drive usability, reduce footprint and minimize downtime. Additionally, we have connected bioassay processes with compound informatics to drive closed loop screening capability and deliver screening process efficiencies. This holistic approach has provided state-of-art capability to keep pace with the ever changing demands of the scientific and technology landscape.
• Modular, fully integrated or collaborative automation — what happens when you want it all?
  Mark Wigglesworth, AstraZeneca — Discovery Sciences
  AstraZeneca has an ambition to create a porous organisation, seamlessly collaborating with external partners. This desire has led to the establishment of the AstraZeneca-MRC UK Centre for Lead Discovery, ultimately located in the future R&D; centre in Cambridge, UK.
• Iterative Design of an Automated Organism Foundry
  Jeff Lou, Ginkgo Bioworks
  Engineering microbes requires a diverse set of activities across many disciplines. Ginkgo Bioworks' organism foundry consolidates many of these activities within a facility that leverages automation and software to simultaneously deliver engineered organisms to customers while learning how to better use these tools. As the pace of development and production in the foundry ramps up, the robotic machinery must not only act as force multipliers for these organism engineers, but also support an ever growing set of activities. Key challenges for automation include the genetic engineering of different types of microbial organisms and measuring salient properties of those organisms. Constructing engineered strains requires managing techniques to handle different types of microbial organisms, cloning and genome integration strategies, and sample logistics, without overwhelming the operator. On the testing side, the foundry must balance the tradeoff between throughput and information content of available methods, while maintaining acceptable CVs and accuracies. Automation engineers also need to evaluate the diverse ecosystem of automated laboratory equipment and then convey this information back to the organism engineers in a constantly evolving dialogue. In this talk I will describe how Ginkgo systematically addresses these challenges through iterative design of robust systems that is crucially informed by process and data analysis.

Automating Phenotypic and Target Based Discovery using Parallel Automated Approaches

Session Chair: Robin Felder, The University of Virginia

• Multiplexed Human Cell Based Assays for Medium Throughput Screening
  Robin Felder, The University of Virginia
  The current success rate of finding lead compounds in medium and high throughput screening is limited by the current knowledge about the interaction between various metabolic pathways that contribute to the mechanism of complex diseases such as hypertension, cancer, osteoarthritis and asthma. Advances are being made in cell based assays and metabolic reporter systems to measure the effect of lead compounds on many simultaneously interacting metabolic pathways. For example, multiplexed cytoplasmic FRET-based biosensors may be transfected into human cells to allow quantifying the relative activity of each metabolic pathway in response to lead compounds. In addition, "label free" technologies can provide a sensitive readout of cell based responses. Collectively, one can see whole cell responses and potentially determine which metabolic pathway is responsible for the cell activity of interest. We are using these techniques to study salt sensitivity to blood pressure, which negatively affects 26% of the adult population. Salt Sensitivity is a complex disease that is manifested in part by genetic variants in various metabolic pathways (SLC4A5 and GRK4) that regulate sodium excretion in the kidney. Discovery of an inhibitor to either or both the products of these genes NBCe2 and the G protein coupled receptor kinase type 4 (GRK4) may lead to selective and effective therapeutics. However, there are many approaches to inhibiting these pathways that regulate salt excretion such as targeting the proteins themselves or the second messengers that regulate their activity. This presentation will discuss the use of human kidney cell-based models obtained from urine specimens obtained from a wide variety of study participants representing ethnic diversity. These cells are grown in a 3D environment for medium throughput screening. Multiple cytoplasmic biosensors are then transfected into these human cell culture models in order to study ion transport, kinase activity, and other metabolic activity in order to find selective and effective lead compounds.
• 3D Shaped Cell Microcarriers for Cell Culture, Manipulation, High-throughput Analysis, and Sorting of Adherent Cells
  Chueh-Yu Wu, University of California, Los Angeles
  There is need for a new paradigm in cell culture and analysis that transitions away from well plate formats to particle-based culture, whereby adherent cells grow and are analyzed on engineered particle microcarriers. This approach has advantages for rapid culture, passaging and analysis without bringing cells into suspension using harsh reagents. However, significant challenges for protecting cells from shear during culture and analysis are present. For analysis, detecting cell morphology, protein localization, cytoskeleton characterization, and cellular force in a large population in high-speed and precise manner can provide efficient and valuable insights into modern disease identification, personal drug development, and foundations of cellular interactions. Current technologies, for example, flow cytometry and high content screening, face challenges for local information lost in the sensing process and relative low-speed detection that microcarriers can solve. To rapidly investigate and study single cells and cell colonies in their adherent state, we developed a 3D microcarrier based on a computationally enabled microparticle design with optimized surface chemistry, carrying cells predominantly in regions that are protected from shear, and possessing a shape that enables self-alignment in channel flows as carriers flow toward a detector. We demonstrate a high-speed microcarrier fabrication process with a production rate of about 36,000 carriers per hour, which can be further highly scaled up with parallelization, using a new setup of optical Transient Liquid Molding (OTLM) published in Adv. Mater 2015. We design the microcarrier shape to be formed by the intersection of a 2D-extruded dumbbell and a slit with notches to protect from shear. Breast cancer cells were shown to adhere and grow on the regions of the microcarrier patterned with extracelluar matrix for three days. We demonstrate that microcarriers with breast cancer cells in protected regions flowing through a rectangular microchannel with channel Re number of ~20 are focused in flow without rotation, enabling promising integration with modern detection and sensing technologies, including fluorescence imaging using radiofrequency-tagged emission (FIRE).
• Phenotypic Screening to identify modulators of immune cell effector functions
  Renate Schnitzer, Boehringer Ingelheim RCV
  The Seminar will inform about multiparametric phenotypic screening approaches to identify compounds which increase the effector function of NK and T cells and induce an improved anti-cancer response. A selective kinase inhibitor library was designed for this purpose, which was screened to discover kinases that play an important role in the inhibitory pathways of NK and T cells. The effect of the library compounds was characterized on cell lines and primary cells. We used the iQueScreener platform to quantify viability of suspension cells and release of several cytokines. Due to the possibility of multiplexed analysis of cells and beads in suspension the different parameters could be measured simultaneously in the same experiment. Identified hits could be nicely confirmed by dose response curves. This approach revealed interesting targets, which are currently investigated in more detail. Strategy and results are delineated and the benefits of the technology for this approach are highlighted.
• 2017 SLAS Innovation Award Top 10 Finalist — Spheroid microarrays to rapidly screen spheroid morphology and protein expression for regenerative medicine, toxicology and cancer research
  Delyan Ivanov, University of Nottingham
  Three-dimensional cell cultures are now entering mainstream drug screens due to the introduction of plate-based platforms for their culture. Drug-response analysis in these assays in mainly confined to simplistic cytotoxicity measurements of volume or metabolic activity (ATP). The next challenge is to develop platforms to rapidly screen spheroid histology and molecular circuitry on the single cell level. The need for higher-throughput analysis of spheroid biology can be addressed by transferring in vitro spheroids to precisely-arranged microarrays for simultaneous analysis of DNA, RNA or protein targets. We have developed a device to arrange up to 66 spheroids in a gel-based array straight from the culture plate for paraffin-embedding and immunohistochemistry. The spheroids are positioned in a sectionable agarose mold at a common plane for cutting sections containing all 66 spheroids for simultaneous staining and analysis. The technique uses 11 times less reagents and consumables compared to embedding technical replicates as separate samples. The common embedding plane allows for automated scanning of all conditions and automated analysis, in contrast to the slow manual task of analyzing randomly distributed spheroids. The device also reduces the chances for sample mix up due to combining all samples in one fixed-configuration array. Spheroid microarrays were reproducibly made from normal neurospheres and tumor spheroids. Spheroids were embedded within 200µm of each other (interquartile range -100 to 50µm, n=195 spheres, 3 experiments). The arrays were sectioned and stained for progenitors (SOX-2), proliferation (Ki-67), and differentiation (βIII-tubulin). Stained slides were scanned and the arrays were automatically scored (ImageJ). Signal variability was quantified for highly-positive (SOX-2, mean=91%, SD=11, n=23), moderately positive (Ki-67=68%, SD=15, n=23) and weakly-positive (βIII-tubulin=1.6%, SD=0.8, n=37) protein targets. We have used the spheroid microarrays to quantify neural differentiation in human brain neurospheres and drug response upon chemotherapy treatment. Ten different media compositions were tested for their ability to promote neural differentiation and the percentage of progenitors and neurons quantified via immunohistochemistry. Mitogen-rich media had the highest number of progenitors (96% SOX-2) and lowest proportion of neurons (4% βIII-tubulin), while neurospheres cultured with serum were made up of 55% progenitors and 39% neurons. Spheroid microarrays enabled the detection of chemotherapy-induced dose-dependent drop in proliferation (Ki-67), accompanied by reduction in spheroid volume. Spheroid microarrays allow users to quickly screen 3D cultures from multiple cell lines for morphology and protein expression differences. We created spheroid microarrays from 11 different cell lines from 7 tissues. Spheroid morphology was different for each cell line showing utility in cell line authentication and phenotype validation. The protein expression profile for each cell lines can serve to validate antibodies and companion diagnostics. This technique will be useful to scientists working on in vitro models of cancer, stem cells and safety profiling.

Using Physiologically-relevant Models for Automated Screens

Session Chair: Shane Horman, GNF (Novartis)

• Reconciling Throughput and Physiological Relevance in High Throughput Screening
  Robert Damoiseaux, UCLA
  High throughput screening typically relies on simplified assays systems to quantify disease modifying activities or processes – often using e.g. either isolated enzyme preparations or a simple cellular background such as well-established cancer cell lines with easy to measure readouts such as fluorescence or luminescence. Traditionally, one trades in physiological relevance for throughput as physiological relevance and throughput are often diametrically opposed. More complex physiological responses that are highly disease relevant are typically harder to quantify because they are the result of the interplay of multiple proteins or even cells. However, creative use of new technologies enables the generation of assays that are amenable to high throughput and also allow for the quantification of these responses: Here we will show examples of how custom made materials, high content screening and large particle sorters can be leveraged to create such high value assay platforms that are high throughput compatible. In a first example we will show how soft lithography can be used to generate deformable soft substrate patterning on the bottom of microtiter plates that enable measurement of cellular forces. Such an assay is useful for the measurement of cellular force involved in many physiological responses that range from muscle contraction all the way to immune responses. In a second example we will review how cell spheroids can offer insight into complex processes. On one hand, patient derived cell spheroids are useful for cancer drug screening and we will show an example where the matrix embedding the spheroids plays a crucial role. In a second example we will highlight the use of neurospheres in order to emulate the effects of Zika virus on complex neurological structures. We will conclude our talk with examples of whole organism screening for small molecule genotoxicity using C.Elegans and show neurotoxicity screening of nanomaterials on zebrafish in high throughput.
• HTS Tumor:Stroma Co-Culture Spheroid Platform Reveals CAF-Specific Chemotherapeutic Targets
  Shane Horman, GNF (Novartis)
  This presentation will be recorded at SLAS2017 and made available free-of-charge to SLAS dues-paying members and full conference attendees post-event.
  
  It has been observed that targeting the mutated drivers of cancer cells may not be sufficient to kill tumors. Indeed, recent evidence points to the stromal microenvironment of solid organ tumors as the dominating influence in tumor progression, metastasis and drug resistance, indicating that environment may be dominant to the intrinsic altered signaling pathways of cancer cells. In order to expand these concepts to early stage drug discovery initiatives we have scrutinized the interactions between normal colon stroma and colorectal carcinoma (CRC) cells in a high content co-culture 3D microtumor spheroid screen. Systematic knockdown of genes within normal colon fibroblasts resulted in the inability of seeded CRC cells to form 3D tumor spheroids. Employing this multicellular spheroid platform in a genomics-based approach enabled the identification of novel candidate stromal genes crucial for paracrine signaling and tumor initiation/maintenance. Furthermore, low molecular weight inhibitors against lead hit targets recapitulated the genomics data indicating certain fibroblast-dispensable targets are amenable to therapeutic intervention. This work may hopefully expand the target space of chemotherapeutics to include cancer cell extrinsic targets that modify the tumor microenvironment to alleviate disease.
• A novel high-throughput multi-parametric drug screening method for 3D tumor spheroids using Celigo image cytometer
  Steven Titus, NCATS/NIH
  Three-dimensional (3D) tumor models have been utilized to screen potential cancer drug compounds. The ability to perform high-throughput screening of 3D tumor spheroids can highly improve the efficiency and cost-effectiveness of discovering potential cancer drug candidates. Although many image-based cytometry instruments have shown the ability image and analyze 3D tumor spheroids, a robust scoring system has not been demonstrated. Previously, the Celigo image cytometer has demonstrated the development and validation of a novel method for high-throughput screening of 3D tumor spheroids. In this work, we demonstrated a novel cancer drug scoring method using multi-parametric analysis to rank the anti-cancer effects of a panel of 1760 drug compounds on Hey tumor spheroids. The multi-parametric analysis was conducted by performing orthogonal assays of growth inhibition, spheroid perimeter cell death, as well as measuring spheroid viability using GFP and propidium iodide (PI). The Celigo image cytometer was used to image and analyze the Hey tumor spheroids after 3 days of incubation with the panel of drug compounds. The parameters exported to investigate the compound effects were spheroid area (µm2), the total area of the spheroid and perimeter dead cells, GFP fluorescent intensities, and PI fluorescent intensities. The spheroid area was used to determine if the drug showed growth inhibitory effects. The ratio of spheroid area/total area showed the drug effect of inducing perimeter cell death, where the spheroid remained small, but showing a layer of dead cells surrounding the spheroid. Finally, spheroid viability was determined by calculating the GFP intensity/PI intensity ratios. Each drug showed various level of effect on the tumor spheroids, and by using 3, 6, and 9 X standard deviations, each drug was assigned a value from 0 — 3 for each orthogonal assay. Finally, the values were summed to generate a drug effect score for each compound. The Celigo image cytometer enabled rapid high-throughput screening of these drugs using multi-parametric analysis. By using this novel drug effect scoring method, the panel of drugs can be quickly and efficiently screened and ranked to determine potential anti-cancer drug candidates.
• microHeart: A screening-ready, physiologically relevant human induced pluripotent stem cell-derived cardiomyocyte platform
  Fabian Zanella, StemoniX, Inc.
  Human induced pluripotent stem cells (hiPSCs) have been perceived as a powerful tool to study organ and system-specific diseases and toxicity. hiPSC-derived cardiomyocytes have been increasingly adopted in cardiac disease modeling and cardiotoxicity research. Despite their inarguable value in current research pipelines, challenges pertaining to correct cell geometry, as well as sarcomeric and cardiac cell junction assembly and organization still hinder a wider adoption of this powerful and refined model. Here describe microHeart, a novel platform formatted into high density (384 and 1536-well) screening plates that contains hiPSC pre-plated in a microstructure that emulates correct cardiac muscle fiber organization. We observed that compared to standard cell cultureware, microHeart leads to improved sarcomeric organization, as well as correct targeting of cardiac cell junction components to distal intercalated discs. We also observe improved gene expression of key components of cardiomyocyte calcium handling pathways, ion channels, cardiac transcription factors, and contractile proteins. In parallel, we observe a reduction in the expression of NPPB, a feature of increased maturity. Altogether we describe a novel hiPSC-derived cardiomyocyte platform with greater physiological relevance that is pre-formatted to high throughput screening.

Automating Novel Analytical Tools for PKA, Drug-Drug Combination and Synergy Assays, Drug Repurposing

Session Chair: Wei Zheng, National Center for Advancing Translational Sciences, NIH

• Unbiased high throughput drug combination screening identifies synergistic drug combinations effective in patient derived melanoma cell lines
  Paul Johnston, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh
  Cumulative evidence from animal and clinical studies indicates that cancer drug combinations are more effective than single-agent therapies. The most effective drug combinations increase tumor cell killing, in either an additive or synergistic fashion, by combining agents with different molecular mechanisms and non-overlapping toxicities to reduce the probability of resistance. Traditionally drug combinations were selected empirically and evaluated in clinical trials. However, even for only 100 FDA approved cancer drugs, there are 4,950 possible pairwise drug combinations which would be prohibitively expensive to evaluate in animal models, let alone clinical trials. Not all of the possible drug combinations could be justified from a mechanistic standpoint. Specific drug combinations might be selected by rational, hypothesis-driven approaches based on the altered oncogenic tumor targets and pathways identified in genomics, proteomics and gene expression studies. An understanding of the intrinsic and adaptive drug resistance mechanisms of tumors might also provide a basis for the selection of specific drug combinations. However, tumors exhibit multiple alterations to genes and signaling pathways and our understanding of drug resistance and drug mechanisms of action are incomplete. Although hypothesis-driven and empiric approaches remain important, the systematic high throughput screening (HTS) of drug combinations against panels of genetically annotated cell lines provides a complimentary strategy to identifying effective combination therapies. An unbiased HTS strategy also has the potential to identify unanticipated combinations. We developed and implemented an unbiased pairwise drug combination pilot HTS of 10 selected anticancer agents in six patient derived melanoma cell lines. Combinations of the SRC inhibitor dasatinib with either B-Raf inhibitors (dabrafenib and vemurafenib) or a MEK (mekinist) inhibitor were flagged synergistic in the screen, and were then subsequently confirmed to be synergistic in human melanoma cell lines, and in two mouse melanoma cell lines that were sensitive or resistant to dabrafenib. These data support our hypothesis that harnessing the throughput and capacity of HTS to prosecute unbiased pairwise drug combination screening campaigns provides an effective strategy for the selection and prioritization of new drug combination therapies that might be more effective in the clinic. In addition the HTS and follow up data can provide guidance for appropriate drug concentrations and ratios to be tested in vivo.
• Drug repurposing identifies drug combinations to combat emerging infectious diseases
  Wei Sun, National Center for Advancing Translational Sciences
  Repositioning of approved drugs has recently gained new momentum for rapid identification of new therapeutics for many diseases including cancer and emerging infectious diseases. However, the main issue in drug repurposing screen is the low potency of identified compounds for new indications. This limits their immediate clinical applications because the known, tolerated plasma drug concentrations are lower than the required therapeutic drug concentrations. We have developed a targeted drug combination approach that uses automated quantitative high-throughput screening to identify synergistic drug combinations. The effective drug combinations with 2- or 3-drug in a combination set can be rapidly identified that have great potential for clinical uses in patients.
• Identification of KRAS Mutant Selective Inhibitors of Pancreatic Cancer Cell Growth Via Parallel Phenotypic High-throughput Screening of Approved Drugs
  Shurong Hou, The Scripps Research Institute Molecular Screening Center, The Scripps Research Institute
  Pancreatic cancer remains a leading cause of cancer-associated death, with a 5-year survival rate less than 10%. Genetic KRAS mutations are found in more than 90% of pancreatic cancer patients. With a move toward precision medicine approaches, this study set out to identify potent and broadly effective antitumor drugs with possible subtype selectivity. To do this we developed a phenotypic 1536-well format high-throughput cell-based assay to test ~3200 FDA, EU and Japanese approved drugs. This was performed in triplicate and in parallel against 6 pancreatic cancer patient-derived cell lines (4 cancer cell lines with different oncogenic mutations and 2 cancer-associated fibroblast lines). Assay quality was notable with Z' averaging 0.81 ± 0.06 across all assays and cell lines. All the active compounds identified against each cell lines were selected to determine their cytotoxicity concentration 50% (CC50) in each of the 6 cell lines. This parallel HTS effort leads to the discovery of 25 compounds among the approved drugs that inhibit the growth of pancreatic cancer cells with observed CC50
• Retrospective Analysis of 18 Activity Based Protein Profiling HTS Assays for the Identification of Selective Inhibitors of Disease Relevant Enzymes
  Virneliz Fernández Vega, Scripps Florida
  Many diseases are related not only to aberrant expression but, also post-translation modification; ultimately effecting enzyme activity. Addressing well characterized and fully functional enzymes utilizing highly tailored substrates is not always achievable, affordable, nor disease relevant. To tackle these issues and to further our understanding of disease relevant enzymes, Scripps has completed 18 HTS campaigns testing the full NIH library which, currently encompasses 375K compounds. The targets studied cover a broad spectrum of diseases including cancer and neurological disorders and in many cases exploit an extremely powerful chemical biology tool incorporating a fluorescent labelled cysteine or serine reactive substrate probe which is used in Fluorescence Polarization (FP) assays to observe activity based enzyme activity. Custom-synthesized fluorescent probes with high affinity to a selected enzyme feature, typically located in the enzyme's active site, were utilized to test a compound's ability to effect the enzyme which at times even identified covalent modifiers. Multiple structural classes with incredible affinity and specificity to a particular enzymes active site were identified. Here, we present a retrospective analysis on the outcomes of the HTS vs. 18 targets that exploit fluorescence polarization- activity-based protein profiling (fluopol-ABPP) and the types of modifiers found. The success of this technology is apparent simply by acknowledging discovery of 10 out of the 18 licensed molecules coming from the Molecular Libraries Probe Center Network initiatives.

In-house Automation: Devices and Software Developed Internally

Session Chair: Rob Keyser, Calico Life Sciences

• Global Innovations and Technologies Team (GLINT): Building (in-house) Advancements for Science and Technology
  Matthew Boeckeler, AstraZeneca PLC
  The Global Innovations and Technologies Team is a global, cross-functional team charged with creating value through the development of advanced, high-impact, innovative solutions to challenging technical and scientific issues. The main challenges to developing in-house solutions are resource availability (funding and staff), and the expertise to execute such projects. The GLINT Team overcomes these obstacles by employing global, cross functional teams of individuals with diverse areas to deliver solutions in an expedited fashion. Global resourcing helps to not only expedite development, but allows for diversity of ideas, approach and solutions in a resource efficient manner. This model helps to maintain momentum for projects, while giving global visibility into the teams activities. Internal expertise in systems design, fabrication and software development help to drive and facilitate in-house development for systems and integrations. The implementation of 3D printing technologies is used to provide in-house manufacturing capabilities, rapid prototyping, as well as production of beta system components. This talk will further outline the processes employed by GLINT to ensure project scope is in alignment with business need, ensures the project deliverables meet requirements, value created by these solutions, and impact of the Team on Global Technologies. Examples such as the in-house development of an In Vitro sampling and quantification system for bacterial based assays and the integration of an automated system for performing plate-based x-ray crystallography. Projects will be presented that show the power and diversity of AstraZeneca's in-house capability for automated platform development, software control systems while showcasing its successes.
• Automated Magnetic Bead Protein Purification
  Justin Provchy, Amgen
  Screening of large panels of cell culture for protein expression results in numerous samples which require removal of the cells, filtration and purification. Amgen's Research and Automation Technologies group partnered with Amgen's Biologics group to implement a novel process and the enabling technologies to employ magnetic beads to greatly accelerate the protein purification workflow. Traditionally, cell culture samples required multiple conditioning steps, such as filtration and clarification, prior to final purification using UPLC. Presented will be a novel platform for automating purification using magnetic beads, allowing for rapid batch sample processing and eliminating sample conditioning and the need for UPLCs. Magnetic bead purification with Magneto is at least 10-fold faster than manual processing and will provide savings on consumables as the process can been completed using the original cell culture vessel. Highlighted will be the novel technologies developed in-house to support this process and their creation using computer assisted design (CAD), 3D printing, laser cutting, CNC machining and programming of control software.
• Lab-Made Technology for NGS Library Construction at the BC Cancer Agency
  Robin Coope, BC Cancer Agency Genome Sciences Centre
  The Instrumentation Group at the BC Cancer Agency's Genome Sciences Centre (GSC) is unusual in having an engineering group and extensive prototyping facilities embedded in a biomedical research and clinical cancer treatment facility. The group has completed numerous pipeline-specific automation projects, and in one case, enabled the GSC to participate in a major international consortium based on our in-house technology. In-house solutions range from simple manual devices to components integrated with liquid handlers, to full robots as well as ongoing programming of the GSC's liquid handlers. This presentation will highlight significant projects and some lessons in efficient prototyping and good software practices that have been learned. Our largest lab-made automation project has been "Barracuda", a 96 channel, gel-based size selection robot that has run ~20,000 samples to date and enabled the GSC to participate in The Cancer Genome Atlas (TCGA) project, performing all the miRNA library construction and sequencing for TCGA. The size selection technology subsequently spun off as Coastal Genomics and is available via a partnership with Hamilton Robotics. Efficient prototyping was key to getting Barracuda from conception to production in five months and is thus a great example of the power of "2D prototyping". This method is centered around water jet cutting, sheet metal bending, and finishing with in house powder coating, all of which is available in our hospital machine shop. In this case we were able to build a unique automation system from recycled linear stages where only one new component, the gel tray, required traditional CNC machining. A challenge revealed by Barracuda was in refilling the gel trays to create the "consumable" and integrating that activity with other production work. Conversely, the project demonstrated the advantage of iterating the software in close collaboration with end user technologists and being able to rapidly respond to evolving lab requirements. The group's other role is programming the GSC's commercial liquid handling robots, working closely with our methods development, quality assurance and production teams. This joint approach, with expert molecular biologists working in tandem with expert programmers and independent QC, has produced robust methods for a variety of sample types. In fact, we have harmonized chemistry and liquid handling protocols to the extent that seven different NGS library types (PCR-free gDNA, FFPE DNA, RNAseq, CHiP, Bisulfite Seq, (exome or other) capture samples and circulating cell free gDNA) run under one main program. This has substantial advantages as well as potential disadvantages which will be described.
• The PetriJet laboratory automation platform — one device to rule them all
  Christoph Otto, University of Technology Dresden
  The field of laboratory automation targets recurring processes in a laboratory environment which are monotonous, time consuming but demanding in quality uniformity. The automation of those processes leads to a higher sample throughput, highly qualitative results and reliable data material. Although culture dishes are currently used in all fields of microbiological research and analysis, only little solutions dealing with dish handling and manipulation have actually been established. Especially for small scaled laboratories with changing requirements there is no adequate system available. To fill this gap the modular culture dish handling platform, called PetriJet, has been developed. The compact device itself represents a high throughput dish processing unit with exchangeable stacks and processing stations. The only manual task the user has to do is to provide filled culture dish stacks to be treated and remove them after a run. And even this step is going to be automated in further developments. When starting the machine, the dish stacks are transported from a storage unit to the platform and detected by a specially developed gripper which transports every single dish to a processing station. The gripper allows the processing of unsealed and sealed dishes of different diameters with a unique separation unit located on the gripper arms which decollates sealed dishes sticking together. The processing station is the key component of the platform. The device provides enough space for several stations, therefore it is possible to pose a complex workflow within one run. At the current developmental stage a fully automated image acquisition station is implemented in the PetriJet platform. The Images of the dishes can be used for documentation, quantitative analysis such as colony counting or qualitative analysis e.g. for positive/negative tests. With the implementation of a real-time automatic image analysis any treated culture dish can be sorted in different release stacks. After the processing is done, the stack is transported back to the storage system. Taken together the device has the capacity to process more than 30 stacks per load, each filled with 20 Petri dishes, so high throughput applications with 600 dishes are possible. Current developments dealing with the labelling and allocation of the dishes to keep the possibility of a correct assignment between image and culture. Beyond that new processing stations are in the stage of conception like an automated nutrient medium filling station. With different processing stations the PetriJet will provide a large variety of functions in one bench-top lab automation device.

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Cellular Technologies Track

Track Chairs: John Doench, the Broad Institute and Benjamin Haley, Genentech

Advances in Genome Editing Technologies

Session Chair: Gregory Davis, MIlliporeSigma

• Comparison of Cas9 activators in multiple species.
  Alejandro Chavez, Wyss Institute at Harvard
  Cas9 has taken the scientific community by storm due to its ease of engineering and its ability to be targeted to nearly any genomic locus. One of the applications of the technology that has received considerable attention is Cas9-based transcriptional activators. To date, there are at least eight different Cas9 activator systems that have been published. Sadly, due to differences in cell line, target gene, method of activator delivery and guide RNAs employed, it remains difficult for the community to gauge the relative efficiencies of the different Cas9 activators. As a result, it is unnecessarily challenging to select the most efficacious system for a given application. To provide much needed guidance, we have undertaken a systematic study in which we directly compare the various Cas9-based activator systems. We find that three in particular &madsh; VPR, SAM, and Suntag — are the most potent Cas9-based activators. We validate these results within several human, mouse, and fly cell lines to ensure that trends we observe are not unique to a given cell line or locus. In addition to our comparative analysis, we also sought to combine the most potent activator systems to determine if a more powerful hybrid activator could be created. Finally, in cases where maximal levels of gene induction are required, we demonstrate that using multiple gRNAs per target is highly effective and functions to increase the amount of activation independent of the Cas9 system employed.
• Genome engineering with targeted recombinases
  Thomas Gaj, University of California, Berkeley
  Site-specific recombinases (SSRs) are tremendously valuable tools for basic research and genetic engineering. By promoting high-fidelity DNA modifications, SSRs have empowered researchers with unprecedented control over diverse biological processes, enabling countless insights into cellular structure and function. The strict specificities demonstrated by SSR systems, however, has limited their adoption in fields that require tools with flexible targeting capabilities. To overcome this, protein engineering has been used to alter SSR specificity. While this strategy enables the isolation of SSR variants with new properties, it can also reduce their specificity and utility. Hybrid recombinases consisting of a serine recombinase catalytic domain fused to either a custom-designed zinc-finger or transcription activator-like effector DNA-binding domain represent a potential solution to this problem. Unlike conventional SSRs, hybrid recombinase specificity is the product of modular site-specific DNA recognition and sequence-dependent catalysis. Thus, new recombinases with distinct targeting capabilities can be generated in a "plug-and-play" manner. We have demonstrated that hybrid recombinases generated by this approach can modify user-defined DNA targets and direct site-specific integration into endogenous genomic loci in human cells.
• Using engineered model organisms and high throughput screening tools to automate and scale the drug discovery process for rare genetic diseases
  Tom Hartl, Perlstein Lab, PBC
  Perlstein Lab, PBC (PLab) is a San Francisco-based biotech startup using model organisms to discover small-molecule drug candidates for individuals with rare genetic diseases. We engineer model organisms such as C. elegans and D. melanogaster to carry rare genetic disease mutations, and use them as the foundation for high throughput phenotypic screens to discover small molecules that rescue the disease. Specifically, we engineer the organisms using CRISPR, phenotype the mutated organisms, develop a scalable screening assay based on the phenotype, screen our 50K compound library, and then validate hits in the organisms and patient cells. We rely heavily on high throughput equipment such as the Echo acoustic liquid handler, the BioSorter for organism dispensing, and the IXM for high throughput imaging for our rapid and efficient hit discovery pipeline. In addition, Modular Science built us a custom imager and analysis tool to automate the D. melanogaster screen readout. Using model organisms and these HT tools for screening, we were able to complete a screen and discover a lead therapeutic candidate for our first disease indication, Niemann Pick Type C, in under one year. In addition, in collaboration with Vium Inc, we are able to quickly move our lead compound into a mouse disease model and uncover novel metrics to monitor disease progression. With our focus on efficient and low-cost phenotypic screening from the engineered model organism to the mouse model, we are able to automate and scale the drug discovery pipeline for rare genetic diseases.
• Genome ad Epigenome Editing with ZFNS and CRISPR
  Gregory Davis, MIlliporeSigma
  Zinc finger nucleases and CRISPR/Cas systems have radically changed questions which can be posed in experimental biology. Mouse genetic models mimicking human disease have been a dominating technology in past decades and can now be verified by CRISPR modification of genes in human cells or in other organisms (rats, pigs, etc.) which may better model certain human disease phenotypes. Despite huge success in creating gene knockouts in a wide array of organisms, the ability to edit genomes to have user-defined genome modifications is still largely limited by the potential of various cell types to integrate synthetic DNA via homologous recombination (HR). Various cell treatments and donor DNA formats to address HR limitations will be discussed. The simplicity and low cost of programming CRISPR-Cas systems has created possibilities for high throughput, genome wide interrogation of gene function. For example, a drug treatment of cells can be characterized by knocking out tens of thousands of genes in parallel to identify pathways that lead to drug resistance. While this mode of genome wide genetic interrogation has been available via RNAi and transposons for years, CRISPR enables are very controlled and effective method of eliminating human gene function. Various new applications of CRISPR screening will be discussed. Beyond editing the genome at the base pair level, new formats have been developed which enable editing of the epigenome. In this talk, a new epi-CRISPR technology will be discussed which allows acetylation of histones to open up chromatin landscapes to allow local gene expression and/or association of enhancers with distant regulatory elements.

Development of Cellular Models for Phenotypic Screening

Session Chair: Joel Klappenbach, Merck & Co., Inc.

• Systematic validation of disease-specific genetic association data in cellular models
  Joel Klappenbach, Merck & Co., Inc.
  Human genetics provide a means to assess the potential efficacy and safety of therapeutic targets prior to investment in drug development in the laboratory. Genetic association data establishes a link between a conserved region of the genome and disease occurrence or phenotype, but rarely a direct casual mechanism. While SNPs identified through GWAS analysis can be directly located in protein-coding genes leading to direct loss-of-function, the majority of GWAS marker SNPs are located in intergenic regions. Intergenic SNPs can affect protein-coding genes by modifying binding sites of transcriptional effector proteins. Predictive bioinformatic analysis can be used to estimate the potential functional consequences of coding and non-coding mutations, yet functional laboratory validation of GWAS data is required to definitively establish casual linkage between genes and disease phenotypes. In vitro cellular models that accurately recapitulate human disease phenotypes are required to validate genetic association data. Within these models, CRISPR/Cas9-mediated genome engineering provides the means to evaluate the effects of single-nucleotide polymorphisms, gene inactivation, inhibition and over-expression on disease phenotypes. We are applying CRISPR gene editing for human genetic target validation using a variety of in vitro cellular systems including immortalized cells, primary cells, iPSCs and organoids. Using arrayed CRISPR-based gene inactivation and overexpression, we are screening for casual gene(s) within significant GWAS loci for diseases including Parkinson's disease and inflammatory bowel disease. Using pooled genome-scale CRISPR screening, we are identifying druggable regulators of genetic targets, causal genes within GWAS loci, and the mechanisms of action for small molecule hits uncovered through phenotypic screening. As the ability to use genome-engineering tools in primary cellular models continues to expand and improve, we anticipate our ability to more rapidly identify causal human genetics will translate into improved therapeutic success in the clinic.
• Calibrating human adipocyte assays for type 2 diabetes target validation
  Amit Majithia, Broad Institute/ Massachusetts general hospital
  A critical challenge in target validation and drug discovery is the development of preclinical assays that predict whether a therapeutic will ultimately succeed in the clinic. In order for a cellular assay to predict clinical response, it must read out a biological function that, if perturbed by a drug, will yield benefit to the patient. For most diseases, preclinical assays are poorly predictive of clinical response, resulting in expensive failures in late-stage clinical trials. Human genetics offers a method to forge a critical connection between human disease phenotypes in vivo and preclinical assays in vitro. By assessing genetic variants both for association to disease in populations, and to functions in the laboratory, it should be possible to triangulate on disease-relevant functions of cells. However, as genes have many variants, and cells have many functions, it can be far from straightforward to sort out which variants and which cellular assays are most relevant to a given disease. Genome sequencing of large cohorts now makes it possible to mine human genome sequence variation for "experiments of nature" that perturb the functions of a wide range of genes. In some cases, these "experiments of nature" can be used to infer a dose-response curve of gene function that indicates how enhancement or suppression of the encoded protein's activity raises or lowers disease risk. In the well known case of PCSK9, for instance, loss-of-function mutations decrease LDL and cardiovascular disease risk, while gain-of-function mutations increase LDL and cardiovascular disease risk. Clinical trials suggest that inhibiting PCSK9's encoded protein is a promising strategy for lowering LDL and preventing cardiovascular disease. Type 2 diabetes and the insulin resistance that underlies it are physiologic disruptions with multi-organ dysfunction. We know that adipocytes, cells that store excess nutrients and signal energy homeostasis to other organs, are key to pathogenesis, but lack adequate cell culture models and assays which have been credentialed to report on disease states in vivo. We have developed a platform centered on human adipocytes consisting of 1) a suite of genetic perturbation technologies, 2) highly parallelized cell culture, and 3) high throughput functional assays to enable credentialing and pharmacologic screening of adipocyte functions. We present an application of this platform deployed to build and calibrate a disease-relevant assay for adipocyte differentiation, a process that predisposes to insulin resistance and diabetes when dysfunctional in vivo.
• Developing assay technologies to realize the potential of iPSC technology in drug discovery
  Han Xu, Amgen Inc
  As iPSC and genome editing technologies go through rapid development and exert their revolutionary effect in the biomedical fields, more and more novel technologies in these two areas are being adopted by drug discovery industry. However, the requirement of drug discovery process presents unique challenges of applying both iPSC and genome editing technologies efficiently. In this presentation, I will use two specific cases to detail these challenges and discuss solutions that can not only overcome these challenges, but also provide new opportunities to further improve their application. The 1st example arises from the need to measure the effect of cardiac modulators on contractile force in addition to Ca2+ flux using iPSC derived cardiomyocytes. To overcome this challenge, we have developed plate-based measurements that perform both measurements simultaneously. The 2nd example comes from the need to perform efficient genome editing in human iPSC that can ensure a homogenous cell population with the targeted gene in order to fully understand disease mechanism through gene expression and function analysis. I will discuss the options we evaluated and solutions developed to increase confidence of homogeneity and decrease time line.
• Toward primary drug screening using iPSC-derived human motor neurons in 1536-well plates
  Zhong-Wei Du, BrainXell, Inc.
  The personal, societal, and economic burden from disorders of the central nervous system (CNS) is enormous, and novel therapeutics are in urgent need. Neurons differentiated from induced pluripotent stem cells (iPSCs) present new opportunities for modeling disease processes and screening drug libraries. Patient iPSC-derived neuronal subtypes provide a naturalistic in vitro high-throughput-screening (HTS) platform to identify promising drug leads. We have developed technologies to generate large quantities (billions in one batch) of highly enriched (~90%) neuronal subtypes from patient iPSCs and to enhance the maturation of these neurons within one week for phenotypic presentation. Toward drug screening, we developed two screening systems using human motor neurons derived from individuals with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Using genome editing techniques on the iPSC lines, the reporter nanoluciferase (NLuc) was fused to endogenous neurofilament light chain (NF-L) for ALS and to the full-length SMN2 for SMA. Nanoluciferase is more sensitive than the traditional luciferase and allows for a simple and quantitative readout of NF-L and SMN2 protein levels. The assays were adapted to meet HTS requirements, including: large batch sizes, 1536-well format, minimal well-to-well variation, short-term culture, plating by automated dispenser, and low reagent volumes. Applying a quantitative HTS approach, we screened the LOPAC, NPC, and MIPE libraries (>6,000 compounds) in a dose dependent manner on both motor neuron lines with a hit rate of ~0.5%. These hits were validated in 96-well format using freshly prepared compounds. This work demonstrates the feasibility of using human neurons to conduct drug screenings in biological systems that more closely resemble the diseases as they exist in man.

Genetic Screens for Target Discovery and Validation

Session Chair: Louis Staudt, National Cancer Institute

• Therapy of Lymphoma Inspired by Functional and Structural Genomics
  Louis Staudt, National Cancer Institute
  Diffuse large B cell lymphoma (DLBCL) is a heterogeneous diagnostic category that is comprised of two prominent molecular subtypes, termed activated B cell-like (ABC) and germinal center B cell-like (GCB), which are now recognized as pathogenetically distinct diseases. Genetic screens using RNA interference and CRISPR-Cas9 have revealed different oncogenic signaling pathways in these DLBCL subtypes can be blocked by targeted therapeutic agents. We defined a "chronic active" form of B cell receptor (BCR) signaling that activates NF-κB in ABC DLBCLs. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as the kinase BTK or components of the BCR itself. Over one fifth of ABC DLBCLs have mutations affecting the CD79B or CD79A subunits of the BCR that augment BCR signaling. To attack chronic active BCR signaling therapeutically, we initiated clinical trials in relapsed/refractory DLBCL of ibrutinib, an irreversible and highly selective inhibitor of BTK. Ibrutinib monotherapy induced a high rate of complete and partial responses in ABC DLBCL, while GCB DLBCL tumors rarely responded. We have also defined other oncogenic signaling pathways in ABC DLBCL that cooperate with BCR signaling to sustain cell survival, including the MyD88 pathway, which is activated by oncogenic MYD88 mutations in ~40% of cases. DLBCL tumors with both MYD88 and CD79B mutations responded frequently to ibrutinib, and other lymphoma types that harbor these two mutations also respond well. We have demonstrated a functional crosstalk between the MYD88 and BCR pathways in ABC DLBCL cases with both mutations, and the mechanisms underlying this phenomenon have been revealed by CRISPR-Cas9 genetic screens, which will be discussed.
• Pooled genome-wide screens as a powerful tool for discovering drug resistance genes in cancer
  Federica Piccioni, Broad Institute of MIT and Harvard
  One of the main causes of failure in the treatment of cancer is the development of drug resistance by the cancer cells. Drug treatments successfully inhibit the tumor growth, but the clinical benefit is transient; the majority of patients ultimately develop resistance treatment. The actual challenge is to identify genes driving the drug resistance, new potential drug targets and develop more effective therapeutic combinations. Pooled genetic screens are an effective technique that offers improvements in speed and scale compared to arrayed-based screening. The power of pooled genetic screens is particularly evident in the setting of positive selection, such as identifying genes that confer drug resistance. Also, the development of CRISPR/Cas9 and ORFs technologies has made possible to perform loss-of-function and gain-of-function studies at genome scale to broadly interrogate mechanisms of drug resistance. A subset of drug targets/cancers, representing a diverse range of cancers chemotherapy and targeted therapies, were screened for causal drug resistance perturbations using genome-scale ORF and CRISPR lentiviral libraries. We have completed over seventy individual functional screens using over ten distinct cell types in presence of different drug panels. Our screens identified classical genes involved in resistance mechanisms as well as novel and potentially druggable genes. The identification of resistance mechanisms to targeted and traditional cytotoxic therapies can be used to evaluate the most effective path forward for resistance studies to new therapies that reduce the incidence of and time to therapeutic relapse within selected tissue/tumor types. Moreover, it helps the understanding of the biology of tumor responsiveness to inhibition of specific targets or cytotoxic drugs.
• Target Devalidation by CRISPR/Cas9 Technology
  Lorenz Mayr, AstraZeneca
  Recent advances in the field of Precise Genome Editing (PGE), particularly around CRISPR/Cas9, Cpf1, and other RNA/DNA-guided endonucleases, enable identification and validation of drug targets at unprecedented ease, speed and precision. It is believed that CRISPR/Cas9 has the potential to substitute the currently used technologies for target validation which are mostly based around RNA-interference (RNAi), such as siRNA, shRNA and their variations. These technologies can cause off-target and/or unspecific systemic effects in cells and animals.
  
  The protein MTH-1 (MutT Homolog 1) has been described by the use of RNAi technologies as a novel anti-cancer drug target (Gad, H. et al. (2014) Nature 508, 215-221; Huber, K.V.M. et al. (2014) Nature 508, 222-227). MTH-1 is a purine nucleoside triphosphatase which hydrolyses oxidised nucleotides into mono-phosphate forms to prevent damaged bases from being incorporated into DNA, hence MTH-1 is believed to play an essential role in the survival of cancer cells. It has been postulated that inhibition of the enzyme will provide a completely novel avenue for the development of new anti-cancer treatments. Hence, these findings have triggered a world-wide race towards further target validation and the generation of novel small-molecule inhibitors for that enzyme.
  
  The current presentation from AstraZeneca will describe the generation of highly potent chemical molecules and the generation of CRISPR/Cas9 gene knock-out cell lines for further validation of MTH-1 as a drug target. During the course of that study, the use of HTS, FBS, structural biology, biophysics and cellular profiling has resulted in pico-molar inhibitors of MTH-1 with cellular activity confirmed by target engagement assays (CETSA). These compounds have been tested in wildtype and CRISPR-generated MTH-1 knockout cellular tumor models. Our studies seem to indicate that MTH-1 inhibition has no effect on the proliferation of cancer cells and question the overall concept of MTH-1 as a drug target (Kettle, J.G. et al (2016) J. Med. Chem 59, 2346-2361). To the best of our knowledge, the present study is the first published example of target devalidation by the use of CRISPR/Cas9 technology.
• High resolution CRISPR screens for mapping the human essentialome
  Jason Moffat, University of Toronto
  The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. Forward genetic screens with CRISPR-Cas9 genome editing enables high-resolution detection of genetic vulnerabilities in cancer cells. To extend the catalog of human core and con-text-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in a small number of human cell lines. Using an improved Bayesian analytical approach, we consistently dis-cover 5-fold more fitness genes than were previously observed and present a list of >1,500 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known onco-genes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. We also conducted genome-wide CRISPR-Cas9 screens in RNF43 mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation, and discovered a unique requirement for a Wnt signaling circuit specifically engaging a single FZD receptor. Our results highlight an underappreciated level of context dependent specificity at the receptor level in RNF43 mutant PDAC cells. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirm that functional specificity cannot be explained by protein expression patterns alone. Moreover, synthetic human anti-FZD antibodies robustly inhibited the growth of RNF43 mutant PDAC cells grown in vitro and as xenografts in vivo, providing strong orthogonal support for the functional specificity observed genetically. Lastly, proliferation of a patient-derived PDAC cell line harboring a RNF43 variant previously associated with PDAC was also selectively inhibited by the anti-FZD antibodies, further demonstrating their use as a potential targeted therapy. Taken together, genome-scale forward genetic screens with pooled CRISPR-Cas9 libraries is an efficient method for identifying therapeutic targets for cancer.

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Data Analysis and Informatics Track

Track Chairs: Lenny Teytelman, Protocols.io and Margaret DiFilippo, Dotmatics

Let There Be Light: Informatics Approaches to Exploring the Dark Genome

Session Chair: Rajarshi Guha, NCATS

• Illuminating the Druggable Proteome
  Tudor Oprea, UNM School of Medicine
  We evaluated, organized and distilled over 80 protein-centric and over 20 gene-centric resources for human proteins, as part of the "Illuminating the Druggable Genome Knowledge Management Center" , IDG-KMC, [1]. We currently focus on four protein families: G-Protein Coupled Receptors, Nuclear Receptors, Ion Channels and Kinases, using data wrangling coupled with algorithmic processing, text mining of drug labels, the patent corpus, medical literature as well as human curation and drug-target ontology development. Tissue expression data from GTEx [2], the Human Proteome Map [3], the Human Protein Atlas [4] and other sources, disease-centric text mining, genome-wide association studies and other resources are integrated using a number of specialized ontologies, e.g., the Brenda Tissue Ontology [5] and the Disease Ontology [6]. IDG-KMC is developing a specific drug-target ontology to better integrate bioactivity [7] and drug information [8], and catalogs 20,186 proteins using metrics from text mining [9], gene reference into function [10], and the number of antibodies [11]. Of these human proteins, 37.6% (7,583) are categorized as "Tdark", i.e., proteins that lack functional information and disease relevance. Another 601 proteins (3%) have a confirmed drug mechanism of action ("Tclin") [12]. Two other categories reflect levels of literature, functional and disease annotations ("Tbio"), as well as knowledge about (potent) small molecules ("Tchem"), respectively. Pharos [13], the IDG-KMC interface portal supports mining and interactive browsing of this multi-dimensional data collection, providing informative summaries for the broader scientific community. Our integrative effort supports the following observations: i) there is a knowledge deficit, i.e., we lack understanding of protein function for over 37% of human proteome; and ii) only 3% of the human proteome is therapeutically addressed by drugs, although this value is higher for the 4 target families outlined here. These observations are confirmed by mining the patent corpus (1981-onrwards), by examining R01 funding patterns (2011-2015), and disease associations. Global drug sales data from 75 countries (2011-2015) provided by IMS Health [14] confirm that GPCRs, ion channels, kinases and nuclear receptors are very lucrative targets. References: [1] IDG-KMC website: http://targetcentral.ws/ [2] GTEx portal: http://www.gtexportal.org/ [3] HPM website: http://humanproteomemap.org/ [4] HPA website: http://www.proteinatlas.org/ [5] Brenda Tissue Ontology available at https://bioportal.bioontology.org/ontologies/BTO [6] Disease Ontology website: http://disease-ontology.org/ [7] ChEMBL portal: https://www.ebi.ac.uk/chembldb/ [8] DrugCentral portal: http://drugcentral.org [9] Text mining resources from JensenLab: http://jensenlab.org/ [10] NCBI Gene Reference into Function: http://www.ncbi.nlm.nih.gov/gene/about-generif [11] Antibodypedia website: http://www.antibodypedia.com/ [12] R. Santos, O. Ursu et al., A Comprehensive Map of Molecular Drug Targets. Nature Rev. Drug Discov, accepted. [13] Pharos user interface portal: https://pharos.nih.gov/ [14] Data from the IMS Institute for Healthcare Informatics, http://www.imshealth.com/
• Open Targets Platform: mining gene-disease evidence for improved drug target selection
  Denise Carvalho-Silva, Open Targets — EMBL-EBI
  Open Targets is a public-private partnership made up of four global leading institutions in the fields of pharmaceuticals, bioinformatics and genomics, GSK, EMBL-EBI, the Wellcome Trust Sanger Institute, and Biogen. We combine large-scale genomic experiments with objective statistical and computational techniques to identify and validate the causal links between targets, pathways and diseases. We have recently designed and developed the Target Validation platform, a web application for data integration and visualisation, which supports both target- and disease-centric workflows. Our platform enables biomedical researchers to discover and prioritise biological targets for new therapies. Targets may be a protein, protein complex or RNA molecule, and we integrate evidence through the target gene. Phenotypes and diseases are integrated through the Experimental Factor Ontology, so that we can include both Mendelian (rare) and common diseases and their phenotypes. We derive evidence of association between a target and a disease from multiple public domain resources, including germline and somatic genetics, known drugs, gene expression profiling, reaction pathways, murine genetic models and the scientific literature. We also provide an association score, which takes into account the observed frequency, the experiment confidence, and the likely strength of the effect of the target on the disease. By drawing on expertise in product and platform development including product testing, UX design and site development, we have created this comprehensive and robust data integration for access and visualisation. In addition, programmatic retrieval of data via REST services is also available. In this talk, I will introduce the Open Targets project, the Target Validation platform and demonstrate how it can be used to visualise and interpret target-disease associations based on the different datasets available.
• Dr. Gene Budger: A Web Application to Predict Drugs to Modulate the Expression of a Specific Gene
  Avi Ma'ayan, Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai
  This presentation will be recorded at SLAS2017 and made available free-of-charge to SLAS dues-paying members and full conference attendees post-event.
  
  Mechanistic molecular studies in biomedical research often discover important genes that are aberrantly expressed in disease. However, manipulating these genes' expression to attempt to improve the disease state is challenging. Here we present Drug Gene Budger (DGB), a web-based application developed to assist investigators with prioritizing small molecules that are predicted to maximally influence the expression of their gene of interest. With DGB, users can enter a gene symbol and specify whether they wish to upregulate or downregulate its expression. The output of the application is a ranked list of small molecules that have been experimentally determined to produce the desired expression effect. The table includes log-transformed fold changes and p-values for each small molecule, reporting the significance of differential expression as determined by the limma method [1]. Relevant links are provided to further explore knowledge about the target gene, the small molecule, and the source of evidence from which the relationship between the small molecule and the target gene was derived. The experimental data contained in DGB is compiled from signatures extracted from the LINCS L1000 dataset and the Gene Expression Omnibus (GEO) [2, 3]. DGB provides a useful preliminary technique for identifying small molecules that can target the expression of single genes in mammalian cells. The prototype application is available at: http://amp.pharm.mssm.edu/DGB.
• Gene-network based predictive modeling to identify biomarkers for high dimensional genomic data
  Viswanath Devanarayan, AbbVie
  Identification of biomarkers with prognostic or predictive power has been an interesting topic in both academia and industry. However, interpretation of biological significance of identified biomarkers has been a challenge. Genes and proteins work in network rather than individually and therefore hub genes within biological network might play a central role in multiple biological processes and functions. Reconstruction of gene regulatory networks can help us identify hub genes. Here we demonstrate how the incorporation of hub genes to the predictive model building process for genomic data can yield more biologically meaningful signatures without compromising predictive performance. We illustrate this using data from a Neuroblastoma study.

The Challenges and Benefits of Collaboration

Session Chair: Farida Kopti, Merck & Co

• How Can Informatics Systems Help External Collaborations Succeed?
  Robert Brown, Dotmatics Inc
  It has now become standard practice for research projects in pharma and biotechs to be conducted with external partners in a variety of arrangements ranging from fee-for-service CRO projects to joint discovery. With such large portions of research budgets being directed towards collaborations, the question now becomes the extent to which these arrangements are demonstrating the appropriate return on investment — whether that is measured by increased innovation, reduced cost, or accelerated cycle times? Evidence is mixed but by some estimates 60-70% of collaborative partnerships are not delivering their intended outcomes. There are a variety of reasons that have been put forward, including cultural and even legal issues. Among them is the lack of a supporting informatics infrastructure to facilitate efficient data sharing and communication within a distributed project team, despite a number of commercial systems become available over the last few years specifically designed to for this purpose. We have commissioned an industry survey to gather evidence on the current success rates in collaborative research, and understand the state of adoption of informatics infrastructure supporting collaborations today in pharma and biotech. In this presentation, we will first report on those survey results, which suggest that there is still a maturity gap between the externalization of research and the provision of dedicated informatics systems to support it. We will then discuss a set of criteria that an organization can use to determine if a system could help increase the ROI on their collaborations, and the characteristics of currently available solutions to illustrate the benefits that can be gained. Finally, we will report our own customer case studies to provide real world examples of the benefits.
• 'Virtual' R&D; in Pharma: What are the Virtues (and Vices)?
  Steven Wesolowski, Xenon Pharmaceuticals
  Drug invention and development are intrinsically collaborative endeavors — unlike other areas of technology, nobody can (legally) run a successful pharmaceutical company single-handedly from concept to drug launch in their garage. "Virtual" R&D groups take collaborations to the logical extreme by relying entirely on partnerships with CROs and collaborators to execute drug discovery and development programs. This talk outlines the structure of one virtual R&D; group and highlights both the benefits and challenges of such an environment.
• How to solve the logistical challenges of collaborating to enable efficient access to innovation and expertise.
  Elizabeth Iorns, Science Exchange
  As research has become increasingly specialized and complex, there has been a significant rise in outsourcing, where research is conducted externally by a highly fragmented network of experts often located at contract research organizations and academic institutions. Identifying appropriate resources and managing these relationships raises significant logistical hurdles for organizations. Utilizing software to connect with and manage external collaborations provides an opportunity to solve some of these logistical hurdles and can enable efficient access to the external global network of innovation and expertise. This will be a competitive differentiator for research organizations and is critical for success in the modern research environment.
• Utilizing Cloud Technology to Solve Challenges in Modern Laboratories
  Marc Daxer, Fraunhofer Institute for Manufacturing Engineering and Automation IPA
  Exchanging data between laboratories can be challenging: internal data management can be facilitated with laboratory information systems (LIMS), however, these are rarely interconnected across companies. Furthermore, migrating processes across laboratories is nearly impossible without effort. Setting up a federated cloud-infrastructure can help to overcome these difficulties: multiple laboratories participate in a federation. Customers order services and may explicitly authorize laboratories to cross-access their data. Ultimately, obliterating the barrier between computational- and wet lab services results in an easy integration of different services to meet the personalized needs of customers: bioinformatics analyses, provided as Software as a Service in the cloud, can be based on data created by experiments conducted in laboratories by different service providers. Permissions management is done by the cloud-platform, which guarantees isolation of data. To facilitate this obliteration of barriers, we demonstrate an easy integration of automated wet-lab processes into a cloud. We explain, how standardized laboratory devices can be integrated into a cloud-lab, which can easily be replicated and modified on demand. Finally, we conclude with a proof-of-concept — demonstrating aforementioned use case and postulating that the time is ready for the emerging of numerous cloud-laboratories.

Enhancing Scientific Reproducibility and Reuse Through Better Workflow and Data Technologies

Session Chair: Timothy Gardner, Riffyn

• Standing up for the quality of our data: A voluntary commitment to research quality assurance.
  Rebecca Davies, University of Minnesota College of Veterinary Medicine
  Strategies to improve the way that scientific research is performed have been initiated by entities that fund and publish research effort. For example, efforts are underway by the National Institutes of Health and prominent publishing groups to identify opportunities to improve research quality and reproducibility (https://www.nih.gov/research-training/rigor-reproducibility). Recently, these entities have provided expanded publication guidelines and grant application instructions as a mechanism for influencing the reproducibility of research outcomes. While these measures are promising, they fail to mitigate the risk associated with inconsistent research data management and record keeping that is typically unaddressed in our research training programs. In addition, these measures may not provide the tools scientists need to demonstrate the quality of their work. Research Quality Assurance (QA) systems are used to monitor and support the processes by which scientific data are generated and managed. Quality systems provide assurance through the generation of credible evidence (documentation) that data are fit for their intended purpose and that the processes under which they have been generated can be reconstructed. Responsible agencies mandate robust research QA systems in formally regulated research environments as a way of supporting data reliability. However, in basic research programs, QA systems are rare, even though the voluntary adoption of research QA best practices (also known as 'Good Research Practices, GRP) have been recommended for many years (Volsen SG Kent JM, et al. 2004. Drug Discovery Today.9:21;903-905; WHO. Quality Practices in Basic Biomedical Research. World Health Organization. Geneva, Switzerland: WHO/TDR). Therefore, most scientists remain unaware of how the use of QA can improve research records and increase the likelihood for successful data reconstruction and research reproducibility. The scarcity of QA training within academia may be attributed to a lack of institutional expertise in QA and the nearly universal lack of targeted funds to support quality systems. However, a commitment to a modest set of good research practices is an achievable goal. Biomedical research scientists must be prepared to demonstrate the quality of their data to ensure that their work stands the test of time and advances human and animal health. In addition, trainees need to leave our institutions prepared to transition into careers where a seamless integration into a QA environment is expected. Models that provide training and infrastructure for the implementation of QA best practices and programs in academic research environments are needed to support the critical work that scientists do. Scientists have the opportunity to promote scientific excellence, improve research training, and stand up for the quality of their work by introducing innovative QA programming and promising best practices into their individual, collegiate or institutional research settings. In this seminar, strategies for integrating QA best practices within non-regulated research settings will be described.
• Cloud-based design and data analytics for reproducible, reusable science
  Timothy Gardner, Riffyn
  Research in life science, material science, and chemistry is burdened by low experimental reproducibility and barriers to effective collaboration. This leads to low-confidence decisions, wasteful rework, missed observations, and delayed or failed technology transfer across sites or scales of development.
• Digitizing Discovery Lab Workflow
  Viral Vyas, Bristol Myers Squibb
  This presentation will focus on how BMS delivered inter-laboratory workflow-based applications across R&D; organizations built on a Business Process Management (BPM) platform. Laboratories that are responsible for running assays are rarely an island. They need to interact with their customers, suppliers, and CRO's to provide visibility into status, progress and results. LIMS enable these organizations to automate their lab processes and workflows. However, LIMS products are typically designed for a specific scientific domain and the workflow within the LIMS is not portable to other domains. For example, a LIMS that manages inventory of compounds can enable requesting of compounds but may not be able to deliver an application for biomarker assay requests. Therefore, BMS took the innovative approach of developing inter lab workflow applications on the Bonita BPM platform, and using the platform's native connector capability to integrate the apps with domain specific LIMS. One of the first BMS implementation on Bonita BPM lead to the digital transformation of analytical assay requesting processes in discovery. As a result of digitizing paper based processes, the following benefits were realized:
  • Time savings equivalent to 5 full-time employees.
  • Errors due to missing and incorrect information were virtually eliminated.
  • Increased transparency into state of the request reduced the constant back-and-forth between Chemists and Analysts.
  • Excel based metrics were replaced with dynamic on demand live metrics.
  In addition, the delivery time for a production-ready application was reduced by half. Since the initial implementation of BPM Platform in 2014, it has been expanded to other organizations: Biologics, Veterinary Sciences, Histology, and High Throughput Screening. As of May 2016, there are 5 applications in production used by 1400 users across US and India. Over 600 new requests are submitted daily. We are currently working on delivering 4 additional workflow applications and expect to deliver 10-15 more over the next 2-3 years.
• Toward quality management in cancer proteomics
  Christopher Kinsinger, National Cancer Institute
  The field of proteomics holds significant potential for advancing prevention, diagnosis, and treatment in cancer medicine. Despite the work of multiple, hard-working standards committees, the proteomics community currently lacks widely adopted data standards. Compared to the volume of proteomic data produced, a relatively small fraction are shared between labs, and an even small amount of analyzed data are shared. Furthermore, questions of reproducibility persist in the field, especially in relation to mass spectrometry. This is, in part, due to several excesses in proteomics: an excess of signal such that some ions go undetected; an excess of spectra increasing the risk of false identifications; an excess of peptides confounding the identification of proteins; and a dearth of statistical power Over the past ten years, the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) has sought to advance proteomics technology to a state where cancer medicine can benefit from this promising field. Through a variety of methods, CPTAC has transitioned from a technology assessment program to a full-production, biomarker development, proteomics pipeline. Following in the footsteps of the genomics field, CPTAC established benchmarks for proteomic technologies through a combination of measurement standards, inter-laboratory studies, and performance metric software. In the current production phase of CPTAC, the program is implementing a quality management system based on guidelines from the Clinical Laboratory Standards Initiative. This system will cover all components of the program from specimen collection, through data production and data analysis. This presentation will summarize the highlights and challenges of establishing quality practices in a rapidly evolving field. The content will include key accomplishments as well as lessons learned from data-directed quality control.

Making Scientific Data 100x Easier to Use

Session Chair: Amy Kallmerten, Merck Research Labs

• A comprehensive Information Management Strategy for the improvement of data and information quality across research
  Amy Kallmerten, Merck Research Labs
  In today's increasingly fast paced world of research, the reuse of data has become critical to success. Unfortunately the silo-ed nature of information sources across the research landscape make it challenging to combine and comprehend related datasources especially when the use case does not reflect the primary intended use of the data. Introducing technologies for data integration and analysis are only one part of the solution — emphasis must be placed on developing high quality information sources through a solid information architecture backbone and cultural appreciation for the value that experimental data brings to an organization. At Merck we are combining technologies and practices established to manage challenges associated specifically with the reuse of scientific data. We have found that although generic data storage and manipulation tools can simplify support and architecture from an IT perspective, they often fall short of enabling experimental work without placing undo overhead on the scientist. Our component based data platform provides flexible acquisition, enhancement, and integration layers focused toward each problem being addressed. This platform is complimented by data capture technologies using common languages and standards to control vocabularies and enhance interoperability between sources. A renewed cultural focus on accessibility and data quality through best stewardship practices ties our information management plan together resulting in a more agile approach to serving the needs of our scientists.
• Managing data-driven automation: Data workflows of an image-based automated cancer screen for cell viability and chemosensitivity
  Timothy Sherrill, Beckman Coulter
  Screening technologies are frequently used to explore biological questions and to identify potential new treatments for cancer and other diseases. The large sample throughput typical of screens introduces challenges when the sample preparation and analysis are straightforward and complexity increases dramatically automating cell-based screens. Cell-based screens typically require numerous interactions across multiple days and these interactions increase the opportunity to introduce errors into the screen not found in one-shot assays. In addition, if automation only executes a program of pre-determined set of steps, the researcher is left to update programs (even for simple changes) and process data with copy and paste. These challenges require a flexible data management plan that uses input data to identify incoming samples in the library, process data to drive automation such as hit-picking, and analytical information to power further analysis. All information brought into the system or produced by the analyzer should be retrievable by database extraction, in Microsoft® Excel®, or in a web report. We will describe how this data management plan can be used on a system to automate all the sample and data workflow steps required for the transfection of a miRNA mimic library into a cancer cell line, subsequent compound and reagent addition, and the acquisition and analysis of cellular images to measure cytotoxicity at multiple time points. This data management plan strengthens the reliability of screen data to ensure the correct hits are identified and resources are not misdirected in hit validation efforts.
• AnIML 1.0: Releasing a universal data format for analytical and biological data
  Burkhard Schaefer, BSSN Software GmbH
  The ASTM subcommittee E13.15 is releasing version 1.0 of the AnIML data standard. Since 2003, the group has been driving a cross-industry effort to standardize a universal data format for analytical chemistry and biological data. The resulting Analytical Information Markup Language (AnIML) Data Standard defines an XML-based format for documentation of laboratory experiments and their results. It is suitable for a wide range of analytical measurement techniques. The AnIML format has been well received by the vendor and user community. It has been developed collaboratively by stakeholders from industry, government, academics and the end user community. The format has been implemented by over 10 vendors to date. As analytical chemistry and biological experiments become increasingly more complex, as the amount of result data and metadata per experiment grows, and as retention requirements for analytical results lengthen, the need to structure and to record such experimental outcomes in a uniform, retrievable, and searchable manner is becoming more and more important. Much of the desired information is either requested by the analytical instrumentation or created by it; however, to date, there is no across-the-board mechanism to transfer what the instrument "knows" about an experiment to a standard, web-friendly report file containing the result data. The Analytical Information Markup Language (AnIML) has been created to provide such methodology. This presentation will describe the current progress of the standard and highlight a number of implementations contributed by vendors and end users. It also describes adoption strategies for end users who are considering rolling out the standard in their laboratories.
• From patient to drug: Developing new drug strategies for patient profiles with text analytics and literature mining
  Brendon Kellner, PerkinElmer
  Personalized medicine relies on understanding the relationships between diseases and genetics to generate target patient profiles. As the number of publications has soared, it is becoming increasingly difficult for experts to develop a deep understanding of the current literature, often relying on purchased curated content for summary information.

Informatics of Drug Design and Compound Life Cycle Management

Session Chair: Dmitry Lupyan, Schrodinger

• Harnessing and Sharing Science, Data and Knowledge Within an Organization
  Dmitry Lupyan, Schrodinger
  Schrödinger Inc. had developed a number of structure-based modeling technologies such as WaterMap, MD, FEP+, and novel scoring methods that have led to increased demand for managing and searching models and hypothesis. Harnessing and sharing data and knowledge within an organization and with the outside collaborators is both the challenge and the reality of today's drug discovery process. This presentation will describe how Schrödinger Inc. is overcoming these challenges by building a platform that allows for collaborative design between molecular modelers and chemists. Solutions with structure- and chemistry-aware databases and their application for structural modeling, prioritization, and triage will be covered. By incorporating the capability for automated execution of scientific software, we will describe how such solution can accelerate discovery and enforces best practices for modeling workflows.
• In silico intervention to improve the quality of decisions in the Design-Make-Test cycle for small molecule drug discovery
  Farida Kopti, Merck & Co
  The design of small-molecule drugs is an iterative process comprised of 6 distinct steps:
  1. Developing a hypothesis
  2. Designing and selecting the best molecules to test that hypothesis
  3. Synthesizing the designed compounds
  4. Purifying and registering them
  5. Testing new compounds in physical, biochemical, and biological assays, and
  6. Analyzing the results, assessing the design hypothesis, and providing the basis for the next round of design
  At Merck we have embarked on a process to implement a sustainable, scalable, and reusable information technology framework for the compound Design and synthesis closed loop which will improve the quality of hypotheses and molecules. The objectives are to enable rational drug design of molecules with desired chemical properties, further increase the molecular understanding of the interactions between the drug and its target in biological systems, and enable high throughput chemistry experimentation.
• The Application of Project Management Tools and Practices to Lead Discovery in an Academic Environment
  Julianne Bryan, St. Jude Children's Research Hospital
  Drug discovery, by its very nature, is a collaborative effort; no one person (or at times, entity,) has all of the necessary knowledge and/or resources to bring a compound from bench to bedside. Over the past ten years, it has become more common for lead discovery activities to be performed in academic and not-for-profit settings. With its world-class research and world-wide network of collaborators, St. Jude Children's Research Hospital (St. Jude) is uniquely positioned for translational research. To facilitate and streamline collaboration, we have implemented and developed a platform of web-based tools and processes for use by cross-functional teams. These tools and processes serve to organize the project. One example is a project site developed by the project management team in collaboration with the project leaders for each individual project. These sites allow all academic researchers at St. Jude to view basic information on the project and key contacts for each project. Additionally, these sites give the project team members a secure area to coordinate project logistics/timelines, manage documents and data, discuss ideas, and record project decisions and action items. These sites facilitate knowledge preservation and transfer within a project team and across programs. Additionally, a workflow-based site for compound management has been developed for collaborators to request purchase, acquisition, shipping, or delivery of individual compounds; delivery of existing compound plates; or custom reformatting and registration of new plates from powders, vendor plates, or our compound library. Ensuring effective and efficient communication of a request is crucial in the compound management delivery process. To this end, we have customized the notification emails to deliver stage specific information, including a graphic visualization on the progress of their request. The best platform in the world is only as valuable as its usage. Concurrent with this platform, we have implemented project management practices to facilitate communication and translation between disciplines and within the cross-functional, inter-departmental teams. The tools of project management are not difficult to learn, however, implementing them effectively takes skill and experience. The mission of the Chemical Biology & Therapeutics' (CBT) Project Management (PM) Team is to implement project management practices into the Lead/Tool Discovery process at St. Jude by enhancing communication and data sharing across inter-departmental project teams, tracking projects, and effectively managing resources. The CBT PM Team utilizes project management methodology, concepts and tools, and educates the St. Jude faculty and project team members on the benefits of using project management practices. To aid in this, the Team has implemented project management principles and developed a number of project management tools mentioned above. We have had a significant impact on the efficient and effective communication and management of collaborative projects.
• Multiple Research Platforms: One Single Data Sharing Portal
  Farid Said, CSols Inc.
  The need for researchers to share their scientific data has increased dramatically in recent years. Using commercially available informatics systems such as ELN, EDMS and SDMS researchers from multiple groups can collaborate and share their data. In this talk, we will explore an example of a pre-clinical DMPK study where the compound analysis involves a workflow that brings together requests and datasets from the study director and the study coordinator, as well as researchers from the chemistry, formulation, in vivo and analytical groups before the final results are approved and published.

The Digital Dark Hole: Handling Large-Scale Data for Use, Reuse, and Sharing

Session Chair: Laurie Goodman, GigaScience

• A Reproducible Science Platform for Translational Bioinformatics
  Andrew Smith, Bristol-Myers Squibb
  The reproducibility crisis in scientific research has motivated diverse organizations to take action. New procedures and technology platforms are being adopted to ensure that all reported results may be repeated and verified. This is especially true for research involving computational or statistical analyses such as those commonly performed in bioinformatics.
• PubChem BioAssay: a continuous effort towards open HTS data sharing
  Yanli Wang, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health
  High-throughput screening (HTS) plays a key role for drug discovery and is routinely conducted by pharmaceutical companies as well as large-capacity screening facilities at academic institutions and universities. Rapid advances in assay development, robot automation, and computer technology have led to the generation of terabytes of data in the screening laboratories. On the other hand, less efforts were devoted to HTS data integration and sharing, and HTS data were hardly made available to the public in general. To fill up this gap, the PubChem BioAssay database (https://www.ncbi.nlm.nih.gov/pcassay) was created in 2004 to serve as an open access archive system for biological results from small molecule and RNAi screening. With over ten years' continuous development effort, PubChem BioAssay now provides a public data repository and open information platform to worldwide researchers supporting drug development, medicinal chemistry study, and chemical biology research. This presentation will provide a review on the HTS data content in PubChem BioAssay including the HTS from the NIH Molecular Libraries Program (MLP) and progress of data deposition in the database to stimulate knowledge discovery and data sharing.
• The Visual Assay™ Platform: Using Mobile Devices and Workflow Technology In the Lab to Capture & Share Assay Data For Improved Data Integrity and Reproducibility
  Mannix Aklian, Label Independent, Inc
  The lack of data reproducibility has been a growing concern across the scientific community. Disparate systems which store and process data lead to fragmentation of the experimental information. This has led to false conclusions, lost time, and wasted money trying to reproduce these data. Therefore, ensuring data reproducibility and data integrity have become key initiatives of NIH, the pharmaceutical industry, and the scientific community at large. The US government 'Moonshot to Cure Cancer' initiative has also placed significant emphasis on reproducibility and data sharing to accelerate discovery. Factors leading to poor data reproducibility include inadequate personnel training, poor documentation of the experimental details including assay flow, reagents used and lot, timing of the experiment, inaccurately linking the data directly to the experiment performed as well as selectively choosing which experiments or which data to include in the notebook and ignoring those that didn't work thus losing potentially valuable information. Visual Assay® is software designed for tablets, mobile devices, and desktop computers. It is a non-browser based software application that continuously saves the assay details, data, and related information and synchronizes with all Visual Assay platforms that are sharing the assay making all experimental details transparent to the researchers and their collaborators. It allows real time joint development of assay protocols and assay data within a lab or across the globe with a gesture-driven, image-intensive interface minimizing the amount of writing necessary and using images to convey information alleviating language barriers. While performing an assay, it captures all data including who did each step of the experiment (with time stamps of each step) and seamlessly captures and links data files to the assay in real time. It enables the user to create and share plate maps which can be used to calculate the data results from within the program. It allows the scientist to capture observations that occurred during the experiment using either written, spoken, or photographic documentation. It enables tracking of experiments that worked and those that didn't work and enables the researchers to overlay those experiments to identify differences. It is auditable and can communicate with external chemical databases and electronic notebooks. It can also show the current progress of multiple assays enabling the manager to reallocate resources if necessary. With a continually evolving platform, Visual Assay should be a workflow tool that can help improve data reproducibility by capturing work as it happens and making it shareable in real-time with colleagues and collaborators. We will present the application followed by a demonstration of the platform as a mobile workflow solution for experiment data capture and sharing.
• Publishing Data: Credit Where Credit is Due
  Laurie Goodman, GigaScience
  Data is the base upon which all scientific discoveries are made. Making data rapidly and broadly shareable can therefore promote scientific discovery and improve reproducibility. With current communication technologies and potential access to "big data" such availability can have an enormous impact on the rate at which advances are made worldwide. Beyond scholarly mandates, there is very little incentive for researchers to share data. The current currency for receiving grants and career promotions is publications, and access to data is what allows discoveries to be made, tested, and published. Thus, in fact, sharing in the current system actively disincentivizes sharing as researchers need to 'get' as many publications as they can out of a dataset before other people can. Given that the overall goal for research is to improve human health and our environment, waiting to release data until after the publication of conceptual findings (sometimes for years) is unacceptable. Data publication, which actually has a long history, has recently been highlighted as a mechanism to promote sharing as it rewards researchers with the currency of a publication. Here I will discuss what data publication is and is not and its role as a reward for the scholarly pursuit of carefully collected, organized, curated, and validated data.

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Drug Target Strategies Track

Track Chairs: David Swinney, iRND3 and Chun-wa Chung, GlaxoSmithKline

Hard Targets — Success Through Collaborations

Session Chair: Chun-Wa Chung, GSK

• Case-study in consortium-based drug discovery: allosteric inhibition of the AAA ATPase p97
  Michelle Arkin, University of California, San Francisco
  This presentation will be recorded at SLAS2017 and made available free-of-charge to SLAS dues-paying members and full conference attendees post-event.
  
  There has been a significant effort over the past decade to bring drug discovery to academia, but this comes with many challenges to balance the potential advantages. In academia, the focus on innovative technologies and novel biological targets encourages us to work on difficult or commercially underpowered problems in healthcare, but how do we develop a strong team with diverse expertise from relatively small and loosely affiliated academic labs? At the UCSF Small Molecule Discovery Center, we have taken various approaches to this challenge, including working with drug discovery consortia. The National Cancer Institute's (NCI's) Chemical Biology Consortium is a consortium of academic, government, and private-sector laboratories working together to find innovative treatments for cancer. Through this consortium, a team of scientists from Caltech, University of Pittsburg, UCLA, University of Minnesota, SRI International, NCI, and UCSF have developed novel inhibitors of the AAA ATPase p97, an exciting, emerging target in cancer. p97 is a master regulator of protein homeostasis, and modulates ubiquitin-dependent degradation as well as membrane fusion activities throughout the cell. These events are directed by a network of protein-protein interactions and ATPase-dependent changes in p97 conformation. The team has designed allosteric and PPI modulators for different aspects of this complex machine and has solved the first high-resolution structures of p97 bound to an inhibitor using cryo-electron microscopy (cryo-EM). We see cryo-EM as a cutting edge technology for structure-guided design in drug discovery. By comparing inhibitors of p97 that act through different mechanisms, we aim to develop new experimental therapeutics and to decipher the complex biological pathways regulated by p97 activity.
• Discovery of Kynurenine Monooxygenase inhibitors for the prevention of multiple organ failure in Severe Acute Pancreatitis — an Industrial-Academic partnership
  Jon Hutchinson, GlaxoSmithKline
  Acute Pancreatitis (AP) is an inflammatory condition of the pancreas which can lead to a systemic inflammatory response followed by multiple organ dysfunction and death. There are currently no treatments to prevent progression to severe disease. In 2011 GSK formed an Industrial-Academic partnership with Mr Damian Mole and colleagues at the University of Edinburgh, under GSK's Discovery Partnerships with Academia (DPAc) initiative. This harnesses the complementary expertise of each partner in an integrated team to deliver innovative medicines. Early results from the Edinburgh group implicated the Kynurenine pathway of tryptophan metabolism, and the pivotal enzyme Kynurenine Monooxygenase (KMO), in the progression to severe disease.
• The Dementia Consortium: building partnerships to address early stage neurodegeneration drug discovery
  Catherine Kettleborough, MRC Technology
  There have been no new treatments for dementia approved for over 10 years, many Pharma companies have withdrawn or scaled back their efforts in this area, and yet at the G8 Dementia summit world health leaders pledged to find a "disease-modifying treatment for dementia by 2025". New mechanisms to address this are needed. One such approach is the Dementia Consortium. The consortium is a partnership bringing together the charity sector, pharma companies and academic scientists to accelerate innovative biology by de-risking targets and building the reagents and data package to initiate drug discovery programs. Launched in March 2014, the consortium consists of Alzheimer's Research UK, AbbVie, Astex, Eisai, Lilly and MSD, and MRC Technology. The partners contribute funding (over £4.5 million is available), access to drug discovery facilities and resources, expertise in dementia drug discovery and project management. There are currently 5 active, hands-hand drug discovery programs running, with academic scientist's bases in UK, USA, and Europe. The presentation will: give examples of how this approach can advance early stage biology closer to the clinic, what is needed to prosecute such projects, the challenges and lessons learnt and plans for the future.
• Collaborative development of advanced bioluminescence resonance energy transfer approaches
  Kevin Pfleger, Harry Perkins Institute of Medical Research and The University of Western Australia
  An ongoing collaboration between the Harry Perkins Institute of Medical Research and The University of Western Australia, The University of Nottingham, Promega, BMG Labtech, Dimerix Limited and The University of Queensland has and continues to be exceptionally successful. It is funded by the Australian Government through both the Australian Research Council Linkage and Researchers in Business grant schemes, with matched industry funding. Outcomes include the development of the NanoBRET ligand binding assay enabling real-time, live cell monitoring of ligand-receptor interactions for both small molecules and peptides [1]. The receptor is N-terminally-tagged with the NanoLuc bioluminescent enzyme and resonance energy transfer to the fluorophore-conjugated ligand is assessed upon substrate addition. Furthermore, the BRET trafficking assay has now been developed extensively, and validated with confocal microscopy in comparison to established subcellular markers. This approach enables real-time, live cell monitoring of luciferase-tagged protein trafficking from the plasma membrane (proximal to the fluorophore-tagged K-ras marker) to various subcellular compartments (proximal to fluorophore-tagged RabGTPase (Rab) markers): Rab5a for early endosomes; Rab4 for early endosome recycling; Rab11 for recycling endosomes; Rab7a for late endosomes/lysosomes; Rab9 for late endosome trafficking to the trans-Golgi network; Rab1 for endoplasmic reticulum trafficking to the cis-Golgi; Rab6 for Golgi apparatus and trans-Golgi network; or Rab8 for trans-Golgi network to plasma membrane [2]. Both the BRET ligand binding and trafficking approaches are very powerful and complement the more established receptor-G protein and receptor-arrestin BRET proximity assays that we also utilize, and have now validated with the latest BRET systems. Particularly hard targets to evaluate are receptor heteromer complexes [3]. Our latest data will be presented, illustrating how we are now applying all of these novel advanced BRET approaches to monitor binding, G protein coupling, arrestin recruitment and subcellular trafficking in live cells and in real-time specifically for these complexes. Such comprehensive kinetic profiling of receptor complexes has not been demonstrated previously, and has significant implications for enabling improved understanding of receptor and ligand bias, with and without the added complexity of heteromerization.

Non-Traditional Modalities as Therapeutics

Session Chair: Stephen Hale, Ensemble Therapeutics

• Design Considerations for Synthetic Macrocycles as Drugs
  Adrian Whitty, Boston University
  Current definitions of druglikeness were developed primarily by analyzing the properties of known drugs or advanced clinical candidates. However, many biologically compelling drug targets fall outside the set of classically targeted protein families. It is increasing clear that many of these so-far unexploited targets are highly challenging to target with conventional small molecule drugs, and require different kinds of compounds to bind to and inhibit them. Despite a wealth of evidence that certain compounds that violate conventional notions of druglikeness can make effective oral drugs, for these non-canonical drug chemotypes until recently there has been little guidance available as to what specific compound structures are likely to possess favorable pharmaceutical properties. I will discuss how analysis of the number, strength and spatial distribution of binding energy hot spots at protein surface sites can be used to establish the druggability of challenging drug targets such as protein-protein interfaces, and to understand how non-canonical drug chemotypes interact at such sites. In particular, I will focus on how large natural product macrocycles bind to proteins, and how the structural and physicochemical properties of oral macrocyclic drugs differ from those of conventional drugs. I will also discuss how macrocycle binding sites on proteins differ from sites that bind conventional drugs, and how these distinctive features might be used to prospectively identify targets that are suitable for the application of macrocyclic ligands.
• Development of an ELT Selection Method for Irreversible Inhibitors
  Zhengrong Zhu, GlaxoSmithKline
  Traditionally drug discovery has been focused on reversible inhibitors; however, irreversible inhibitor may offer advantages for some therapeutic targets. High biochemical efficiency of irreversible inhibitors may translate into lower dose and reduced off-target effects. Uncoupling pharmacokinetics and pharmacodynamics and prolonging duration of action by irreversible inhibitors may result in less-frequent drug dosing. So it is not surprising many approved drugs are irreversible inhibitors. Encoded library technology (ELT) is a powerful technology platform for identifying small molecule compounds that bind protein targets using DNA tagged combinatorial libraries. Standard ELT selection process is very effective in finding reversible inhibitors but may not be so for irreversible inhibitors. In this project we used tool compounds with DNA tag to develop a new ELT selection method for irreversible inhibitors. ELT selection conditions were optimized for this new method. The new method was validated by identifying tool compounds that were spiked in an ELT compound library at the same concentration of individual compound in the library. This new method of ELT selection will offer an enabling tool for drug discovery targets that may be undruggable for reversible inhibitors.
• Small Libraries, Big Impact: Leveraging focused screens for rapid identification of target space/vulnerabilities.
  Robert Hills, Wistar Institute
  The standard model in drug screening is bigger and faster. The more compounds you push, the better your odds of finding a hit. This paradigm works well for complex uncharacterized targets, but for validated targets and pathways, smaller, more focused compound sets offer value in terms of time and cost associated with a screen. We have been assembling sub-libraries comprised of well-defined chemical probes spanning multiple pathways and targets of interest to Wistar researchers. Many of the compounds in these libraries are currently marketed medications or in late stage clinical development, therefore representing "safe" well characterized mechanisms of action and potentially allowing for rapid translation into the clinic. This last point being critical in the potential role screening can play in personalized medicine where small sample sizes are the norm and clinical translatability is of great value. Libraries are divided into class based on mechanism or pathway. An initial single point screen is performed with hits then being arrayed in 10 point dose response for confirmation. Confirmed hits are then bucketed into mechanism/target and a new more target centric library is assembled and tested. The entire process from initial screen to target class identification can be performed in less than 5 days, allowing for rapid hypothesis testing and generation of publication quality data. We will present data from select campaigns focused on viral latency and oncology.
• Messenger RNA as a novel therapeutic platform
  Tej Pavoor, Moderna Therapeutics
  Messenger RNA has been studied extensively as part of the core dogma of molecular biology. Only recently, has its applications as a novel therapeutic platform been investigated. At Moderna, we are focused on bringing mRNA therapies for a wide array of disease areas to patients. The Moderna platform develops novel engineered mRNAs that are utilized by the Moderna eco-system composed of partners and ventures. Moderna has built its own in-house mRNA synthesis capacity to serve the mRNA needs of the eco-system. In this presentation, we will introduce the technology, its applications, and discuss the preclinical mRNA synthesis capability based on high throughput automation and custom robotics.

Uniting Phenotypic and Target-Based Drug Discovery

Session Chair: Fabien Vincent, Pfizer

• Combining of Phenotypic and Target-based Approaches Helped Identification and Characterization of Antiviral Activity of GS-5734.
  Veronica Soloveva, USAMRIID/HJFM
  The 2014-15 EBOV outbreak in West Africa brought to light the critical need for a safe, effective, and readily available antiviral therapy for Ebola Virus Disease (EVD). A small library of nucleotide analogs from Gilead R&D; was evaluated at USAMRIID's Therapeutic Development Center (TDC) using in vitro phenotypic assays applying high content imaging of the immuno-stained viral structural proteins expressed after infection of cells with authentic viral isolates in 384-plate format. The cells physiologically relevant to EVD were used with authentic Ebola-Zaire and Ebola-Makona viral infections in addition to regular transformed cell lines. Phenotypic data with sub-micro molar potency, an selectivity index (SI) >100 in multiple cell lines including PBMC-derived macrophages clearly warranted advancement of the selected compounds into in vivo efficacy testing. However, the additional target specific information about GS5734 was critical for success. The physiologically active triphosphorylated form of GS-5734 was efficiently produced during in vitro by endothelial cells and macrophages and as well as systemically in PBMCs following intravenous administration of GS-5734 to rhesus monkeys. In vivo tests were possible in the NHP but not the rodent Ebola infection model due to high serum activity of rodent esterase that would adversely degrade the GS-5734 and impact in vivo exposure. Resulted intravenous administration of GS-5734 in the NHP Ebola-Makona challenge model provided 100% protection even when delivered 3 days after initial EBOV exposure. The Phase II evaluation of GS-5734 in PREVAIL treatment trial for EVD survivor men with persistent Ebola viral RNA is currently ongoing. In summary, the combination of phenotypic approaches and target-driven specific analysis leads to success in development new anti-Ebola therapy.
• Combining Target-Based and Phenotypic Discovery Assays for Drug Repurposing
  Sharlene Velichko, DiscoverX, BioSeek Division
  Drug repurposing can be a cost-effective means of addressing unmet medical needs. For drugs such as kinase inhibitors that can have multiple targets, a strategy that combines both target-based and phenotypic screening can be highly efficient at identifying optimal repurposing opportunities. Here we applied phenotypic profiling for the repurposing of approved multi-kinase inhibitors as anti-fibrotic drugs. We tested the hypothesis that disease-relevant phenotypic activities common to drugs approved for idiopathic pulmonary fibrosis (IPF) could be used to evaluate other compounds as potential therapeutics. For this, we tested kinase inhibitors from a number of target classes, including angiokinase, BCR-Abl, and Flt-3 across a panel of human primary cell based disease and tissue models. This phenotypic panel, the BioMAP® Diversity PLUS panel of 12 assay systems, contains assays modeling vascular, immune and epithelial biology as well as inflammatory and tissue remodeling responses. Profiling of the two drugs recently approved for IPF, nintenanib and pirfenidone, identified a number of activities relevant to efficacy in IPF. These activities were used to score the profiles generated for a number of clinical-stage multi-kinase inhibitors. Based on this analysis, the BCR-Abl inhibitor, dasatinib, was prioritized over other drugs, including sunitinib, bosutinib and ponatinib, as potential therapeutics for IPF. We confirmed the activities of dasatinib in a focused panel of fibrosis-related assay systems (BioMAP Fibrosis Panel) that incorporate renal and small airway lung epithelial/fibroblast co-cultures. Combined analysis of target selectivity data and phenotypic profiling data, including analysis for both efficacy and safety, enabled an optimal selectivity profile to be defined. Combining target selectivity and phenotypic profiling provides an efficient approach for identifying repurposing opportunities for approved drugs which can also be applied to new drug discovery.
• Phenotypic screening identifies a small molecule that inhibits PCSK9 protein translation and directly binds to the human 80S ribosome
  Paula Loria, Pfizer
  Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that down-regulates low density lipoprotein receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C) and promotion of atherosclerosis. Human genetic associations of PCSK9 activity with LDL-C levels are compelling and neutralizing antibody therapeutics targeting PCSK9 show robust lowering of LDL-C in patients. Despite these implications, no small molecule inhibitor of PCSK9 has been developed to date. Here we report the discovery of a small molecule inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), identified through a high throughput phenotypic screen. In Huh7 cells that endogenously express PCSK9, R-IMPP behaved as an anti-secretagogue of PCSK9, increasing cell surface levels of LDL-R and promoting uptake of LDL-C. R-IMPP had no effect on the secretion of transferrin from Huh7 cells, indicating that the compound was not a general inhibitor of protein secretion. The enantiomer, S-IMPP, did not decrease PCSK9 secretion, enabling the use of the enantiomer pair as a tool for further dissection of the mechanism of action. A systematic investigation of R-IMPP cellular activity revealed that the compound does not decrease PCSK9 transcription nor increase its degradation, but instead causes transcript-dependent inhibition of PCSK9 translation. In support of this surprising mechanism of action, we show that R-IMPP is able to directly bind to human 80S ribosomes. R-IMPP represents one of the first examples, to our knowledge, of a compound that can selectively target protein translation in eukaryotic cells. Our results suggest a new approach for small-molecule-mediated modulation of challenging targets such as PCSK9.
• Differences in the Therapeutic Hypotheses associated with Phenotypic and Target-Based screening.
  David Swinney, Institute for Rare and Neglected Diseases Drug Discovery
  In this talk I will address and compare the roles of phenotypic/empirical screening (PDD) and target-based screening (PDD) to establishing and testing a therapeutic hypothesis. A therapeutic hypothesis connects the findings from preclinical discovery to patients through testing clinical candidates in human clinical studies. The goal of both PDD and TDD is to identify lead molecules that, after optimization, can be used in the clinic to test a therapeutic hypothesis. It can be argued that the high attrition rates in drug discovery are due in part to inaccurate therapeutic hypotheses, e.g. the candidate medicines do not work in the hypothesized manner to provide efficacy at a safe dose. Accordingly, it is important to identify ways to improve the reliability of therapeutic hypotheses. An important difference between PDD and TDD is that the target is the therapeutic hypothesis for TDD, whereas the clinical candidate is the therapeutic hypothesis for PDD. For TDD the clinical candidate is used to test the therapeutic hypothesis that the target and mechanism are valid for the disease, in contrast the therapeutic hypothesis tested for PDD is that the clinical candidate will be effective, regardless of its mechanism of action. This difference in approaches is reflected in the stages associated with drug discovery using TDD and PDD. Drug discovery and development can be considered to involve four stages: 1) basic research to provide a sufficient understanding of a disease to facilitate development of tools for discovery, 2) discovery/invention to identify a potential therapeutic lead that can be optimized to test a therapeutic hypothesis, 3) optimization of the lead to a clinical candidate (this includes pharmaceutical development, ADME and safety), and 4) clinical testing of the therapeutic hypothesis using the clinical candidate. The differences in PDD and TDD are most notable in the first stages (Stages 1 & 2). Under what circumstances would a mechanistic therapeutic hypothesis (TDD) or pharmacology (PDD) dependent therapeutic hypothesis be warranted? To address this question, the mechanisms of action of first-in-class NMEs approved by the US FDA between 1999 and 2015 were analyzed and categorized by type of discovery; e.g. PDD, TDD, modification of natural substances and biologics. The majority of biologics were antibodies against extracellular receptors and cytokines, TDD discovered modulators of enzymes (majority kinases) and receptors (GPCRs), natural substances included modified peptide agonists, enzyme replacement therapy and metabolites, and PDD discovered medicines with by far the greatest number of different novel mechanisms of action. A conclusion from this analysis is that therapeutic hypotheses from PDD have a greater potential to identify novel, innovative mechanisms of action, whereas the success of specific therapeutic hypotheses associated with TDD, biologics and modification of natural substances were associated with specific mechanisms of action.

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Micro- and Nanotechnologies Track

Track Chairs: Sindy Tang, Stanford University and Andrew deMello, Institute for Chemical and Bioengineering

Digital and Droplet Microfluidics

Session Chair: Adam Abate, UCSF

• Drop Based Microfluidics and the Quantitative Biology Revolution
  Assaf Rotem, Harvard University
  Modern measurements in biology such as high throughput sequencing are loading bioinformatics with exhaustive data that are increasingly difficult to process and interpret. Instead, novel sample preparations have the opportunity to circumvent this problem by first increasing the specificity and sensitivity of the measurement. One technology enabling this concept is Drop Based Microfluidics (DBM) — the encapsulation of biological samples in micron size drops that can be incubated, merged, split, detected and sorted at kHz rates in precisely designed microfluidic devices. For example, "Single-drop labeling", a method that pre-labels assays in drops before measurement, enables sequencing samples at single cell resolution. DBM is also used to perform isolated evolution experiments of millions of single microbes by compartmentalizing each lineage in a single drop. I will describe such platforms and how they revolutionize the way we measure and think of biology.
• Droplet based functional assay for detecting protease secretion from circulating tumor cells
  Manjima Dhar, UCLA
  Isolating circulating tumor cells (CTCs) from whole blood and analyzing single cell behavior towards personalizing treatments and understanding cancer progression remains a challenge. We have developed an integrated device that combines vortex trapping of circulating tumor cells and a novel extreme throughput droplet generator, to measure proteases secreted by individual CTCs, which may serve as an improved measure of metastatic potential. Cell-secreted proteolytic enzymes that cleave extracellular matrix (ECM) proteins are hypothesized to play a key role in enabling extravasation. Analysis of metastatic tumors and patient blood serum has shown significantly higher levels of MMPs, suggesting that CTCs may release MMPs that allow them to degrade the ECM, and that the level of MMPs could serve as a prognostic marker. In order to test this, measurements of MMP secretion by individual CTCs are necessary, however, this is extremely challenging given the rarity of CTCs and the dilution of few enzymes secreted by single cells into bulk solution. Our platform addresses these challenges with an integrated system that can automatically isolate and encapsulate purified CTCs into a small number of microdroplets to interrogate MMPs secreted at the single-CTC level for the first time. The vortex HE chip isolates CTCs in a label free manner from diluted blood. Importantly, the vortex device washes out background molecules, and can exchange solution around captured cells, allowing this assay to be highly specific to secretions from captured cells. After CTC isolation and solution exchange with a FRET MMP substrate, we divert captured cells to a second outlet via a valve that leads to a droplet generator. The droplet generator creates a monolayer of droplets and operates at 1µl/min per channel with 100 parallel channels, without oil co-flow. It is relatively insensitive to changes in flow rate that occur during the release step. Encapsulation increases the concentration of the analyte by 105, which increases the sensitivity of our system. The MMP substrate exhibits low nonspecific signal, because of the integrated solution exchange step. Thus only droplets with cells actively secreting MMPs fluoresce after incubation. Total analysis from sample input to secretion assay takes minutes, making this system compatible with studying live cells while they retain physiologic conditions. Protease release patterns of lung cancer cell lines and healthy leukocytes and endothelial cells show cancer cells release more proteases with higher variance than healthy cells. The ability to purify and encapsulate CTCs in droplets from whole blood opens the door for single CTC genomic sequencing. These new assays better characterize CTCs enabling improved prognostic measures, and personalized therapy selection.
• Rapid access to libraries of non-rule-of-five chemotypes via DNA-encoded solid-phase combinatorial synthesis
  Marie Malone, The Scripps Research Institute
  DNA-encoded libraries provide an efficient and vast source of diversity for screening and are often deployed against targets that are unsuited for, or have failed to produce ligands using traditional HTS style screens. Generated combinatorially, DNA-encoded libraries tend to be prepared via 2-3 cycles of iterative coupling, and seldom comply with the traditional "rule-of-five." We have demonstrated the synthesis of 3 unique one-bead-one-compound (OBOC) DNA-encoded solid-phase libraries for the discovery of ligands to 3 non-traditional targets (mutant HIV-1 protease, serum antibody binding, RNA) via multiple screening strategies. The low-diversity (29k member) HIV-1 protease inhibitor library, composed of variations on known FDA-approved inhibitor themes, was designed to screen for inhibitors of 6 drug resistant HIV-1 protease mutants in order to generate pan-library structure activity relationship profiles against a rapidly mutating viral target. Serum-antibody binding libraries, designed to mimic unknown antigens, featured much larger diversity (500k members) and benefited from truncations and reaction side-products to further increase library diversity. The RNA-binding library was designed to utilize known RNA binding modules for efficient identification of optimal module display scaffolds (47k members). DNA-encoded solid-phase libraries were then either screened for target-binding to beads by FACS (serum screening) or for modulation of activity using functional assays (HIV-1 protease or Drosha activity) in microfluidic droplets. The hit beads were collected in each case, the DNA encoding tags amplified in PCR, and the resulting amplicons were sequenced in bulk. The next-generation sequencing output was used to elucidate hit structures and prioritize hits for resynthesis based on homology and redundancy. DNA-encoded solid-phase combinatorial libraries and miniaturizing high-throughput screening (by microfluidics or FACS) provide a distributable and economical platform for small molecule discovery and facilitate efficient exploration of unconventional targets and chemical spaces.
• Stability of water-in-oil droplets as micro-reactors for ultrahigh-throughput droplet microfluidics applications
  Sindy Tang, Stanford University
  Droplet microfluidics, in which micro-droplets serve as individual reactors, has enabled a range of high-throughput biochemical processes. Although the physics of single drops has been studied extensively, the flow of crowded drops or concentrated emulsions — where droplet volume fraction exceeds ~80% — is relatively unexplored in microfluidics. Ability to leverage concentrated emulsions is critical for further increasing the throughput of droplet applications. Prior work on concentrated emulsions focused on their bulk rheological properties. The behavior of individual drops within the emulsion is not well understood, but is important as each droplet carries a different reaction. Motivated by applications in rapid disease diagnostics and bio-manufacturing, this talk examines the stability of drops in a concentrated emulsion by tracking the dynamics and the fate of individual drops within the emulsion. At the fast flow limit, we show that droplet breakup within the emulsion is stochastic. This contrasts the deterministic breakup in classical single-drop studies. We further demonstrate that the breakup probability is described by dimensionless numbers including the capillary number and confinement factor, and the stochasticity originates from the time-varying packing configuration of the drops. Practically, the results provide a direct guide for the rational design of microchannels and the choice of operation parameters to increase the throughput of the droplet interrogation step while preserving droplet integrity and assay accuracy. To mitigate breakup, we design and show that novel amphiphilic nanoparticles are more effective than surfactant molecules as droplet stabilizers.

Perspectives on Commercialization of Integrated Micro and Nanofluidic Devices

Session Chair: Sammy Datwani, Labcyte Inc.

• 2017 SLAS Innovation Award Top 10 Finalist — Optimizing Clinical Combination Therapy Using a Phenotypic Personalized Medicine Technology Platform
  Dean Ho, UCLA Bioengineering and The Weintraub Center
  The emergence of personalized and precision medicine approaches to develop combination therapies and nanotherapies for oncology, infectious disease, cardiovascular, and other indications may result in improved treatment outcomes. However, identifying the optimal drug-dose ratio during combination therapy for both individualized regimens and population-optimized fixed dose formulations has been virtually impossible. This is due to the infinite parameter space that exists when attempting to pinpoint these ratios. Also, every patient responds differently to chemotherapy, their drug regimens are constantly changing due to comorbidities (e.g. infection, additional surgical procedures, etc.) and drug synergy and antagonism can change within a single patient during the course of treatment. To address this major barrier that has challenged the persistent actionability of personalized and precision medicine, we have developed a powerful digital medicine platform named Phenotypic Personalized Medicine (PPM). PPM is a calibration technology that correlates quantifiable phenotypic endpoints, or physical traits (e.g. tumor burden, viral/bacterial load, safety markers, etc.) with therapeutic inputs (e.g. drug dosages) to optimize treatment independent of disease mechanism. In addition, PPM input-output calibration can be visualized by constructing patient-specific response surfaces and 3-dimensional drug interaction maps to deterministically pinpoint the best drug-dose ratios without modeling or algorithms. Importantly, these response surfaces/maps change over time as patients are given additional therapies or surgical procedures. PPM continuously recalibrates patient responses to drug inputs, optimizing treatment efficacy and safety based on phenotypic outputs (e.g. faster tumor burden decrease, rapidly reaching target levels for safety/tolerance markers such as creatinine or white blood cell count, etc.), for the entire duration of combination therapy.
• 2017 SLAS Innovation Award Top 10 Finalist — Transcriptional profiling of single cells using droplet microfluidics
  Tobias Wheeler, 10X Genomics
  To further the understanding of complex, multicellular biological systems (e.g., the immune system and heterogeneous tumors), new tools are required that provide transcriptome profiling of 1000s of single cells. Existing single cell RNA-sequencing (scRNA-seq) technologies are limited in throughput, have low cell capture efficiencies, or are difficult to implement, and therefore cannot meet this need. We have developed a microfluidic system that enables 3' mRNA counting for up to 48,000 single cells (6,000 per sample), in a single run. The technology is based on nanoliter-sized droplets that contain both a single cell and a single hydrogel bead. Each bead contains primers having a single, unique sequence of oligonucleotides with which the mRNA from a particular cell is labelled during reverse transcription. Once barcoded, the mRNA sequences in each droplet are pooled into a single bulk volume for amplification and Illumina sequencing library preparation. To characterize the performance of the system, we used a mixture of human (Jurkat) and mouse (3T3) cell lines. Experiments show that the system captures ~50% of loaded cells with a multiplet rate of ~1% (per 1,000 cells recovered), thus potentially enabling the system to be used for the profiling of cells of limited quantity. At 100k reads/cell, the system detected a median of ~4,500 genes in each human and mouse cell, indicating comparable sensitivity to other droplet-based platforms. To demonstrate its ability to detect cells in heterogeneous populations, we used the system to profile >100,000 peripheral blood mononuclear cells from healthy donors; all major subpopulations were detected at the expected proportions, from both fresh and cryopreserved cells. Finally, we used the single cell resolution of the system to detect variations in the gene profiles of bone marrow mononuclear cells of transplant patients. These variations give insight into host and donor chimerism and other complex interactions between donor and host cells, and provide a means of monitoring response to transplant treatments. Through its performance and robustness, we envision that this technology will enable the widespread adoption of high-throughput single cell mRNA analysis, and will advance significantly the study of diverse developmental systems and tumor samples in basic and clinical research.
• Microfluidics without Borders: Commercialization Perspectives of High Throughput Applications for Acoustic Droplet Ejection Technology
  Sammy Datwani, Labcyte Inc.
  This presentation addresses the commercialization perspectives of integrating on the micro and nanoscale acoustic droplet ejection (ADE) for high throughput (HT) applications with mass spectrometry (MS) and gel electrophoresis (GE). First highlighted are the physical principles of ADE and the key technologies that enable robust acoustic liquid handling operations — namely the ability to: probe the properties of the fluid; adjust in real-time to account for dynamic surface tension and viscosity variations, determine the acoustic energy required for ejection; and to impart charge on the droplet to aid in droplet transfer.
  
  Next, we explore the commercialization perspectives for HT applications in four different modalities; three modalities explore directly acoustically loading samples into MS, enabling true HT screening in a label-free format, and the fourth modality explores the low volume transfer of proteins in solution into a microfluidic gel device for a GE assay. For the GE assay, we demonstrate 100 nL per well transfers of free antigen-binding (FAB) fragments in solution; Kd is extracted; and >80% improvement in assay precision and throughput compared to manual operation.
  
  The first of the three MS modalities utilizes electro spray ionization (ESI) whereby ADE delivers up to 500 spray events per second from an assay well in a microplate into the MS. A sample kinase assay was able to tolerate the changes to buffer conditions without impacting enzyme activity or kinetics. The utility of this integrated system as a biochemical HT platform in this format was demonstrated on a focused kinase 70,000 sample screen through a traditional ADP accumulation assay. The second MS modality explores another promising method of ionization of the sample prior to loading in the MS using direct analysis in real time (DART). ADE delivers up to 500 nL sample per second onto a substrate where heated gas containing metastable helium atoms flows from the source, vaporizes the sample, and ionizes the analyte via gas-phase collisions. In single-ion recording mode, an interleaved 384-well plate of pure water and aqueous 100 µM caffeine executed at 3 seconds per sample produced 100% true positive and 0% false negative hits. The entire peak for a 100 nL sample requires 500 milliseconds and represents 10 pM caffeine. The third MS modality explores the integration of an Open-Port Probe (OPP) with ADE-MS whereby the ADE delivers up to 500 nL sample per second into an inverted OPP with a subcritical vortex interface stabilized by nebulizer gas and methanol. Experimental results are presented for increased sample volume with baseline resolution, linearity, and response.
  
  In conclusion, commercialization perspectives are addressed with these high throughput applications with ADE and the need to integrate "big data" collection to support further HT applications in the development of microfluidic devices.
• 2017 SLAS Innovation Award Top 10 Finalist — Vortex Biosciences technology for fast and label-free isolation of circulating tumor cells from blood samples
  Elodie Sollier-Christen, Vortex Biosciences, Inc.
  Current tumor tissue biopsies are invasive procedures that can be limited by small sample size and difficulty accessing the tumor site. Moreover, single-site tumor biopsies may not recapitulate intra-tumor heterogeneity and may fail to reflect the genetic diversity of a patient. These limitations can be overcome with a liquid biopsy. Liquid biopsies are non-invasive blood tests that aim to isolate and analyze circulating tumor cells and/or cell-free DNA that are continuously shed into the bloodstream by both primary and metastatic lesions. Liquid biopsies are readily amenable to serial sampling and could provide real-time and more representative information on tumor evolution, treatment effectiveness and cancer metastatic risks. Ultimately, liquid biopsies may allow earlier detection and more personalized treatment of cancer. Vortex Biosciences has developed VTX-1, a fast and simple platform to isolate and collect intact circulating tumor cells (CTCs) directly from whole blood in less than 1.5 hours. Based on inertial microfluidics and capture in microscale vortices, our CTC isolation process is label-free, contact-free, and high-throughput, providing intact CTCs that can be collected in suspension within various containers. Besides, samples processed by the Vortex VTX-1 system have minimal white blood cell contamination, resulting in a highly enriched CTC sample. In preliminary clinical studies, CTCs were isolated with high purity (from 1.4 to 92.5 WBCs per mL blood) from patients with metastatic breast (median 40.68 CTCs per 7.5mL; n=22), colorectal (median 12.23 CTCs per 7.5 mL, n=41), non-small cell lung (NSCLC) (median 26.25 CTCs per 7.5 mL, n=15), and prostate (median 5.63 CTCs per 7.5mL, n=20) cancers. This CTC technology offers significant advantages for downstream analysis: (i) Isolated CTCs are representative of the patient's status and remain unbiased by molecular characteristics, as confirmed by immunofluorescence staining and enumeration. (ii) CTCs collected at higher purity increase the accuracy and sensitivity of downstream assays, such as cytology, next-generation sequencing and Sanger sequencing. For example, using Papanicoalou staining, atypical cells were detected in 15 out of 16 NSCLC samples, with morphological similarities observed in corresponding primary tumor. In another study, KRAS, BRAF, PIK3CA mutations were detected by Sanger sequencing, revealing concordance between CTCs and liver metastasis for 7 out of 9 colorectal cancer patients. (iii) CTCs are unaltered and undamaged by labels or reagents, making them ideal for cell culture experiments and live cell assays, such as CDX model, RNA sequencing, or protein assays (western-blot, Epispot assay). Several case studies with patient samples of metastatic breast, lung, colon and prostate cancer will be presented to illustrate these advantages of the system.

Making Micro-Volume Biology Work: Tools, Techniques & Secrets

Session Chair: Daniel Sipes, GNF

• Optically Actuated Micro-fluidics: Applications in BioMedical R&D
  Kevin Chapman, Berkeley Lights, Inc.
  We have developed an optically actuated micro-fluidic environment for cloning, culturing, assaying, and exporting mammalian cells. The system enables opto-electronic positioning (OEP) for manipulation of cells in cell culture media, and opto-electrowetting (OEW) for manipulation of droplets in oil. Optical actuation allows movement of cells and droplets into and out of the microfluidic path in a fundamentally interactive way, and under computer control. In this talk, I will discuss applications of this system to BioPharmaceutical, genomic, and cell therapy work-flows.
• 2017 SLAS Innovation Award Top 10 Finalist — Dynamic Profiling of Anti-tumor Immune Response at the Single-Cell Resolution by Microfluidic Cell Pairing
  Tania Konry, Northeastern University/HMS
  New approaches to single cell analyses and live cell-cell interaction on a single cell level are needed to uncover fundamental biological principles and ultimately improve the detection and treatment of disease. We have developed a new Lab-on-a-Chip (LOC) microfluidic technology to acquire live functional phenotyping of single cells of human immune system to monitor and regulate their interactions with tumor cells in hematologic cancer-relevant system. In particular, the droplet based microfluidic sensor technology developed herein allows: 1) conducting dynamic and simultaneous multi-parameter analysis of both cell surface and secretions, 2) controlled delivery of regulatory agents and therapeutics to study their effect on functional phenotype of the cell and 3) monitoring of cell-cell interactions on a single cell based level. In this platform, we investigated the functional responses of activated effector immune cells dynamically. We observed strong heterogeneity in DC-T cell conjugation irrespective of the activation status of DCs. The downstream effector functions of activated T cells were modeled by assessing CD8+ T cell-mediated cytolysis of cancer cells. T cells induced either fast or slow target death and in specific instances, sequential contact with target cells. We evaluated the functionality of our platform in characterizing the efficacy of DC-based vaccines against cancer cells by co-encapsulating three types of cells within a droplet. These results suggest that the droplet array-based microfluidic platform is a powerful technique for dynamic phenotypic screening and applicable for preclinical evaluation of effector immune functions. Furthermore, this approach should have a broad impact on diverse biological systems for the characterization of cell surface and secretion proteins as potential biomarkers and targets for diagnostics and therapeutics as well as cell-cell interactions in immune response to malignancy, autoimmune diseases, immunotherapy and biomarker discovery.
• Monoclonal Cell Line Generation and CRISPR/Cas9 Gene Knockout using Automated Single-Cell Electroporation
  Vincent Lemaitre
  Stably-transfected monoclonal cell lines are widely used in drug discovery and biological research. However, the process of generating these lines requires time-consuming dilution techniques in order to avoid the presence of a genetically-mixed population of cells. Here, an efficient method for producing stably-transfected cell lines was developed using the Nano Fountain Probe (NFP) single-cell electroporation system in conjunction with a pre-patterned array of cell colonies. The NFP, consisting of a flexible cantilever beam with embedded micro-channel connected to a reservoir, was implemented with precise positioning using a micro-manipulator with submicron resolution and resistance-based feedback control. Experiments conducted with HEK, HeLa, macrophages, and stem cells showed a transfection efficiency of more than 90% and equal cell viability. In monoclonal cell line generation experiments, HEK293 cells were grown in an array of equally-spaced colonies of around 10 to 20 cells each. This confinement was achieved by micro-stamping a thin layer of fibronectin (allowing cell adhesion) onto a polystyrene substrate, using a PDMS stamp, followed by passivation of the untreated surface of the well with pluronic acid. The size and spacing of the micro-stamp were optimized to maintain a healthy environment for the cells. In each colony, a single cell was transfected with a plasmid encoding the green fluorescent protein (GFP) and resistance to an antibiotic (Zeocin). GFP-expressing colonies, originating from single transfected cells, were selected after antibiotic treatment and expanded as stably-transfected clonal lines. Using this method, monoclonal cell lines were generated in five weeks, with 6.9% of the transfected cells forming GFP-positive colonies. The technique, which is amenable of full automation, can generate clonal cell lines efficiently and without the need to perform tedious limited dilutions. Our model of arrayed colonies and single-cell electroporation was also used to perform a specific gene knockout using the CRISPR/Cas9 system. Genetically-engineered HEK293 cells containing a single copy of the GFP gene were grown into colonies as described above. All cells of the colony were transfected with Cas9 nuclease and a specific single guide RNA targeting the GFP coding sequence, in a buffer Tris-HCl 10mM, EDTA 0.25 mM, pH7.4. After two days, fluorescence microscopy demonstrated loss of GFP expression in transfected cells, consistent with gene knockout by Cas9. By contrast, non-transfected control cells exhibited green fluorescence. In summary, these examples show that iNfinitesimal's NFP technology of single-cell electroporation provides novel ways to generate clonal cells lines and CRISPR/Cas9 gene editing for cell engineering with applications to synthetic biology, stem cell research, and development of novel therapeutics.
• Arrayed force-phenotyping of single-cells for high-throughput screening in drug discovery
  Ivan Pushkarsky, University of California, Los Angeles
  Cell-generated forces play key functional roles within many physiological systems, such as regulating local vascular resistance, producing cardiac contractions, and propelling intestinal contents. Additionally, cellular forces are necessary to critical processes at the cellular level such as phagocytosis of pathogens and remodeling of extracellular matrix (ECM) fibers. As a result, a multitude of disorders are caused directly by malfunctioning cellular force generation including chronic conditions such as asthma, hypertension, and bowel disease. Such disorders place a significant burden on patient health and there is a paucity of effective drugs, arguably due to a lack of adequate tools for evaluating cellular force in early drug discovery. Herein, we describe a high-throughput phenotypic screening platform that meets this need by directly measuring the contractile forces generated by single-cells, which is implemented in both the 96- and 384- SLAS standard wellplate formats. The platform consists of a glass-backed elastomeric surface that is arrayed with uniformly shaped adhesive and fluorescently-labeled biomolecular micropatterns to which single-cells adhere to and displace via contractile forces. The fabrication process, which we developed, can be used to stably embed virtually any ECM protein into the elastomer, enabling us to simulate a variety of physiological environments. Custom image analysis algorithms measure the displacements of the micropatterns adhered to by single-cells, comparing them to the unperturbed (cell-less) micropatterns present in the same image set, which serve as internal controls, and displays a visual report of the population distribution of this phenotype. The effects of force-modulating compounds are directly visualized and quantified by monitoring the changing dimensions of these micropatterns. The elastomeric surface can be mounted and sealed onto a bottomless multi-wellplate for integration with standard automation equipment to facilitate high-throughput screens. Using this assay, we performed dose-response analyses of a known inhibitor of contraction and evaluated the potency of a panel of bronchoconstrictive agents involved in asthma, demonstrating its utility in quantifying cellular force. Furthermore, we validated the platform's drug discovery potential by assessing the bronchodilation effects of the long-acting β2 agonist, formoterol, (commonly used for asthma management), and demonstrating for the first time the bronchodilatory potential of a phosphoinositide 3-kinase inhibitor, which we found to have comparable potency to formoterol. The platform will be used in full scale screens to identify additional compounds that reverse agonist-induced bronchoconstriction in both healthy and asthmatics airway smooth muscle cells. This functional assay is compatible with a wide range of cell types and, in principle, can be applied to any disease arising from faulty force generation. As such, it is poised to strengthen all drug development efforts focusing on the restoration of normal cellular force generation at the i) high-throughput screening, ii) lead generation, and iii) toxicity screening stages.

Single Cell Analyses

Session Chair: Daniel Chiu, UW

• Precision Immunology Through Deeper, Single Cell Profiling
  Pratip Chattopadhyay, Precision Medicine Incubator, Vaccine Research Center, NIH
  This presentation will be recorded at SLAS2017 and made available free-of-charge to SLAS dues-paying members and full conference attendees post-event.
  
  Three trends have dominated biomedical research over the last decade. The first, the NIH Roadmap's Single Cell Analysis Program, was founded on the principle that cells are extremely heterogenous, and that this heterogeneity is important in health and disease. For this reason, cells must be characterized individually, rather than by insensitive and misleading analysis of bulk cell populations. This trend renewed appreciation for cellular heterogeneity, and incited a revolution of new technologies that could comprehensively analyze single cells (the second trend, deep profiling). Finally, a third biomedical research trend was sparked by President Obama's Precision Medicine Initiative, which aims to define genomic and proteomic differences between patient groups, and use this information to inform treatment decisions.
  
  In this talk, I will discuss my work at the intersection of these three trends, and demonstrate the value of new technologies for comprehensive and complete cellular analysis. I will provide examples of how deep knowledge about immune responses can be attained, using examples drawn from our recent work in HIV vaccine settings, immunotherapy, and fundamental immunology. This talk will highlight our work developing 30 parameter flow cytometry, single cell RNA sequencing, and new bioinformatic tools and include some discussion of how microfluidics and nanotechnologies can fit into a pipeline that includes the above technologies.
• 2017 SLAS Innovation Award Top 10 Finalist — A High Throughput Technology for the Isolation of Single Cells Using a Nanoliter Dispenser
  Michael Klinger, Fraunhofer Institute for Manufacturing Engineering and Automation IPA
  With the single-cell analyses gaining more and more importance in cell biology, technologies for the rapid isolation and patterning of single cells from heterogeneous suspensions are highly demanded. Speed, reliability and cell viability are crucial parameters for these systems, however, market-ready technologies do not yet meet the high demands of today's cell research and production. Addressing this issue, we developed a novel technology for the rapid isolation of single cells, using the fully automated nanoliter dispenser I-DOT. Recent studies have demonstrated, that the dispensing technology itself is well suited for cell printing, leading to a cell viability of more than 80% for all tested cell types, such as MCF-7 or hMSCs. With the help of the newly developed optical cell detection system, droplets are analyzed on the fly, whether they contain a fluorescence labeled cell or not. This allows the I-DOT system to dispense single cells onto virtually any culture surface, such as glass slides or multiwell plates. Comprehensive experiments with cell sized fluorescent polystyrene microspheres have shown a cell detection rate of 100% in a set of 100 droplets. The challenge remains solely in the adjustment of the initial cell concentration, preempting to enclose two or more cells within one droplet. Given the I-DOTs dispensing frequency of up to 400 drops per second, a significant increase of the cell isolation rate can be achieved compared to existing technologies. A 96 well plate can be filled with single cells (one cell per well) in less than 3 minutes in single channel mode, and in approximately 20 seconds utilizing the 8 channel parallel printing mode.
• Open the Doors of Perception: Reproducible data reduction and deep learning bring diagnostic intra-sample comparison to n-dimensional single cell data from cytometry and single cell sequencing.
  Michael Stadnisky, FlowJo, LLC
  The single cell is the basic unit of disease, but emerging technologies in cytometry and single cell sequencing are held back by myopic, time-consuming, sequential manual steps or computationally expensive, non-deterministic data reduction approaches. Herein, we describe phiSNE, an approach which combines a powerful nonlinear yet non-deterministic data reduction technique (t-SNE) and random forest regression which breaks critical limitations of t-SNE to enable the reproducible comparison of patient samples in n-dimensional space. We show the limitations of current PCA-based approaches in identifying cell subsets in single cell data from flow cytometry and single cell sequencing, and use our approach to discover a previously undescribed CD8+ T cell population. This diagnostic deep phenotyping approach allowed, for the first time, to use 25 parameters to discover and to diagnose a cell subset unidentified by an expert's analysis driving the preponderance of the difference in vaccine responses of patients in a publicly available data set. In addition, we leverage the open API of a leading single cell analysis platform to give bench scientists access to this tool with one click. We extended this work in two important dimensions. First, we used our phiSNE approach with single cell sequencing of whole melanoma metastases to compare the T-cell gene expression programs of 19 patients and reveal similarities and differences in their exhaustion. Furthermore, using both cell subset protein ontology for flow and mass cytometry and gene tables for single cell sequencing, we use deep learning to generate phenotype and gene expression templates which provide the first high-fidelity, shareable, scalable, truly automated, and interpretable identification methodology. Thus, our approach overcomes critical limitations in single cell biology -- we unlock n-dimensional data for use as a diagnostic tool, reduce manual analysis time, provide tools which rapidly reveal unidentified cell populations, and create the opportunity to compare large single cell studies.
• Novel method to dissociate solid tumors into viable single cells using Bulk Lateral Ultrasonic energy
  Daniel Holley, Microsonic Systems Inc.
  Molecular analysis of tumor tissue is one of the newer diagnostic approaches that has improved cancer outcome. Dissociation of viable single cells from solid tumors is an emerging methodology in clinical cancer diagnosis. The methodology should produce viable single cells from solid tumors without affecting the phenotype or genotype and provides an unmet need for comprehensive analysis of the tumor microenvironment to understand the heterogeneity at an individual cell level. A solution must simplify the workflow, save time, and be able to provide higher throughput thus enabling widespread adoption and development of routine application in clinical settings. we have pioneered the use of a novel ultrasonic technology "Bulk Lateral Ultrasonic™ (BLU) Energy", to dissociate solid tumors into viable single cells in less than five minutes. Our data show how this new form of ultrasonic energy is used to generate single cell suspensions from fresh, and formalin fixed paraffin embedded (FFPE) xenograft (PDX) tumor biopsies derived from research animals, including: 1) The analysis of single cell suspension from fresh tissues to evaluate cell yield and viability, 2) Flow Cytometry analysis of single cell suspension from fresh tissues to assess cell size and complexity, 3) Phenotypic evaluation by flow cytometry after staining the single cells from fresh PDX tumor tissues with monoclonal antibody panels specific for either tumor or immune cells, 4) Analysis of single cell suspension from FFPE tumor tissues to investigate cell yield and cell integrity, and 5) Comparative analysis of single cell data generated using the BLU energy technology and the reference method (manual and enzymatic tissue dissociation of solid tumors). Optimization of Bulk Lateral Ultrasonic energy for use on solid tissues has led to a novel single cell isolation tool which is scalable, provides higher throughput, saves time, is cost effective, simplifies the workflow, and offers a standardized protocol for fresh and FFPE solid tumor tissues.

Bioprinting: Multidimensional Microscale Cellular/Tissue Engineering

Session Chair: Markus Rimann, Zurich University of Applied Sciences

• 3D Bioprinted tissue models for substance testing
  Markus Rimann, Zurich University of Applied Sciences
  Bioprinting is a scaffold-based additive manufacturing technology to produce three-dimensional (3D) tissues with spatial control of cells, bioactive materials and molecules to reflect the inherent complexity of native tissues to a high degree. In precompetitive and industry-driven research projects we are developing bioprinting solutions. The current bioprinting setup includes: i) inkjet- and extruder-based printheads with temperature control for cell jetting and contact printing into well plates, ii) different ECM-like printable matrices (bioinks), iii) a photopolymerization unit to crosslink bioinks with UV-LED (365 nm) and iv) a cell mixing unit to avoid cell sedimentation in the print cartridge while printing. For tissue generation alternating layers of bioink and cells are printed to produce a multi-layered 3D tissue construct. In an industry research project we were developing an all-in-one solution to produce and analyse bioprinted in vitro muscle-tendon tissues in a specialized 24 well plate. The goal is to replace the cumbersome, time consuming and not much reliable animal experiments with isolated muscle-tendon tissues and to find new treatments for muscle-tendon related diseases. The wells in the plate contain two posts to build muscle-tendon tissues around and in between them. Precursor cells are printed, co-cultured and differentiated into muscle-tendon tissues. To setup the test system, monocultures of primary human myoblasts and primary rat tenocytes were printed separately in a dumbbell-shape around the posts. After cell differentiation the myoblasts were stained positive for myosin heavy chain (MHC) and myotubes developed and for tendon the characteristic collagen I-distribution around the cell nuclei was detected. Biological functionality was shown by muscle contraction on electrical stimulation. In the future electrodes will be integrated into the well plate to stimulate muscle contraction and at the same time measure the effect optically in the well plate. In a precompetitive research project we are elaborating the possibility to rebuild parts of the kidney, the so called proximal tubulus, to analyse nephrotoxicity as a major cause for effective drugs not being marketed. In preliminary experiments we printed the proximal tubule of the kidney and the adjacent blood vessel with sacrificial bioinks that were later seeded with proximal tubule epithelial cells and endothelial cells to cultivate them under physiological flow conditions. We demonstrated the ability to culture the cells under flow for several days in a newly developed PDMS-based flow device. The development of standardized 3D in vitro tissue models combined with suitable read-outs is a prerequisite for the future success of 3D tissues in substance testing and medicine in general. Funding: CTI, Project Numbers: 14331.1, 16313.1, SNF No.: 20PC21_161566 / 1
• 2017 SLAS Innovation Award Top 10 Finalist — Rapid Multi-Material Extrusion Bioprinting
  Y. Shrike Zhang, Brigham and Women's Hospital, Harvard Medical School, Harvard-MIT Health Sciences and Technology
  Bioprinting has emerged as an enabling technique with unprecedented flexibility in generating three-dimensional (3D) architectures. Despite recent development in 3D bioprinting technologies, which have significantly facilitated the precise control over the spatial deposition of bioinks into pre-defined structures, until now, most of the current bioprinting modalities have been limited by the use of a single bioink during each deposition process and thus cannot achieve rapid bioprinting of sophisticated compositional structures. To overcome this major hurdle, here, we report the development of a novel multi-material bioprinting platform that is capable of extruding multiple coded bioinks in a continuous manner with extremely fast and smooth switching among different reservoirs for rapid fabrication of complex (tissue) constructs. We have provided a proof-of-concept demonstration by mounting a single printhead consisting of a bundle of 7 equal-sized capillaries, each connected to a unique bioink reservoir that could be individually actuated by digitally controlled pneumatic pressure. The ejection process, when synergized with the movement of the motorized stage, allows for rapid deposition of two-dimensional (2D) patterns and 3D architectures composed of multiple desired bioinks in a spatially defined manner, at a speed up to 15 times faster than existing bioprinting modalities based on extrusion. Using this novel rapid continuous multi-material bioprinting technology, we were able to create a series of printed structures possessing unprecedentedly high levels of complexity with much reduced durations. For example, we bioprinted human organ models with multiple bioinks to mimic the hierarchical architecture of those in vivo. Notably, cells encapsulated in the bioprinted constructs showed strong viability and proliferation. In addition, we ascertained that these organoids were not only surviving but also actively interacting with the bioprinted heterogeneous microenvironment, by quantitatively analyzing their responses when grown on the constructs with gradually changing bioactive properties. Furthermore, we also demonstrated broad applications of our continuous multi-material bioprinter to the construction of high-throughput point-of-care devices as well as bioelectronic circuits composed of gradually changing concentrations of conductive bioinks. The proposed technology is likely to advance the field of bioprinting by offering unprecedented capacity in printing speed and continuity, which is compatible with a wide variety of bioinks ranging from shear-thinning biomaterials to photocrosslinkable hydrogels and conductive bioinks. Most of all, our 3D bioprinting platform may be conveniently extended to any numbers of bioinks necessary for engineering highly complex functional biomaterials, tissues, and devices. It is anticipated that our rapid continuous multi-material extrusion bioprinter will find widespread applications in biomedicine.
• Overcoming chemoresistance from heterotypic cellular communication and physical stress in bioprinted and microfluidic 3D cancer models
  Imran Rizvi, Department of Dermatology, Massachusetts General Hospital, Harvard Medical School
  Identifying the molecular, cellular, and microenvironmental cues that lead to heterogeneity and treatment resistance in tumors is critical to developing strategies to target unresponsive populations of stubborn disease. Identifying effective combinations requires a multi-faceted approach that includes the development of bioengineered cancer models and corresponding image analysis tools. Here, 3D in vitro models that restore the architectural features, physical stress and heterocellular signaling experienced by tumors in vivo are described in the context of metastatic ovarian cancer, the leading cause of death among gynecologic malignancies. The potential value, and challenges, associated with developing bioprinted and microfluidic cell-based models that restore 3D architecture, integrate communication with stromal partners and account for physical stress, will be presented. A particular focus will be on using image-based quantification to characterize variability in response to conventional agents and the ability of rationally-designed combinations to overcome this heterogeneity.
• Precisely Engineered Cellular Arrayed Microenvironments for Highly Selective Cellular Activation and Screening
  Michael Floren, University of Colorado Anschutz Medical Campus
  Preparation of 3D synthetic cellular microenvironments which capture physico-chemical-mechanical parameters of the natural in vivo environment with high fidelity, poses significant challenges yet opportunities to the development of novel cell therapy and disease modeling. 3D cellular models produced with current in vitro technologies lack precisely tunable properties such as mimetic tissue stiffness, geometry and biomolecular signal presentation. To overcome these limitations, we have developed an enabling technology of engineered cellular arrayed microenvironments (CAMs) to screen and investigate multivariate tissue environments, which is being used to elucidate complex matrix signaling mechanisms accredited to drug actions, tissue homeostasis, repair or regeneration, providing key insights into disease modeling for targeted therapy. Our platform consists of precisely-controlled nano/micro-fibrous 3D hydrogel materials based on photoclickable thiol-ene poly(ethylene glycol), which integrate a variety of spatially-defined molecule signals via array printing technique. Thiol-ene polymerizations proceed by an orthogonal, step-growth mechanism where one thiol reacts with one ene leading to a highly homogenous distribution in crosslinks, thus imparting tunable substrate stiffness as well as permitting subsequent covalent post-functionalization with engineered peptides with high reactivity and specificity. Combining with microarray contact printing, we have created CAM environments with highly selective and combinatorial biomolecule presentation where traditional engineered microenvironments often fail. Here we demonstrate the power of such tailored environments by the selective attachment of a diversity of cells, including primary and stem cell lines, in precise geometries based on selective ligand functionalization of our platform. Further, we show that through judicious tailoring of our substrates that specific engineered ligands may be exploited to develop advanced co-culture arrays, with cell populations chosen by the end user. In particular, co-culture assays of fibroblasts and macrophage lines will be highlighted to demonstrate utility towards exploring complex inflammation environments over short length scales. Taken together our technology introduces a novel screening device which enables precise control over critical tissue parameters resulting in improved 3D in vitro representation, including enhanced tissue modeling, with broad applications in regenerative medicine, disease modeling, and drug discovery. Physiological in vitro systems reflecting anatomical and functional characteristics of the human condition could be instrumental towards developing effective, robust therapies of the future.

Microphysiological Systems

Session Chair: Dan Huh, Penn

• Organs-on-Chips: Micro-engineered environments for studying human tissues functions
  Ville Kujala, Emulate Inc
  "Organs-on-Chips" use microscale engineering technologies that when combined with cultured living human cells create microengineered systems that recapitulate individual organ functions and organ-organ interactions. Human "Organs-on-Chips" offer the potential to develop specialized in vitro human disease models that could revolutionize drug discovery and development. These microengineered systems enable the study of human physiology, tissue specific metabolic function, and disease development. Each Organ-on-Chip is composed of a clear flexible polymer about the size of a USB memory stick that contains hollow channels lined by living human cells. The cells are cultured under continuous flow and mechanical forces thereby recreating key mechanical forces known to influence cell function in vivo. Our Organs-on-Chips are designed to fully recreate the complex, dynamic state in which living cells function within a real human organ: substrate (extracellular matrix), tissue-tissue interface, mechanical forces, immune cells and blood components, and biochemical surroundings. Working closely with collaborators in academia, regulatory bodies and the industry, we develop micromechanical environments and quantitative readouts specific to particular organs and disease states. Here we present examples of Organs-on- Chips that recapitulate micromechanical environments and quantitative readouts specific to particular organs and disease states in humans.
• A Microfluidic Model of Kidney and Other Transport Tissues for 96-Well Format Screening of Therapeutics
  Joseph Charest, Draper
  In vitro screening for therapeutic effects in humans can be made more predictive by creating in vitro models which express organ- or tissue-specific function in platform with a high level of throughput. The organ- or tissue-specific function can then be evaluated across many samples, doses, and replicates and extrapolated to a clinical measure impacted by the organ- or tissue-specific function. We create a microfluidics-based model of kidney proximal tubule tissue which expresses active reabsorption function. The model uses controlled microfluidic fluid flow, cell-substrate topography, and cell-cell cues to influence human renal proximal tubule epithelial cells and human microvascular endothelial cells. The resulting tissue can be evaluated within the microfluidic architecture for barrier function and active transport function. To achieve higher levels of throughput, the model can be replicated within a 96-well format while still maintaining the unique characteristic of controlled flow. To improve data collection, integrated electrical traces measure trans-epithelial/endothelial electrical resistance (TEER) in near real-time and provide a means to create additional sensing capabilities in the future. The microfluidic model demonstrates the ability to generate tissue in vitro with tissue-specific function, provides near real-time feedback on tissue barrier function, and can scale to relevant levels of throughput resulting in a screening tool for drug interaction with transport tissues.
• Mechanically actuatable airway disease-on-a-chip: a novel platform to study biomechanical basis of human lung disease
  Jungwook Paek, Department of Bioengineering, University of Pennsylvania
  The lung is arguably the most mechanically active organ in the human body that continually experiences dynamic tissue deformation and fluid flow throughout life. Various types of mechanical forces arising from this dynamic environment are essential to the homeostasis and physiological function of the respiratory system. Clinical evidence has suggested that abnormal alterations in the mechanics of the lung may play a causative role in many respiratory diseases. Research efforts to delineate the fundamentals of this significant clinical association, however, have been greatly hampered by the technical challenges of modeling complex changes in the structure and mechanical microenvironment of the respiratory tract during disease progression. Here we describe a novel microengineering strategy to tackle this long-standing, critical challenge in respiratory biology and medicine. This approach is based on microphysiological three-dimensional (3D) cell culture integrated with programmable actuation of soft elastomeric microstructures to emulate i) cellular heterogeneity and complex microarchitecture of native lung tissue and ii) disease-induced mechanical forces and resultant pathophysiological tissue distortion in the lung. Specifically, we have created a microengineered disease model that reconstitutes the constriction of conducting airways in the distal lung, which is a hallmark of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease. As the first step to establish this model, we constructed a compartmentalized microfluidic device that enabled co-culture of primary human small airway epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in a physiologically relevant spatial arrangement. Fabrication of this device was achieved by utilizing removable templates and surface tension-induced pinning effects to form an airway compartment enclosed by an extracellular matrix hydrogel scaffold that contained a perfusable microchannel. Cell culture in this system produced multilayered tissue constructs consisting of the small airway epithelium supported by the underlying stroma laden with fibroblasts and perfused through an endothelialized vascular tube. To mimic pathophysiological obstruction of small airways during disease, we integrated our cell culture platform with a microfabricated soft elastomeric actuator capable of converting pneumatic pressure into precisely controlled inflation of microchannel walls. Actuation of this component exerted compressive forces on the cell culture chamber and induced the microfluidic airway tissue to undergo 3D structural distortion reminiscent of airway constriction in vivo. Our small airway-on-a-chip represents a major advance in the development of new technologies to address the critical unmet need for human-relevant lung disease models. This system provides a novel platform to investigate how mechanical abnormalities contribute to disease processes in the distal airways that have recently emerged as a key player in the development and progression of various lung diseases. Our microengineered model has the potential for broad impact in pulmonary research and may play an integral role in improving our fundamental understanding of respiratory health and disease.
• Perfused intestinal Caco-2 tubules suitable for high throughput screening
  Remko van Vught, MIMETAS B.V.
  The human epithelial Caco-2 cell line has been widely used as an intestinal barrier model. Caco-2 is usually cultured on Transwell® inserts to study compound transfer across an epithelial barrier. However, these systems often fail to produce mucus layer and microvilli formation, which are essential to the physiological relevance of a human intestinal model. Hereby, we want to introduce the OrganoPlate®, a microfluidic platform which enables the culture of membrane-free cell barriers into a tubular structure1. The OrganoPlate® is based on a 384-well microtiter plate resulting in up to 96 data points per plate (Fig.1). An extracellular matrix (ECM) layer is patterned using the Mimetas phaseguide™, a small ridge present between the channels that acts as a capillary pressure barrier. After gelation of the ECM, a suspension of Caco-2 cells is added to the adjacent channel to form a membrane-free barrier model mimicking in vivo intestinal environment (Fig.2). The Caco-2 tubular cultures were characterized by immunofluorescence staining at day 4 and 11, showing cell polarization, tight junction formation and expression of key receptors. After 4 days, the Caco-2 tubes showed dome formation and were positively stained with ZO-1 (tight junction marker) and acetylated tubulin (polarization marker). The presence of the glucose receptor (Glut-2 staining) and epidermal growth factor receptors (ErbB1&2 staining) on the basolateral side indicating polarization of the tubules. Most importantly, the presence of intestinal villi and the formation of a mucus layer were detected using Ezrin and Muc-2 staining, respectively. After 11 days, invagination patterns were observed and stained positive for MRP-2 (drug transporter) on the apical side and Glut-2 on the basolateral side. These advanced characterizations show that our OrganoPlate® culture system offers a better physiologically relevant Gut-on-a-chip model, providing a powerful tool for high throughput compound screening in pharmaceutical industry.

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Special Sessions

Panel Discussion: Whose Responsibility is Research Reproducibility?

Session Chairs: Lenny Teytelman, Protocols and Cathy Tralau-Stewart, Catalyst & Associate Professor Therapeutics (AJ), University of California San Francisco

Moderator: Richard Harris, NPR

Panelists:
Cathy Tralau-Stewart, Catalyst & Associate Professor Therapeutics (AJ), University of California San Francisco
Richard Neve, Gilead
Ivan Oransky, Retraction Watch
Tara Schwetz, National Institutes of Health
Elizabeth Iorns, Science Exchange
Veronique Kiermer, PLOS

There is a broad consensus among academic and industry researchers, funders, and the lung is arguably the most mechanically active organ in the human body that continually experiences dynamic tissue deformation and fluid flow throughout life. Various types of mechanical forces arising from this dynamic environment are essential to the homeostasis and physiological function of the respiratory system. Clinical evidence has suggested that abnormal alterations in the mechanics of the lung may play a causative role in many respiratory diseases. Research efforts to delineate the fundamentals of this significant clinical association, however, have been greatly hampered by the technical challenges of modeling complex changes in the structure and mechanical microenvironment of the respiratory tract during disease progression. Here we describe a novel microengineering strategy to tackle this long-standing, critical challenge in respiratory biology and medicine. This approach is based on microphysiological three-dimensional (3D) cell culture integrated with programmable actuation of soft elastomeric microstructures to emulate i) cellular heterogeneity and complex microarchitecture of native lung tissue and ii) disease-induced mechanical forces and resultant pathophysiological tissue distortion in the lung. Specifically, we have created a microengineered disease model that reconstitutes the constriction of conducting airways in the distal lung, which is a hallmark of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease. As the first step to establish this model, we constructed a compartmentalized microfluidic device that enabled co-culture of primary human small airway epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in a physiologically relevant spatial arrangement. Fabrication of this device was achieved by utilizing removable templates and surface tension-induced pinning effects to form an airway compartment enclosed by an extracellular matrix hydrogel scaffold that contained a perfusable microchannel. Cell culture in this system produced multilayered tissue constructs consisting of the small airway epithelium supported by the underlying stroma laden with fibroblasts and perfused through an endothelialized vascular tube. To mimic pathophysiological obstruction of small airways during disease, we integrated our cell culture platform with a microfabricated soft elastomeric actuator capable of converting pneumatic pressure into precisely controlled inflation of microchannel walls. Actuation of this component exerted compressive forces on the cell culture chamber and induced the microfluidic airway tissue to undergo 3D structural distortion reminiscent of airway constriction in vivo. Our small airway-on-a-chip represents a major advance in the development of new technologies to address the critical unmet need for human-relevant lung disease models. This system provides a novel platform to investigate how mechanical abnormalities contribute to disease processes in the distal airways that have recently emerged as a key player in the development and progression of various lung diseases. Our microengineered model has the potential for broad impact in pulmonary research and may play an integral role in improving our fundamental understanding of respiratory health and disease.stakeholders that increasing reproducibility of published research is an important goal. However, questions of who should be responsible for validating research results are tricky; industry and academia naturally diverge in answering these. Moreover, specific proposals for improving reproducibility are frequently contentious with fears of unintended consequences for the research enterprise. The goal of this panel is to have a conversation with both industry and academic perspectives on this challenging issue.

Regenerative Medicine: Next Generation Treatments

Session Chairs: Marcie Glicksman, Orig3n, Inc. and G. Sitta Sittampalam, NIH/NCATS

• Progress and Challenges in Translational iPS Cell Research
  Ilyas Singec, NIH/NCATS
  Epigenetic reprogramming of somatic cells into induced pluripotent stem (iPS) cells with defined factors holds great promise to transform drug discovery and personalized regenerative medicine. Patient- and disease-specific iPS cells can self-renew indefinitely and differentiated into any cell type of the human body. As the generation of new iPS cell lines has become a routine approach across many different laboratories over the last decade, there are still key challenges that need to be addressed in order to move this technology closer to the patient. In particular, clinically relevant applications of iPS cells require deeper insights and control of pluripotency, cell differentiation, and functional maturation of well-characterized cell types. The NIH Regenerative Medicine Program (RMP) and the National Center for Advancing Regenerative Medicine (NCATS) lead a new multidisciplinary effort to leverage these scientific questions and establish rigorous procedures and references for the utilization of iPS cells. This presentation will highlight the goals of this new program and provide examples on how to shed new light on pluripotency and directed differentiation by using quantitative and high-throughput methods.
• Angiopellosis as an alternative mechanism of cell extravasation
  Ke Cheng, NC State University/UNC-Chapel Hill
  Stem cells possess the ability to home in and travel to damaged tissue when injected intravenously. For the cells to exert their therapeutic effect, they must cross the blood vessel wall and enter the surrounding tissues. The mechanism of extravasation injected stem cells employ for exit has yet to be characterized. Using intravital microscopy and a transgenic zebrafish line tg(Fli1a:egpf) with GFP-expressing vasculature, we documented the detailed extravasation processes in vivo for injected stem cells in comparison to white blood cells (WBCs). While WBCs left the blood vessels by the standard diapedesis process, injected cardiac and mesenchymal stem cells underwent a distinct method of extravasation that was markedly different from diapedesis. Here, the vascular wall undergoes an extensive remodeling to allow the cell to exit the lumen, while the injected cell remains distinctively passive in activity. We termed this process Angio-pello-sis, which represents an alternative mechanism of cell extravasation to the prevailing theory of diapedesis.
• Modeling developmental brain disorders using patient-derived induced pluripotent stem cells
  Hongjun Song, Johns Hopkins University School of Medicine
  Psychiatric disorders are heterogeneous disorders characterized by complex genetics, variable symptomatology, and anatomically distributed pathology, all of which present challenges for effective treatment. Current treatments are often blunt tools used to ameliorate the most severe symptoms, often at the risk of disrupting functional neural systems, thus there is a pressing need to develop rational therapeutics. Induced pluripotent stem cells (iPSCs) reprogrammed from patient somatic cells offers an unprecedented opportunity to recapitulate both normal and pathologic human tissue and organ development, and provides new approaches for understanding disease mechanisms and for drug discovery with higher predictability of their effects in humans. I will present our study on using patient-derived iPSCs to investigate the cellular and molecular mechanisms underlying genetic risk factors for developmental brain disorders and our effects to use iPSC-derived neurons for drug discovery.
• Standardized generation of patient-specific and gene-corrected induced pluripotent stem cell lines for disease modeling and drug screening
  Lise Munsie, CCRM
  The use of pluripotent stem cell-derived cell types for disease modeling, drug screening and regenerative medicine is an exciting area of activity in health research. Prior to the availability of pluripotent stem cells and associated methods for generating many lineage specific cell subtypes in vitro, relevant and affected primary cells were hard to obtain and frequently only been accessible post-mortem. Induced pluripotent stem cell (iPSC) technology enables the creation of patient-specific pluripotent cell lines, making personalized medicine approaches a possibility for people working in health research. Furthermore, recent advances in genome editing technologies promise the efficient creation of genetically corrected iPSC lines. At CCRM, we have established an iPSC production facility focused on generating high quality pluripotent cell lines from patient samples for academic researchers and clinicians. Fully operational for three years, CCRM has delivered over 100 patient iPSC lines that are being used for disease modelling, and in drug screening initiatives, at Institutes across Canada. Specializing in non-integrative reprogramming technologies, we have developed SOPs to reprogram many common cell types in feeder-free conditions, including dermal fibroblasts, bone marrow stromal cells, cord and peripheral blood, and endothelial cells. We are additionally developing the capabilities to perform genome editing in iPSCs using the newest and most efficient technologies, including TALENs and CRISPR/Cas9. These technologies can be used to induce a mutation of interest in control pluripotent cells or correct a genetic mutation in a patient-derived iPSC line. Access to patient-derived iPSCs and the associated isogenic line as a control would allow for the discovery of novel disease-associated phenotypes and therapeutic targets for specific patient subpopulations.

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