Primary Cells in Screening

Drug Discovery, Manchester 2011

Angela DunneMolecular Pharmacology Group Leader

GlaxoSmithKline, R&D, Singapore

GSK R&D Centre in Singapore

GSK’s first pre-clinical R&D facility in Asia-Pacific established in 2005

Singapore Neural Pathways Discovery Performance Unit at Biopolis

– R&D China since 2008

– 50 scientific staff: Biologists, Chemists & DMPK

Overview: Primary Cells in Screening @ GSK

Placing Primary Cell Screening assays in a program critical path

Current assay technologies amenable to primary phenotypic screening

Examples of Drug Discovery Campaigns using primary cells at Hit ID

and Lead Optimisation

Future Directions

Recombinant Compound Screening

Reagent Generation

(Isolated Target Protein/Recombinant Cell Lines)

Target Go/No Go assay

Phenotypic Primary cells

Animal Model Studies

<5 compounds

Selectivity 1 Selectivity 2 Enabler 1 Enabler 2

<20 compounds

HTS

2M Compounds

SpecificityXC50

<500 compounds

Recombinant cells

The Attrition Problem in Drug Discovery

The attrition rate for drug discovery programs from target to clinic is unacceptably high

– Causes a lack of new medicines to treat diseases of high unmet need

– Results in inefficiency of drug discovery organizations, requiring large target portfolios to ensure sufficient medicine output

Organisations that improve attrition rates will grow and thrive

– If attrition is 95%, then a modest increase to 90% equals a doubling of productivity in terms of medicines to patients who need them

Significant contributors to attrition post-candidate are lack of efficacy and/or toxicity

– Deployment of more physiological screens earlier in the drug discovery process should make a significant impact in the overall attrition rate

Goal is to ensure all candidates are safe and demonstrably exhibit the desired effect at the target

– So that the main test is whether the target modulation is effective in the whole disease setting

Primary Cells in Screening

Reagent Generation

Primary Cells

HTS in Target Go/No Go Assay

2M Compounds

SpecificityXC50

<500 compounds

Phenotypic

Target Go/No Go assay

Animal Model Studies

<5 compoundsPhenotypic

MOA 1 MOA 2 Enabler 1 Enabler 2

<20 compoundsRecombinant

Focussed Screen

~ 100k CompoundsOR

What are the perceived challenges?

Screening with primary cells

– Cell supply is difficult

– Costly

– Low throughput

– Complex assay development

– Non specific actives

– Data highly variable

– Legal obligations (human)

……………limited to secondary assays in screening cascade

Cell supply?

What does the

data mean?

Assay opportunities in Primary Cells

Phenotypic & Mechanistic

– Sandwich ELISA based methods (MSD/AlphaLISA)

Secreted and intracellular signalling events

– High content/Imaging

Intracellular signalling events, mechanistic markers

– Flow cytometry

Non-secreted analyte detection in complex mixtures (e.g. Blood)

– Chemotaxis

Clinically meaningful endpoint for many inflammatory targets

– Label free technologies

Non-invasive techniques to measure target engagement and effect

Existing assay technologies applied to screening primary cells improving relevance

Screening with Primary Cells at GSKExample 1

– Inhibition of IFN cytokine release for inflammatory diseases

– Assay supported from frozen human PBMCs isolated from blood – 1.2 M compound HTS

Example 2:

– Rescreen due to lack of success with recombinant approach

– Secondary PBMC assay moved up cascade to rescreen phenotypically – enabled successful

Candidate Selection

Example 3:

– Primary Organelle screening using isolated mitochondria from rat liver

– Phenotypic end point assay assisting translation of binding to function and decision making in

absence of recombinant cellular approach

Example 4:

– Chemotaxis assay screening allows MOA elucidation and relevance from HTS outputs using

different signalling approaches

Example 5:

– Make the right decision – where recombinant and primary cellular assays work together

Example 1 : HTS for inhibitors of IFN Release from PBMCs

• IFN is a potential target for inflammatory diseases such as

Inflammatory bowel disease, Rheumatoid arthritis and asthma

• Secreted from T cell

subpopulation of PBMCs

• High expression levels of

IFN

• Number of potential kinase

targets identified for

regulation of IFN

HTS Supported with Frozen Human Primary Cells

Batch Testing v.s. Screening Data

0

100000

200000

300000

400000

500000

0 100000 200000 300000 400000 500000

Batch Testing Data - Average DMSO Counts

Scre

en

ing

Data

- A

vera

ge D

MS

O C

ou

nts

IFN assay

Capture Ab

IFN

Labelled

Detection Ab

Electrode

• Plate based ELISA assay

• 200 GSK blood donors

• 3 day assay protocol

• 150 x 384 well plates/day

• 4 FTE, 2 Readers

MesoScaleDiscovery Assay

IFN HTS DataBar Chart

Binned RESPONSE

1 1 1 1 1 5 7 11 20 26 40 66 93 175

293

434

765

1213

2054

3131

4051

4864

5132

4927

4066

3361

2505

1930

1455

1068

754 708

561 467 423

338 347 262 257 219 194 176 195 152 153 141 154 195 226

847

-125.6 -114 -10... -90.8 -79.2 -67.6 -56 -44.4 -32.8 -21.2 -9.6 2 13.6 25.2 36.8 48.4 60 71.6 83.2 94.80

1000

2000

3000

4000

5000Distribution of

Primary Screen Compounds

20k selected hits for Confirmation n=2

PRIMARY SCREENAverage Z Prime = 0.66 +/- 0.10

(passed plates)

Failure rate = 22.4%

40 475 actives identified

CONFIRMATION44% Confirmation Rate

r = 0.724

4

5

6

7

8

4 5 6 7 8 9

IFN Assay pIC50 Set 1

Se

lec

tivit

y A

ss

ay p

IC5

0 S

et

1

XC50IFN Assay + Selectivity

SELECTIVE LEADSwith cellular efficacy

Correlation Specificity Set C vs Set D

pEC50 Set A

4 4.5 5 5.5 6 6.5 7

4

4.5

5

5.5

6

6.5

Recombinant HTS Output

• Aim: Pathway selective receptor agonism driving release of immunodulatory

cytokine vs pro-inflammatory

• Cellular recombinant reporter HTS assay limited by reporter construct and over

expression of receptor

• HTS output: non specific actives

Example 2: Immunomodulation of Cytokine Release for Allergy

pIC50 Recombinant Receptor Assay

pIC

50 S

pecif

icit

y A

ssa

y

Receptor signalling

in transfectants Isoform Selectivity

Cytokines in PBMCs

in vitro animal model

PBMCs

in vivo model

Pre-candidate(s)

HTS Rescreen and Lead Optimisation Supported with Phenotypic Assay

Z' Trend

Result_Created_Time

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Testset Eval by PS

LNB_Ref

R9527/ 112/ 1 SS104368-036A1 U23104/ 37/ 1 U23119/ 19/ 1

5

5.5

6

6.5

7

Challenging assay – existence of non

responders in donor pool

Phenotypic primary cell assay allowed for HIT

ID and screening in larger biological space

Successful Candidate to FTIH

– Appropriate immunomodulatory cytokine profile

– Pathway activation selectivity

– Plasma Cytokine induction and selectivity

demonstrated in 14 day monkey study

Example 3 : Enzyme Inhibitor for Mitochondrial Stress Target

Purified Protein

Competition Assay

Whole Cell

MPTP/Cytoprotective

Assays

ISOLATED

MITOCHONDRIA

FROM RAT LIVER

Isolated Mitochondrial Assays

SWELLING CALCIUM RETENTION

CAPACITY

Rat liver

Gradient isolation

Approx 300mg mitochondria

12 x 96 well assays96 compounds conc response n=1

384 well development

Use of primary

organelles

providing decision

making data

Isolated Mitochondrial Assays

SWELLING CALCIUM RETENTION

CAPACITY

Rat liver

Gradient isolation

Approx 300mg mitochondria

12 x 96 well assays96 compounds conc response n=1

Detail Graph

Time (Seconds)

550 600 650 700 750 800

Rela

tive L

ight U

nits

1000

2000

3000

4000

PIC50 Mean FLUORESC FORMAT4 4.5 5 5.5 6 6.5 7

4

4.2

4.4

4.6

4.8

5

5.2

5.4

5.6

Recombinant Assays can be misleading

43% confirmed: selected 4k for dose response

500 actives: pIC50>4.5

Compound SPA Radioligand Binding

(pKi)

FP Cy3B

Binding (pIC50)

Isolated Mitochondria (pIC50)

Swelling Calcium Retention

TOOL 6.8 7.8 7.1 7.8

CPD 1 5.9 (n=3) 4.2 No effect No effect

CPD 2 Some activity at high conc <4 No effect No effect

CPD 3 Some activity at high conc 5.3 No effect Some activity at high conc

CPD4 Some activity at high conc 4.4 No effect No effect

CPD 5 Some activity at high conc <4 Some activity at high conc Some activity at high conc

CPD 6 Some activity at high conc 5.5 (I) No effect No effect

CPD 7 Some activity at high conc <4 No effect No effect

CPD 8 6.5 (n=3) 4.7 No effect No effect

CPD 9 Some activity at high conc 4.5 No effect No effect

~2M cmpds @ 10uM (FP-fluoroscein);

20k to confirmation

ß-Arrestin hits

GTP S hits

Chemokine Receptor Antagonist for

Asthma

Th2 cell expression

Eosinophil recruitment

Airway inflammation

Inhibit Th2 cell chemotaxis

Example 4 : Chemotaxis Assay - linking HTS Output to Efficacy

6

7

8

6 7 8

ChemoTXTM Transwell Chemotaxis Assay

Addition of chemoattractant to lower plate

Framed filter confines each cell-suspension sample to its site on top of the filter by the presence of a hydrophobic mask.

Scatter Plot

PIC50 n=1 33uM

4.6 4.8 5 5.2 5.4 5.6 5.8 6

4.6

4.8

5

5.2

5.4

5.6

5.8

6

Requirement for dual inhibition of GTP S and arrestin for blockade of chemotaxis

GTP S -arrestin

54 6348

No

reproducible

inhibition of

T cell

chemotaxis

No

reproducible

inhibition of

T cell

chemotaxis

Potency Correlation of Dual GTP S/ -

arrestin actives in CR1 chemotaxis assay

33% compounds

identified as dual GTP S

and -arrestin actives

displayed reproducible

activity in chemotaxis

assay

Approx 1 log

less potent in

chemotaxis

assay

compared to

GTP S/ -

arrestin

Screening for Donor Selective Response can determine MOA and compound progression

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

15 30 45 60

PIC

50

Time (mins)

Pharmacological standard

Donor 1

Donor 2

4

4.5

5

5.5

6

6.5

7

7.5

8

15 30 45 60

PIC

50

Time (mins)

Compound B

Donor 1

Donor 2

Understand relationship of phenotype to mechanism

Better equipped to determine translational plan

Build compound profile with data from multiple screens: toxicity

96 well

(n=6)

384 well

(n=3)

Standard 6.6 +/- 0.79 6.7 +/- 0.21

Compound B 6.7 +/- 0.44 6.9 +/- 0.16

2wks, 30K primary cortical neurons

-12 -10 -8 -6 -40

10

20

30

40

Rotenone,7.9

NMDA,6.0

Insults, pIC50

log[Insult, M]

Po

st-

syn

ap

se (

PS

D95)

co

un

ts

per

neu

ron

4div

7div

10div

14div

17div

22div

0

10

20

30

Neu

rite

syn

ap

se p

er

neu

ron

Example 5: Inhibition of Oxidative Stress for Neuroprotection

2wk, 30K cortical neurons

Nuclei (Hoechst)

Neurons/Neurites (MAP-2)

Synapses (PSD-95)

Some primary cells and phenotypic

end points are challenging

In vitro compound protection against

neuronal insult

Measure Reactive Oxygen Species (ROS) in HL-60 cells

Use the right/sensible choice of assay to

make compound progression decisions

WB Assay

pIC50

Predicted % Efficacy

in PD Assay

Cmax/IC50 (uM)

% efficacy PD

Assay

100mg/kg

Compound W 5.66 38 30

Compound X 4.71 17 30

Compound Y 6.17 96 100

Compound Z 6.17 97 100

Biochemical Assay

Cellular HL-60 Assay

Whole Blood Assay

In vitro Neuroprotection

Assay

In vivo Pharmacodynamic

Assay

CANDIDATE

HL60 PMA dose response - various cell density - using L-012

-12 -10 -8 -6 -40

100000

200000

300000100k/well

50k/well

25k/well

10k/well

Conc (M)

RL

U

HL-60 ROS Detection using L-012 HL60 PMA dose response - various cell density - using L-012

-12 -10 -8 -6 -40

100000

200000

300000100k/well

50k/well

25k/well

10k/well

Conc (M)

RL

U

Scatter Plot

WB pIC50

4 4.5 5 5.5 6

4

4.5

5

5.5

6

6.5

Primary Cells for ScreeningOverall Conclusions

Phenotypic assays, supported by human PBMCs, can be delivered on the HTS scale

The biological supply chain is critical

– Biological variability causes ~10-20% failure of the assay

The ‘best’ molecules may not have been found by the recombinant route

Thorough planning of the hit validation cascade is key to success

– It may not be necessary to confirm MOA

– Recombinant assays can be used in conjunction with primary cell assays

Future DirectionsAvailability of primary cells for screening is a challenge that can be offset by miniaturisation and improvements in assay technology sensitivity

Increased sensitivity in plate based ELISA

384 well Chemotaxis to allow for Hit ID

Movement of disease relevant tissue assays up program critical path

Advance in stem cell technology

Substitute human tissue for animal tissue

Multiple approaches lead to largest reduction in attrition

Disease tissue, 3D co-cultures, primary cells, stem cells

Acknowledgements

Steve ReesSteve LudbrookJenny StablesMike JowettGareth WayneCaroline BroughAndrew BrownAugustine MzumaraKirsten SearleAdeline CheungSri MulyanidewiTiger BeeMahmood AhmedRichard Rutter