University of Pennsylvania
Perelman School of Medicine
High-Throughput Screening Core
Sara Cherry, Ph. D.
David C. Schultz, Ph. D.
dschultz@mail.med.upenn.edu(215) 573-9641
67 John Morgan BuildingMission
Provide the PSOM community with HTS resources.
• To educate and assist with HTS assay development, optimization,miniaturization, and validation• To provide laboratory robotics infrastructure and technically trained staff forHTS• To provide libraries of small molecule and genetic tools for HTS• To facilitate small-scale screens from user-defined gene-sets

Develop novel technology

Seed collaborative research programs and grants.



Educate the SOM on utility and uses of HTS

SOM Screening Core
› Libraries› Maintenance› Distribution› Liquid handling› Janus MDT/Verispan 8-tip› Bulk reagent dispensors› ELx405 microplate washer› Assay Detection› EnVision multi-mode microplate reader› ImageXpress Micro› FLIPR screening system› BSL2 Tissue Culture capabilities› Hood› Incubators› Informatics› Automated image analysis› Statistical analysis› Validation analysis

Automation: ScreeningAutomatedpippeting station

384w plates spottedwith cDNAs or siRNAs

Dispenser***** **HT Multimode Reader*“Hit”

Automated Microscopy
Slide courtesy of J.HogeneschCell-based functional screening approaches
Gain of function

cDNAHighActivity

Low

Small molecules

Loss of function
RNAi
Slide courtesy of J.Hogenesch

SOM Screening Core Chemical Libraries
Bioactives, FDA approved, and FDA-like compounds• SelleckChem Bioactives (~2100)• Kinase Inhibitors, Epigenetic Inhibitors, Cancer
compounds, GPCR/Ion Channel, Metabolism,
Microbiology, FDA approved/FDA-like• LOPAC (1280): Library of Pharmacologically Active
Compounds
Natural Products• Microsource Purified Natural Products (800)
Diversity set
Chembridge (32,000 total)
20,000 from Core set and 12,000 from Express Pick set

ChemDiv (12,000 total)
SMART libraryChemical Libraries:
SelleckChem Bioactive Library (2083 cmpds)•Kinase Inhibitors, Epigenetic Inhibitors, Cancer compounds, GPCR/Ion Channel,Metabolism, Microbiology, FDA approved/FDA-likekinase373
Other699

Cancer246

Epigenetic
96
Microbiology
172
Protease
Ion Channel
56
78

Metabolism
83
GPCR
280

http://www.selleckchem.com/screening/chemical-library.html

Chemical Libraries:
LOPAC1280: Library of Pharmacologically Active Compounds
1,280 FDA approved, marketed drugs with annotated biological activities,
predictable activities and proven scaffolds directed against a wide range of drugtargets

http://www.sigmaaldrich.com/catalog/product/sigma/lo1280?lang=en&region=US

Diversity Library: cherry picked 44K from…

• Started with ~200,000 compounds• Used modified Lipinski parameters to filter the set.
• Removed Reactive groups (e.g. Michael acceptors) and compoundswith undesirable functionalities.
• Duplicates were removed.
• Lead Finder Clustering and MACCS fingerprints were generated.
• PAINS1, PAINS2 and PAINS3 substructure filters were applied.
• REOS filters were applied.
• Property Based Selection of 50K cpds was performed• Vendor re-supply validated

Joe Salvino & Dora Schnur, Drexel SOM

44K Diversity Set Characteristics

44K Diversity Set Characteristics-II

SOM Screening Core Genetic Libraries
siRNA• human genome-wide, human drugable genome, human GO
categories• user-defined human and mouse
Non-coding RNAs
• lncRNAs (human)• miRNA mimics/antagonists (human V20)Human TRC 2.0 and Mouse TRC1.0 Lentivirus shRNA library• Screening pools: GO categories; user-defined sets
• Order groups/individuals
MGC cDNA collection (CMV-driven)• 18,000 full length, sequenced, mouse and human (arrayed);• user-defined sets
• Order groups/individuals

Genetic Libraries:Ambion Silencer Select siRNA & miRNA mimics/inh.
A. Human genome Silencer Select siRNA
Genes
siRNAs siRNAs/GT
formatDruggable genome^
9032
27093
3
pooledDruggable genome Ext.
1383
4149
3
pooledRest of Genome
11170
33510
3
pooled^ Further organized into GO categories (e.g. kinases, NHRs, GPCRs)# plates*26434

B. Human non-coding RNA siRNA (lncs) & miRNA mimics/inhibitors
long non-coding RNAs (lncs)
miRNA mimics (human V20)
miRNA inhibitors (humanV20)

Targets

siRNAssiRNAs/Tarformat# plates*

2220
2555
2555

6660
2555
2555

31
1

pooledindividual
individual

888* All assay plates are pre-spotted with siRNAs/miRNAs, including controls

Control siRNAs
Control
Neg1
Neg2
GAPDH

siRNA_ID
S813
S814
S815

Type
Negative
Negative
PositivemammalianPositive
Non-mammalian

Library
Genome, lnc
Genome, lnc
Genome, lnc

GFP

s229097

Luciferase

s229095

Positive
Non-mammalian

genome

Kif11

S7903

Positive
Genome, lnc, mir
Mammalian (death)

MALAT1

s239370

Positive
Mammalian* Controls are pre-spotted in all assay plates

genomeLnc

Custom siRNA Libraries
 Custom siRNA libraries are provided by Ambion/Lifetechnologies
 Minimum order of 20 siRNAs REQUIRED 0.1 nmol of Silencer Select siRNA (human) 1 nmol of Silencer siRNAs (human and mouse) siRNAs are received in microtiter plates (96 or 384)
 All orders are submitted through the HTSCCustom Library Ordering• Complete the order form
 Customer information (26 digit Penn fund no)(REQUIRED) NCBI Gene ID, Gene Symbol, # of siRNAs/gene target, species (REQUIRED) Email to dschultz@upenn.edu

Custom Library Ordering (cont) Costs•

HTS/Omics validation set‒ 100 gene targets x 3 siRNAs is ~$4,500•

Custom library (Epigenetics/metabolism)‒ 500 gene targets x 3 siRNAs is ~$12,000 Turnaround
Order size

Order to deliver20-250 siRNAs

5-15 business days

251-500 siRNAs

16-25 business days

501-1000 siRNAs

20-30 business days

1001-2500 siRNAs

25-30 business days

Genetic libraries:
TRC human/mouse LV-shRNA library
Human complete
Mouse V1.0
Totals

GeneTargets
20,01815,96035,978clones

Validated% val

129,695
77,819207,51443,47020,56464,0343426

http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/libraryinformation.html

shRNA library services
 Distribution of Individual clones• $100.00 per Gene Target (5+ clones)• Investigator should sequence confirm shRNA sequence• Investigator responsible for validating knock-down
 Preparation of Custom Gene Sets
• Cherry-pick and array clones of interest into new plates‒ GO categories (e.g. Epigenetic targets)‒ User-defined sets (e.g. ‘omics validation)• Glycerol stock plate + pooled plasmid DNA• 150 gene Targets/625 clones: $2000• 600 gene Targets/3000 clones: $5000

Ordering shRNA clones• Complete the order form
• Customer information (26 digit Penn fund no) (REQUIRED)• NCBI Gen ID, Gene Symbol, Refseq no., species (REQUIRED)• Email to dschultz@upenn.edu

Genetic libraries:MGC cDNA collection• Obtained by J.Hogenesch• 18,000 full length, sequenced, mouse and human cDNAs pre-cloned intopCMV-SPORT6• Insert is fully sequenced and guaranteed to match corresponding BCAccession Number• Expression-ready vectors eliminate additional cloning steps• Robust CMV promoters drive cDNA expression• Gateway sites flanking coding sequence allow for additional flexibility• Stored as bacterial glycerol stocks, arrayed in 96 well microtiter platesScreen:• Complete library• user-defined sets (e.g. Interferon
Stimulated Genes-ISGs)• Order individual clones or groups• $50.00/clone

Automation: Screening

Automatedpippeting station

384w plates spottedwith cDNAs or siRNAsDispenser

****
* **
HT Multimode Reader*“Hit”

Automated Microscopy
Slide courtesy of J.Hogenesch

Envision Xcite: multi-mode microplate reader
A. Measurement Technologies
••
•
•
•
•

absorbance,flouresence intensity (FI),fluorescence polarization (FP),time-resolved fluorescence (HTRF)
ultra-luminesence
AlphaScreen (Amplified Luminescence
Proximity Homogeneous Assay)

B. Assays
Anything that requires a plate reader!
• Luciferase Reporter Gene Assays (RGAs)• Viability assays (e.g. Cell-titer Glo)
• Enzymatic assays (FI)• Protein: nucleic acid interaction (FP, HTRF,alphascreen)• Protein: protein interactions (FP, HTRF, alphascreen)• AlphaLISA

ImageXpress Micro XLS
A. Enables automated acquisition of multi-channel
fluorescence images in microtiter plates• 5 colors

B. Phenotypic assays upto 40X• Physical properties of cells (size, shape, etc.)• Puncta formation (autophagy, lipid droplets, etc)
• Activity (migration, invasion, neurite outgrowth)• Expression and localization of native proteins• Measurement of fluorescent labels (antibody stains,EdU/BrdU, Calcein, PI uptake, mitochondria, etc.)• FRET

C. Image analysis: MetaXpress– Easy analysis pipelines in-house– Computational analysis of images enables measurement of multiple
cellular properties of interest at the object/cell level and/or population level.

High-content Screening Assays
Neurite Outgrowth
control+NGF

3D tumor spheroids (Invasion/viability)

Migration/scratch assay
controlagonistLipid droplets/puncta

FLIPR Tetra: Flouresence Imaging Plate Reader
A. Measurement Technology:• Integrates liquid handling with rapid whole plate imaging offluorescence and luminescence• Can read at sub-second intervals, which enables the kinetics ofthe response to be captured• Integrated pipettor enables successive liquid additions,providing an opportunity to detect agonists, antagonists, andallosteric modulators all in one assay

B. Assays:
1. Intracellular Ca2+ flux: cell permeable
Ca2+ sensitive fluorescent dye (e.g.
cardiotoxicity);
2. Fatty acid or neurotransmitter uptake offluorescence dyes.
3. Membrane potential: lipophilic, anionic,fluorescent dye that partitions acrossthe cytoplasmic membrane of live cells
based membrane potential.

FLIPR Assays
Monitoring Intracellular Ca2+ Mobilization

No agonist

AgonistsblankThe Assay Development Process~ 1-6 months• Idea• Phenotypes• Reporters, cell types• Controls• Existing infrastructure

DesignDevelopment• Controls: Z-factors, S/N,CVs• Solvent; Timing; TX
reagents• Cell type, numbers• Day to day; Plate to plate• Test set (e.g. 1K set)
• Data analysis• Scale up

POC~ 1 hr to a monthSlide courtesy of J.HogeneschAssay Design and Development• Is the nature of the response clearly defined?
• Is the response dependent only on the activity of thecompound being tested or is it conditioned by anotherstimulus?
• What is the duration of the response?
• What sort of ‘secondary assays’ exist to confirm activity
and determine selectivity of newly identified probes?
• What structural classes of known actives exist and what
are the limitations to their use?
Goal: To design an assay that is robust and sensitive

What biology and assays arereadily screened?
• Enzymatic• Protein: protein interactions• Protein: nucleic acid interactions• Reporter Gene Assay• Sensitivity to drugs• Simple design and robust signals…

Biochemical Assays• Enzymatic assays– Luminescence (e.g. ADP-Glo)– Fluorescence (e.g. QFRET, AmplexRed)– Transcreener• Protein-protein interactions– ALPHAscreen– HTRF– ELISA (e.g. luminescence)• Protein-nucleic acid interactions– ALPHAscreen– HTRF• Ligand binding– Differential Scanning Flourimetry/Thermal Stability– SPR

Profiling and Sensitivities• Phenotypic profiling of cell lines– FDA and FDA-likes– Annotated gene family (e.g. kinome)– Synthetic lethality screens– Synergy studies (combinations gene-gene; gene-drug;drug-drug)• Differentiated iPSC from individuals• Across tumors (e.g. melanomas)• Of a particular patient tumor line (to defineresponsiveness)

Pharmacological Response of GeneticallyDefined Tumor Cells

Cell-based Assays• Anything you can read in a plate reader– Reporter Gene Assays (e.g. luciferase)– Survival (e.g. Cell-titer Glo, Alomar Blue, MTS)
– Signaling (e.g. alphascreen, alphaLISA)• Microscopy assays– EdU/BrDU incorporation– Autophagy (e.g. LC3-GFP/RFP)– Migration assays (e.g. scratch assay)– Neurite Outgrowth– Infection– Nuclear/cytoplasmic shuttling– Antigen localization/staining• Signaling (e.g. Ca2+, membrane potential)

Cell types• Chemical screens- all cells amenable• Genetic screens– Transformed cells– Many primary cells• Fibroblasts, endothelial, epithelial, macrophages, etc• Lymphocytes challenging• Stem cells– Mixed cultures• Perturb one cell type and assay another• e.g. siRNA in fibroblast but read tumor cell biology; siRNA inmacrophage but read T cell biology

What scale is useful to you?
• Do you have a defined set of genes orpathways you are interested in?
– Functional study of ‘OMICS data
• RNAseq• CHIPseq• GWAS• Exome– Interested in Kinases or another ‘category’Services• Consultations (per hour)– Assay development– Assay optimization– Assay validation– Grant submissions• Equipment usage (per hour)– With help
– Without help

• Small scale screens– User-defined (siRNAs,shRNAs, cDNAs, chemicals)– Library plates (e.g. kinome)• Large scale screens– Library plates• Data Analysis– Normalization, annotation– HCA analysis sequence dev.
– Screen reports• Reagents– Transfection– Plastics• Tips• Plates• siRNAs, shRNAs, cDNAs– User defined sets
– Individual clones

Funding Opportunities•

NIH– PAR-13-364 Development of Assays for High-Throughput screening for use in
Probe and Pre-therapeutic Discovery (R01)– PAR-14-283/PAR-14-284, High-Throughput Screening (HTS) to Discover
Chemical Probes (R21/R01)– PAR-14-279, Discovery of in vivo Chemical Probes (R01)– PAR-13-049/PAR-13-048, Drug Discovery for Nervous System Disorders(RO1/R21)– PAR-13-007, Early-stage Pharmacological Validation of novel Targets andAccompanying Pre-therapeutic Leads for Diseases of Interest to the NIDDK(RO1)– PAR-14-006, Seeding Collaborations for Translational Research to Discover andDevelop New Therapies for Diseases and Conditions within NIDDK's Mission(RO1)– PAR-15-056, Building on High Impact Neurobiology Through Assay
Development: Advancing Tools for Therapeutic Development (RO1)– PAR-15-070, Innovation Grants to Nurture Initial Translational Efforts (IGNITE):
Assay Development and Therapeutic Agent Identification and Characterization
to Support Therapeutic Discovery (R21/R33)– PAR-15-071, Innovation Grants to Nurture Initial Translational Efforts (IGNITE):
Pharmacodynamics and In vivo Efficacy Studies for Small Molecules andBiologics/Biotechnology Products (R21/R33)

Funding Opportunities (part II)•NIH (continued)– PAR-13-267, Novel NeuroAIDS Therapeutics: IntegratedPreclinical/Clinical Program (P01)– PAR-15-041, Targeting Persistent HIV Reservoirs (TaPHIR)(R21/R33)•NCAT/TRND opportunities••

Foundations (e.g. Welcome Trust, Melanoma Research Foundation,Leukemia/Lymphoma Society, Gates, Cystic Fibrosis)
Commercial (e.g. Bayer Grants4targets, Astrazeneca Openinnovation)•Institute/Center/Program Pilot project funds••••

Institute for Immunology
Center for Orphan Disease ResearchInstitute for Regenerative Medicine
Epigenetics ProgramInput from the Community
We are expanding based on SOM needs….
– FLIPR from Physiology– cDNA library from J. Hogenesch (Pharm)– Mouse shRNA library• Contributions: CHOP, Cancer Biology and IRM– Small molecule libraries• Diversity library contributions: CDB, Biochemistry andMicrobiology• LOPAC from J. Hogenesch (Pharm)

What libraries or functionality would be
useful to you?

SHARED FACILITIESHOW TO GET STARTED?
•Contact David Schultz at dschultz@mail.med.upenn.edu or215-573-9641 for an initial consultation
 Define the project Determine if the facility has relevant
expertise/technology to pursue the project Develop a management plan Set expectations
 Get started!