Primary Cells in Screening - ELRIG · Primary Cells in Screening Drug Discovery, Manchester 2011...
Transcript of Primary Cells in Screening - ELRIG · Primary Cells in Screening Drug Discovery, Manchester 2011...
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