Drug discovery targeting Malaria Protein production and assay development Imperial academic and DDC Target Identification and Validation Imperial College/ Sanger Centre Lead Optimisation DDC and Sanger Centre Crystallogr aphy Oxford University Screening Campaign Dundee, imperial DDC and GSK, Sanger Centre Medicinal Chemistry Imperial-DDC and CRO-India DDC Expertise (Project management)
Drug discovery targeting Malaria. Target Identification and Validation Imperial College/ Sanger Centre. Crystallography Oxford University. Protein production and assay development Imperial academic and DDC. DDC Expertise (Project management). Screening Campaign - PowerPoint PPT Presentation
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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
CaCa
CaCa
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 signalo 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
CDPK1CDPK4CDPK5
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?
• 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)
Development of robust, reproducible biochemical assay to allow primary screening of large compound setsScreened 3 diverse compound setsIdentification of several chemical series with varied selectivity profilesIdentification of compounds for lead optimization and tools purposesIdentification of inhibitor mode of action- both ATP competitive and not competitive available.
Acknowledgements
Imperial, DDC
Albert Jaxa- ChamiecCaroline LowCathy Tralau StewartHayley CordingleyMichelle HeathcoteOjay OkaImperial College funding
University Of Hyderabad
Prabhat Arya
Oxford University
Ailsa PowellJane Endicott
Sanger Centre
Julian RaynerOliver Billker
Wellcome Trust
MMV
GSK Tres Cantos
Javier Gamo-Benito Jose Francisco Garcia-Bustos Jose Miguel Coteron-LopezMaria Jose Lafuente-MonasterioMalaria 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