Tecan 2008Biologics: From Benchtop to ProductionEnabling Technologies for a new era

Yale Laboratory for High Throughput Cell Biology

Yale Laboratory forHigh Throughput Cell Biology

Established July 2008, 5,500 nsf

Goals:-Screen the human genome and identify new components of signalingpathways-Characterize genes from a biological standpoint (Biological Profiling)-Establish functional relationship of genes and phenotype

Resources:-HTS suit for both enzymatic assays and cell based assays-Large repertoire of cell-based assays-Core competences in cell-biology, virology, image analysis, gene delivery techniques, biochemistry, chemistry and automation

Screening from Columbia to Yale;How to set up two HTS labs in 2 years

Or “How to get an ulcer and 30 extra pounds in 2 years…”

Estimated max HTS throughput: 1,000,000 wells/weekEstimated max High resolution imaging: 500,000 images/week

”Three Dimensional” Biological Profiling

Compound/gene/siRNA

Biological Function/Pathway

Cell type

Yale Automation• Estimated max capacity in excess of

1,000,000 wells per week• BSL-2+ facility• BSL-2 enclosures• 4 autonomous units• 3 robotic arms per unit• 96-384-1536 well capability• 20nl-2.5ml volume range• -80 ˚C carrier on platform• Infinity plate readers on platform• Automatic protocol optimization• -25 ˚C up to +200 ˚C heated stages• Opera and Ultraview Imaging platforms

High Content Screening at Yale

EPAS1RPS6KA2RPS6KA1SPHK1 HSPA1ATRAF2APPARHA-1ARHA-2CSRP2 PPP3R1RGS4TRADDRIPK3 NR3C1AP1RE CRE E2FRE GRE HSERE ISRE MYCRENFATRE NFKBRE pRARENFATC1SNX2PKIA

SRE EREp53RE TREAP1(PMA)RE RbREMAPK9RIPK2 PPARGNCK1PRKCB1 HSPCA GSK3BESR2HPCA RGS8RGS2PTK2 FGF2 SPHK2 STAT6 CEF1REETSREGASRELEF1REPSCD2GYS1DGKA

PPARaREAP2REARRE C/EBPRE CBFRECdxA/NKX2.1-2.5RECRE(2) E2F(1)RE E2F(2)RE FAST-1RE FKHRREGATA 1RE GATA 2RE HNF-1AREHNF-3REIRF1REKTP1REMEF1REMEF2REMRRE Oct-1(1)REOct-1(2)REPPARgREPRRERXRREARPTENCDKN1B

Sp1RESRFRESTAT 1RESTAT3(1)RETCF/LEFREVDRREYY1REPLD2CDC25BVSNL1CSNK2A2HYAL2E2F4SUMO HSF2CAMK2A PARGMAPK14MAPK14TNF LBRLMN AACTBTubulinSNX1NR3C2ARNT TULP1

TULP2TULP3HSF1JAK3MAPKAPK2 NR3C1MADH9 AKT1 MADH2 STAT3 HGS (FYVE domain x2)PLCD1 (PH domain only)FOXO1A SLC2A4 BTK CREB1 LNPEP PRKCZ RPS6KB2 TP53C-fosp38ERK1/2MapkapK2 (1)MapkapK2 (2)ATF2HSP27

ASSAYS IN SELECTION

JNK (1)JNK (2)junPKCCREBp90RSKMnk1C-mycAP-1 REPMA RECREE2F RESRESTAT1 (1)STAT1 (2)STAT 2 STAT 6STAT 5STAT1 (1)STAT1 (2)STAT 2 STAT 6STAT 5

STAT 2 STAT 6STAT 5Caspase 3Caspase 6Caspase 7Caspase 8Cell Divisionp53CHK2HistoneH3p38ATF6 REARE REFakActin (1)Actin (2)ActinPaxillin

Informatics Hardware & SoftwareData processing•IBM x3850 db server•IBM x3650 app server•Keck Facility Pipeline Pilot server

Storage•IBM N3300 SAN server•Currently ~10TB disk space scalable to 80TB on present controllersIBM x336 for primary backup mgmt – tape

Backup•IBM x336 for primary backup mgmt•Tape backup: IBM TS3200

Archive•Plasmon optical archival system 

•Retire image data after six months on near line storage•Blu‐Ray discs – 30 GB/disc.  •Slow R/W•Permanent (1000 yr shelf life)•Scalable ‐ upgradable UDO drives

Infrastructure •1 Gb/s fiber West Campus network•10 Gb/s dark fiber – data center to YSM campus

Users•Apple Mac Pro user machinesVMWare Fusion for PC needs

Future Projects:•Mirror online disk array at remote data center

•Google collaboration: move first tier data to RAM

Software‐ActivityBase‐ActivityBase XE‐Pipeline Pilot

‐Acapella

‐Genego MetaCore‐Ingenuity Pathways Analysis

‐BakBone (backup mgmt)

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NFkB translocation assayNon-stimulated cells no translocation (negative control)

Inhibitior

TNF-stimulated cells (positive control)

H NS

OO

N

O

N O 2

OSO

H NN

SID 857745

NHN

S

O

OO

NH N

S

S

C l

OO H N

SO

O

N

NO 2

H NS

O

O

N

O 2 N

NHN

SOO

C l

H NS

OO

N

O

SulfonamidesHighest Observed potency: 0.3 μMActivity mode: Inhibitor

Inhibitors of NFkB Pathway

Inhibitors of NFkB PathwaySID 857745

Alignment of 6 active Sulfonamide Analogsbased on SID 857745

SNH

N

OO

N

N

NN

S

CH3O

OOMe

CU-00000000160MW 366.4AC50 = 230nM

Activator of NFkB Translocation

Potentiates TNFα stimulation (~2-fold @10μM)Non-toxic at highest tested concentration (10μM @ 4hr)

N

N

NN

S

S

O

O

CH3

OMeN

N

S

N

O

OMe

N

N

N

S

N

O

OMe

N

N

N

S

O

O

CH3

CH3

N

N

S

O

N

Cl

CH3

Inactive analogs

100

120

140

160

180

200

220

240

1.00E-08 1.00E-07 1.00E-06 1.00E-05

Concentration

% A

ctiv

atio

n

100.1 10.01

Huntington’s Disease Assay

• Rationale: Huntington’s disease is a devastating neurological disorder linked to an expansion of a polyglutamine tract in the huntingtin protein (htt).

• Clearance of mutant htt aggregation is linked to symptomatic reversal of the disease.

• Assay: Use a stable cell line that conditionally expresses the N-terminus of htt (first 17 amino acids) with 103 glutamines fused to monomeric CFP. The presence of aggregates can be visualized and quantitated using high-content image analysis.

• Screen for small molecules that activate or inhibit clearance of htt aggregates.

S

S

O

ONO

Huntington’s disease: potential probes

Estimated IC50: 2 μM

Estimated IC50: 1 μM

N

N

Cl

HN

Cl

N

N

Cl

NH

HO

N

N

Cl

N

N

O

O

Estimated IC50: 0.3 μMEstimated IC50: 0.1μM

Estimated IC50: 1.25 μM

Estimated IC50: 1 μM

Estimated IC50: 4 μM

SID 4264628Estimated IC50: 0.75 μM

Estimated IC50: 0.7 μM

Estimated IC50: 0.5 μM

1. 2. 3.

4. 5. 6.

7. 8. 9.

Mw range:228-427Activity mode: promotion

of inclusion clearance

LYP• Rationale: LYP is a lymphoid-specific phosphatase. In T cells,

LYP deactivates the T cell receptor.• A mutant allele is associated with high risk of autoimmune

disease in humans.

LYP

TCR

Inhibitors of LYPPrimary Screen: 103 hits

Cluster Analysis: Groups Compoundsby structure andactivity

Soluble Epoxide Hydrolase

Inhibitors of soluble Epoxide Hydrolase

Primary screen: 310 hits

• Rationale: TNFα activation of NFκB induces VCAM-1 expression at 24 h.

• VCAM-1 promotes adherence and migration of monocytes during inflammation and atheriosclerosis.

• Adherence and migration of monocytes requires that VCAM-1 be attached to F-actin stress fibers.

• Loss of either

Multiplexing VCAM-1 Assay

MonocyteVCAM-1 or actinfibers preventsmonocyte adherence.

VCAM-1

Multiplex VCAM-1 and Cytoskeleton

Anti-VCAM-1and Phalloidin Anti-VCAM-1 Phalloidin (actin fibers)

Assay: Cells: HUVECAntibody detection of VCAM-1Phalloidin binds actin fibers

Compounds synthesized

1,026 compounds synthesized for 4 probes

NFkB: 2HD: 1LYP: 1

EPAS1RPS6KA2RPS6KA1SPHK1 HSPA1ATRAF2APPARHA-1ARHA-2CSRP2 PPP3R1RGS4TRADDRIPK3 NR3C1AP1RE CRE E2FRE GRE HSERE ISRE MYCRENFATRE NFKBRE pRARENFATC1SNX2PKIA

SRE EREp53RE TREAP1(PMA)RE RbREMAPK9RIPK2 PPARGNCK1PRKCB1 HSPCA GSK3BESR2HPCA RGS8RGS2PTK2 FGF2 SPHK2 STAT6 CEF1REETSREGASRELEF1REPSCD2GYS1DGKA

PPARaREAP2REARRE C/EBPRE CBFRECdxA/NKX2.1-2.5RECRE(2) E2F(1)RE E2F(2)RE FAST-1RE FKHRREGATA 1RE GATA 2RE HNF-1AREHNF-3REIRF1REKTP1REMEF1REMEF2REMRRE Oct-1(1)REOct-1(2)REPPARgREPRRERXRREARPTENCDKN1B

Sp1RESRFRESTAT 1RESTAT3(1)RETCF/LEFREVDRREYY1REPLD2CDC25BVSNL1CSNK2A2HYAL2E2F4SUMO HSF2CAMK2A PARGMAPK14MAPK14TNF LBRLMN AACTBTubulinSNX1NR3C2ARNT TULP1

TULP2TULP3HSF1JAK3MAPKAPK2 NR3C1MADH9 AKT1 MADH2 STAT3 HGS (FYVE domain x2)PLCD1 (PH domain only)FOXO1A SLC2A4 BTK CREB1 LNPEP PRKCZ RPS6KB2 TP53C-fosp38ERK1/2MapkapK2 (1)MapkapK2 (2)ATF2HSP27

ASSAYS IN SELECTION

JNK (1)JNK (2)junPKCCREBp90RSKMnk1C-mycAP-1 REPMA RECREE2F RESRESTAT1 (1)STAT1 (2)STAT 2 STAT 6STAT 5STAT1 (1)STAT1 (2)STAT 2 STAT 6STAT 5

STAT 2 STAT 6STAT 5Caspase 3Caspase 6Caspase 7Caspase 8Cell Divisionp53CHK2HistoneH3p38ATF6 REARE REFakActin (1)Actin (2)ActinPaxillin

Signaling PathwayComponents

A B C D EDomains

Amino acid sequence

Select Medline Keywords

Splice variants

Diseases

Biological function

Domains

Amino acid sequence

Select Medline Keywords

Splice variants

Diseases

Biological function

Domains

Amino acid sequence

Select Medline Keywords

Splice variants

Diseases

Biological function

Domains

Amino acid sequence

Select Medline Keywords

Splice variants

Diseases

Biological function

Domains

Amino acid sequence

Select Medline Keywords

Splice variants

Diseases

Biological function

Biological Profiling hits, assays 1,4,10 and 19

Domains Significance ScorePH 0.10Phosphatase 0.35Kinase 0.22Transmembrane 0.78

AA sequence Significance ScoreA.1 0.20A.5 0.11B.3 0.43E.2 0.21

Medline keywords Significance ScoreApoptosis 0.84Mitochondria 0.65Potential 0.54Ion channel 0.43

Splice variants Significance Score1 0.212 0.113 0.454 0.765 0.20

Diseases Significance ScoreCrohn’s disease 0.20Rematoid Arthritis 0.34GVH 0.76

Biological function Significance ScoreApoptosis 0.87Spindel formation 0.56

Biological Profiling hits & report

Create significance lists

Accomplishments to date• Built 2 HTS Centers from scratch• Core strength in high throughput/high content cell-based

screening, while also demonstrating the ability to handle diverse assay formats

• 200,000 wells per week screened during last month of MLSCN project including complex cellular assays

• Developed and implemented over 180 cellular assays that provide a strong platform for secondary screening to test for bioactivity, selectivity, novel activities, and mechanism of action of hit/lead compounds identified in primary screens

• Initiated design of BioInformatics tool enabling automatic processing of data from profiling

Yale Screening, 2008

Yale’s expertise is in high-content, high-throughput screening.

Although phenotypic screens can be more difficult and time-consuming than target-based screens, the advantages are:

• Compounds in cell-based screens must function effectively in a cellular system andare therefore bioavailable.

• Since the target for high content assays is unknown, hits can be pursued that may otherwise have been missed in a target-based screen. This may yield useful results.

Furthermore, Yale’s extensive array of secondary assays allows us to study the biological mechanism of action.

Yale further benefits from being able to multiplex read-outs for each assay used as a primary screen.

Acknowledgments• Center Director

– Lars Branden

• Assay Development– Deborah Smith– Michael Wyler– Yan Song– Anthony Raffo– Leena Vishnar

• Cell culture– Ashima Bhan

• Informatics– Adrian Poffenberger– Marie‐Aude Guié– Phillip Williams

• Automation– Lori Ortoleva‐Donnelly – Jason Ignatius

• Lab Operations– Steven Berman

• Admin Support– Trisha D’Ericco