Primary Neural Stem Cell-based High Content Phenotypic Screen for Multiple Sclerosis Mei Zhang MD, PhD Small Molecule Platform (SMP) EMD Serono Research & Development Institute Merck KGaA www. emdserono.com

Overview

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Disease biology_Multiple sclerosis Medical Rationale Phenotypic based HCS

Screening cascade & technical feasibility HT-HCS

Target deconvolution strategy Summary & Conclusion

Multiple sclerosis (MS) damages myelin sheath, reduces oligodendrocytes and eventually neurons

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Remyelination may be a good strategy for MS

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Relapsing-Remitting Secondary Progressive

Brain Volume

Clinical Disability

Inflammation

Axonal Loss

SPMS • 35% of total MS patients • RRMS becomes SPMS

•50% in 10 years •90% in 25 years

Lack of remyelinating drug in the clinic/market

- Despite significant progress in understanding OL differentiation, treatment option lacks for remyelination process.

- Few or no good targets are known.

Nat Rev Drug Discov October 2010

Why a phenotypic screen?

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● Myelination is mediated by oligodendrocytes with a spectacular

celluar phenotype which can be utilized for screen.

● Phenotypic screen aims to the identification of functional compounds.

● Phenotypes may be resulted from different genotypes thus provide

potential to identify novel therapeutic targets.

How were new medicines discovered?

David C. Swinney & Jason Anthony

Nature Reviews Drug Discovery 10, 507-519 (July 2011)

Some known signaling pathways on oligodendrocyte differentiation

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OL Differentiation

Notch1

PI3K

Akt

mTor Rapamycin

PPAR∂

PPAR∂ agonist

Adenosine

Purinergic receptors

Corticoid hormone receptor

Dexamethasone γ-secretase inhib.

Ras Raf

MEK

ERK

PKC

PKA

cAMP

Retinoic acid

5’-N-Ethlycarboxamido- adenosine (NECA)

cJun

Phenotypic platforms deeply rooted at EMD/Merck Serono

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More than 100 robust biologically relevant cellular assays have been developed to screen the secretome, small molecules, and discover biotherapeutics in the areas of:

o Neurology o Immunology o Cancer o Reproductive Health o Metabolic endocrinology o Detailed info can be found: Cellular imaging in drug discovery. Nature Reviews Drug Discovery. Paul Lang, et al. 5, 343-35, 2006

• Oli-neu , a murine oligodendrocytic cell line • Searching for biologics inducing OPC terminal differentiation • Positive control is d-butyric cAMP

• 2000 proteins against 90 biologically relevant cell based assays • Combination of biochemistry and cellular imaging

Mitogen Stimulation

A pioneering functional genomics approach searched for biologics from the secretome.

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Primary neural stem cells for phenotypic screening

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Neural Progenitor

Oligodendrocyte Progenitor

Mature Oligodendrocyte

EGF media bFGF, PDGF

Day 3 0 6 9

x4 T25 T75 x4 x4

T175

(P1) (P2)

~50

x4 T175

+

(P3)

EGF

EGF media EGF, bFGF, PDGF

The behaviors of neural spheres

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(Calcium 5)

Spontaneous differentiation EGF

PDGF bFGF

(anti-O4 ICC in Green)

Characterization of cultured neural sphere-derived oligodendrocyte precursors

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24 h

48 h

NG2 +++ PDGFRα +++

RIP ++ NG2 + PDGFRα +

72 h RIP +++ O4 ++ O1 ++ MBP ++ PLP ++

Lineage progression after plating as single cells

Time OPC/OL marker

96 h MBP +++ PLP +++ O4 +++ O1 +++ MOG +

Chemical dissociation

Embryonic neural progenitors In EGF as neurospheres:

EGF-Nph P2-P6

Single cells PDGF/bFGF >95% OPC

Methodology

Partial differentiation EGF

PDGF bFGF

(anti-O4)

Factors considered for the assay development for the Screen

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1. Cell numbers, plate format

2. ICC assay vs live cell assay 3. “Live stain and wash” vs “add and read” 4. Final condition summary:

• 384 format with Calcein AM plus Hoechst33342 staining

Probes Live Cell Need wash? Intensity Favorite Rank

Probe 1 √ Yes Good 6

Probe 2 √ Yes Good 5

Probe 3 √ May Good 4

Probe 4 √ May OK 3

FLIPR probe √ No OK 2

Calcein AM √ No Very Good 1

Cell plating, treatment, labeling and imaging

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Day 1 Plate progenitors

Day 2 Add

Compounds

Day 5 Imaging

24 hr Neural stem cells from neurospheres

Plating (384 PDL)

72hr Start cpd treatment

CaAM + Hoechst Imaging with 4x objective

Image analysis

Image analysis: MetaXpress, MDCStore, and database

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Offline MX Station(s)

Acquire

MDCStore

(Oracle)

IXMicro / MX

Data management

Remotely monitor progress from Laptop (office/home network)

Neurite Outgrowth (NO) Module Auto Run analysis

Analyze

Data from MDCStore (Oracle)

Phenotypic screen workflow process (Feasibility test)

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1246 compounds screened at single dose (10uM)

Measure morphology - Branch#, Process#, and Total Outgrowth Length per cell

Select compounds with Branch#/cell significant increased (80% increase over DMSO treatment)

10 points DRC

Decide whether to advance to HT-HCS with 96K cpds

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Untreated well (Calcien AM)

Positive response sample 1 (Calcien AM)

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Compound A

OPC HTS DRC

0.283686681955626Final C.(uM)

0.001 0.1 10

Bra

nch/

Cel

l%ab

oveD

MS

O4

-10

20

50

80

110

140

170

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Compound B

OPC HTS DRC

3.51702343860096Final C.(uM)

0.001 0.1 10

Bra

nch/

Cel

l%ab

oveD

MS

O4

0

40

80

120

160

200

Positive response sample 2 (Calcien AM)

Neurite Outgrowth Analysis

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Negative contrl (DMSO)

Positive control

Positive hit

Compound A

Concentration (nM)0.1 10 1000

Nor

mal

ized

Bra

nch

Mea

sure

men

t

90

110

130

150

170

190

Compound A

Concentration (nM)0.1 10 1000

Nor

mal

ized

Out

grow

thM

easu

rem

ent

95

105

115

125

135

145

155

165

Branch numbers

Total outgrowth

Positive Control

Feasibility Screen Summary Summary Items Results Note Compounds screened 1246 cpds Focus library Number of positive compounds from 1 dose screen

98 cpds (hit rate 7.8%)

80% branch# increase over DMSO mean

Number of postive compounds from 10 points DRC

25 cpds (confirmation rate 25.5%)

10 pts DRC

Scaffold analysis results 2-3 distinct chemical structures found

Additional 6 singletons identified

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… so we are moving up.

The high-throuput HCS Campaign

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Primary screen assay Neurosphere-derived oligodendrocyte differentiation assay

Analytics (QC)

Confirmed Hits

Qualified Hits

Primary Hits/Series

DRC confirmation assay Proliferation assay

Series Analysis and Evaluation

Validated Hits

Hit Characterization

In vitro ADME

Including brain penetration (membrane

permeability, brain fraction unbound)

Orthogonal cellular assay Myelin marker

expression

Compound selection rationale for the HT-HCS - Design of a representative subset of the Corporate Screening Set (CSS)

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Size:

96 000 compounds (cherry picked) ⇒ 300 plates

Compound criteria (MS_NO level):

• Compound purity > 90%

• rule-of-5 < 2 and no BFG

Selection criteria:

Maintain Murcko-Fragmentation profile (structural diversity)

Maintain profile of MW, logP/logD, tPSA (BBB penetration)

HT-HCS Hit Selection and QC Criteria

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Hit definition parameters:

Phenotype-inducing activity threshold: Branching ≥ Mean DMSO value + 3xSD, or Outgrowth ≥ Mean DMSO value + 3xSD

Plate QC: Signal Window ≥ 2

We obtained ‘normal’ HTS hit rate…

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A B

HTS (96,000 cpds)

1138

A: Total Outgrowth B: Branches 2sw: hits positive by a signal of window ≥ 2

26 136

A(2sw) -> B(2sw) = 1138 ~ 1.2% hit rate

Mouse Primary assay: Morphology

Rat Secondary assay: MBP production

Myelination Proliferation

Oligodendrocyte progenitors, OPC

Mature oligodendrocytes, OL

PDGFRa NG2 A2B5

O4 O1 GalC MBP

O1 O4 MBP PLP

MBP PLP Mog Mag CNPase

MBP: OL; Neurofilament: axons

Cel

l mar

kers

PDGFRα: OPC; β-tubulin: cytoskeleton

Pre-myelinating oligodendrocytes

MBP:OPC; β-tubulin: cytoskeleton

Differentiation

Confirmation screening assay: MBP ELISA with rat OPC.

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DMSO Compound M

Primary assay Secondary assay

EC50: 0.04 n=3 EC50: 1.6 n=4

EC50: 1.5 n=4

Cpd M

Cpd M

Effective in primary and secondary assays (EC50)

BATCH 1

BATCH 2

Compound M: first identified HTS series confirmed with MBP ELISA

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Cpd M

EC50: 0.03

A

C

EC50: 0.05

E

EC50: 0.09

B

D

F

EC50: 1.5

Parent

EC50: 0.09

EC50: 0.10

EC50: 0.88

Compounds with nanomolar range EC50

MBP ELISA in rOPC

Cpd M’s analogs in rab MBP ELISA assay

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Analogs are predicted BBB penetrants, molecular weight < 300

Summary for the primary and secondary screens of the HTS

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From the 1138 positive hits we confirmed the following: • 29 compounds are confirmed with DRC in phenotypic assay. • 7 compounds are positive in secondary assay: MBP ELISA with

rat OPC. • More analogs are being further analyzed (continuing)

Deconvolution strategy

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Broad assessment of selected series representative on panel of enzymes and receptors (Cerep)

Proteomic/biochemical approaches: • Affinity chromatography and mass-spec and peptide sequencing • DNA fishing technology

General strategy:

• Analyze cell phenotype

• Find signaling pathway

• Find target(s)

Resource Intensity

Confirmation of target using gene knockdown and/or reference compounds

1. From the HT-HCS, we have obtained a small number of hits that are able to induce myelination in primary neuronal cells from 2 different species.

2. Under extensive evaluation, our screen has been able to progress to the next stage of discovery.

3. In conclusion, HT-HCS can be carried out on primary neural stem cells in search of small molecules that are able to induce software recognizable morphology changes in HT format in a reasonable time frame. More robust assays with specific markers for later differentiation stages would produce more biologically meaningful hits.

Conclusions

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Acknowledgement

Carlos Pedraza Christopher Taylor

Michelle Seng Albertina Pereira

Mike Webb Jean Merrill

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Tom Noonan Mohanraj Dhanabal

Brian Healey Susan Zhang

Paul Lang

Stephane Genoud Chantal Alliod Aurelie Baguet Fanny Schmidt

Rosanna Pescini Gobert Marie-Jose Frossard

Sandrine Pouly Rob Hooft

Lesley Liu-Bujalski Theresa Johnson

Michael Krug Henry Yu

Andreas Goutopoulos

Multiple Sclerosis Molecular Pharmacology

Chemistry