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Drug discovery targeting Malaria
Protein production and assay
developmentImperial academic and
DDC
Target Identification and ValidationImperial College/
Sanger Centre
Lead Optimisation
DDC and Sanger Centre
Crystallography
Oxford University
Screening CampaignDundee, imperial DDC
and GSK, Sanger Centre
Medicinal ChemistryImperial-DDC and CRO-
India
DDC Expertise(Project
management)
Protein production
• pfCDPK1 full length
• pfCDPK4 full length
• pfCDPK5 full length
• HTRF assay technology (Cisbio)
ATP
Ca
Ca
Ca
Ca
CDPK
Robust signal , Reproducible Pharmacology
Assay optimisation
Endpoint Assay o Measure in the initial rate
Optimal ATP and Peptideo Run at Km to prevent desensitisation to certain
mode of action inhibitors• Km is the concentration of substrate that
leads to half-maximal velocity.
Reagent optimisation o Ca ions, Mg ions, DMSO, Detergent
• Majority of enzymes are saturated with Ca. Unlikely to identify compounds that compete at the Ca site
Km c. 25uM for CDPK5
Quality Control
Robust signal
o Identify the dynamic range
Z’= 1-(3*STDEV HC+ 3*STDEV LC) (Mean HC-Mean LC)
Standard compounds
o Measured by pIC50• pIC50=-Log.IC50
o Staurosporine• broad spectrum kinase inhibitor
0 5 10 15 20 25 30 35 40 45 500.000
0.200
0.400
0.600
0.800
1.000
CDPK1
CDPK4
CDPK5
Plate number
Z'
Screening
Single shotTarget assay
pIC50 confirmation
Hit compound
Chemical re-synthesis/ purchase
Selectivity
Cell assay/ Phenotypic assay
Cut off selection
Compound Set
Target Validation
Cut off selection
Lead Optimization
N=1, N=2 single concentration 100 uM-1uM
Mean Inhibition + 3SDArbitrary cut offTo get a certain number of compounds
11 point curve, serial dilution in target assay
Biochemical assay usually pIC50 greater than 5 (10uM)
Look for correlation between cell and target assay
Cut off selection
Inhibitor identification for CDPK’s
Screening using 3 diverse sets
• 7000 compound Kinase set at Dundee
• 1500 compound DDC biologically active compounds
• 13000 compound Anti-malarial set GSK
Diverse biological active compounds, 2010
SS against pfCDPK1FL
pIC50 confirmation
Hit identification
Purchase Compounds
1500 Biologically active compounds
SS against pfCDPK4FL
SS against pfCDPK5FL
5.6% hit rate
Compound Identification
PHA 665752pIC50
CDPK1 <4CDPK4 8.31CDPK5 <4CDPK5KD <4Role in inhibiting transmission?
QuercetinpIC50
CDPK1 6.17CDPK4 6.06CDPK5 5.78CDPK5KD 6.31Natural product
Resveratrol- and analogues
pIC50CDPK1 <4CDPK4 <4CDPK5 4.59CDPK5KD 4.63Natural product- Shown to kill Parasite
PP1pIC50
CDPK1 7.34CDPK4 7.49CDPK5 <4CDPK5KD <4(Ojo et al., 2010)
PP1
PP2
1 NM PP1
3 BR PP1
3 MB PP1
1 NA PP1
3 1B PP1
~200 Quercetin and Resveratrol analogue compounds kindly donated by Prabhat Arya (Hyderabad University) to be screened
Anti-malarial set, GSK- 2010
Anti-malarial set, GSK- 2010
GSK compound Library
Parasitic proliferation assay
SS against pfCDPK1FL
SS against pfCDPK4FL
SS against pfCDPK5FL
Hit identification13,000 Hits
Performed at GSK
Single Shot Screening
• 242 Compounds tested for IC50 based on <80% inhibition for CDPK1 or 4, < 50% inhibition for CDPK5FL
CDPK1
1.27%
CDPK4
0.53%
CDPK5 FL 0.16%
Hit rate at 75 % inhibition
Anti-malarial set, GSK- 2010
GSK compound Library
pIC50 confirmation
Parasitic proliferation assay
Cut off selection
Cut off selection
SS against pfCDPK1FL
SS against pfCDPK4FL
SS against pfCDPK5FL
Hit identification13000 Hits
Run in ATP and ATP desensitised mode
Performed at GSK
Results
Selection criteria from anti malaria set NumberActive compounds in at least one assay 157Active compounds in all 4 assays (>5) 18Non ATP competitive compounds 1
ATP competitive Not ATP competitive
Published Data (Gamo et al., 2010)
Selection criteria NumberpXC50_3D7 > 8 1pXC50_3D7 > 7 10pXC50_3D7 > 6 132Total number of different sets as defined by Graph frame cluster characterisation 53
• pXC50_3D7- Parasite Growth assessment
• Graph Frame Cluster_ Broad frame work clustering of compounds
• All data was based on CDPK actives only (157 compounds)
Graph Frame Cluster
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11
11
11
1111111133365151515353
5369696969
696969868690
9199
9999
9999
9999
99100
104104
104104
104104104104104104104104104104104106106106106106111112112112112115122125128128128132138151151151170176
182188
188188
190206
208217
227228
228228
229229230232232233233
233233233233233
235236237242242242243246246246248255255256
256270
281281
281281
281281281281281294305305316323364367367367367367
0
2
4
6
8
parasite proliferation
pIC50Data sorted on Graph frame cluster
Assay
Graph Frame Cluster
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11
11
11
1111111133365151515353
5369696969
696969868690
9199
9999
9999
9999
99100
104104
104104
104104104104104104104104104104104106106106106106111112112112112115122125128128128132138151151151170176
182188
188188
190206
208217
227228
228228
229229230232232233233
233233233233233
235236237242242242243246246246248255255256
256270
281281
281281
281281281281281294305305316323364367367367367367
0
2
4
6
8
CDPK5FL
parasite proliferation
pIC50Data sorted on Graph frame cluster
Assay
Graph Frame Cluster
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11
11
11
1111111133365151515353
5369696969
696969868690
9199
9999
9999
9999
99100
104104
104104
104104104104104104104104104104104106106106106106111112112112112115122125128128128132138151151151170176
182188
188188
190206
208217
227228
228228
229229230232232233233
233233233233233
235236237242242242243246246246248255255256
256270
281281
281281
281281281281281294305305316323364367367367367367
0
2
4
6
8
CDPK4FLCDPK5FLparasite proliferation
pIC50Data sorted on Graph frame cluster
Assay
Graph Frame Cluster
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11
11
11
1111111133365151515353
5369696969
696969868690
9199
9999
9999
9999
99100
104104
104104
104104104104104104104104104104104106106106106106111112112112112115122125128128128132138151151151170176
182188
188188
190206
208217
227228
228228
229229230232232233233
233233233233233
235236237242242242243246246246248255255256
256270
281281
281281
281281281281281294305305316323364367367367367367
0
2
4
6
8
CDPK1FLCDPK4FLCDPK5FLparasite proliferation
pIC50Data sorted on Graph frame cluster
Assay
Anti-malarial set, GSK- 2010
GSK compound Library
pIC50 confirmation
Parasitic proliferation assay
Cut off selection
Target Validation
Cut off selection
SS against pfCDPK1FL
SS against pfCDPK4FL
SS against pfCDPK5FL
Hit identification13000 Hits
Lead Optimization
Run in ATP and ATP desensitised mode
Summary
Development of robust, reproducible biochemical assay to allow primary screening of large compound sets
Screened 3 diverse compound sets
Identification of several chemical series with varied selectivity profiles
Identification of compounds for lead optimization and tools purposes
Identification of inhibitor mode of action- both ATP competitive and not competitive available.
Acknowledgements
Imperial, DDC
Albert Jaxa- Chamiec
Caroline Low
Cathy Tralau Stewart
Hayley Cordingley
Michelle Heathcote
Ojay Oka
Imperial College funding
University Of Hyderabad
Prabhat Arya
Oxford University
Ailsa Powell
Jane Endicott
Sanger Centre
Julian Rayner
Oliver Billker
Wellcome Trust
MMV
GSK Tres Cantos
Javier Gamo-Benito
Jose Francisco Garcia-Bustos
Jose Miguel Coteron-Lopez
Maria Jose Lafuente-Monasterio
Malaria DPU
HTRF peptide assay
• Homogeneous Time resolved FRET
• Low false positive rate
• Time delay between excitation and emission
• Red spectrum reduces
• Compatible with detergent
• High-throughput format
• 384 well plate 10 ul assay volume
P
EuXL665Biotin
BiotinATP ADP
Ca
Ca
Ca
Ca
CDPK
Robust signal , Reproducible Pharmacology
Diverse biological active compounds, 2010
SS against pfCDPK1FL
pIC50 confirmation
Hit identification
Purchase Compounds
Parasitic proliferation assay
Cut off selection
1500 Biologically active compounds
Target Validation
Cut off selection
SS against pfCDPK4FL
SS against pfCDPK5FL
Selectivity Published data
Cut off selection
In progress