1. 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 Crystallography Oxford University Screening Campaign Dundee, imperial DDC and GSK, Sanger Centre Medicinal Chemistry Imperial-DDC and CRO- India DDC Expertise (Project management) DDC Expertise (Project management)

2. Protein production pfCDPK1 full length pfCDPK4 full length pfCDPK5 full length HTRF assay technology (Cisbio) ATP Ca CDPK Robust signal, Reproducible Pharmacology

3. Assay optimisation Endpoint Assay o Measure in the initial rate Optimal ATP and Peptide o 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

4. 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

5. Screening Single shot Target assay Single shot Target 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 + 3SD Arbitrary cut off To 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

6. 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

7. 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

8. Compound Identification PHA 665752 pIC 50 CDPK1<4 CDPK48.31 CDPK5<4 CDPK5KD<4 Role in inhibiting transmission? Quercetin pIC 50 CDPK16.17 CDPK46.06 CDPK55.78 CDPK5KD6.31 Natural product Resveratrol- and analogues pIC 50 CDPK1<4 CDPK4<4 CDPK54.59 CDPK5KD4.63 Natural product- Shown to kill Parasite PP1 pIC 50 CDPK17.34 CDPK47.49 CDPK5<4 CDPK5KD<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

9. Anti-malarial set, GSK- 2010

10. GSK compound Library Parasitic proliferation assay SS against pfCDPK1FL SS against pfCDPK4FL SS against pfCDPK5FL Hit identification 13,000 Hits Performed at GSK

11. 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

12. Anti-malarial set, GSK- 2010 GSK compound Library pIC50 confirmation Parasitic proliferation assay Cut off selection SS against pfCDPK1FL SS against pfCDPK4FL SS against pfCDPK5FL Hit identification 13000 Hits Run in ATP and ATP desensitised mode Performed at GSK

13. Results Selection criteria from anti malaria setNumber Active compounds in at least one assay157 Active compounds in all 4 assays (>5)18 Non ATP competitive compounds1 ATP competitiveNot ATP competitive

14. Published Data (Gamo et al., 2010) Selection criteriaNumber pXC50_3D7 > 81 pXC50_3D7 > 710 pXC50_3D7 > 6132 Total 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)

15. Graph Frame Cluster pIC50 Data sorted on Graph frame cluster Assay

16. Graph Frame Cluster pIC50 Data sorted on Graph frame cluster Assay

17. Graph Frame Cluster pIC50 Data sorted on Graph frame cluster Assay

18. Graph Frame Cluster pIC50 Data sorted on Graph frame cluster Assay

19. 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 identification 13000 Hits Lead Optimization Run in ATP and ATP desensitised mode

20. 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.

21. 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

22. 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 Eu XL665 Biotin ATP ADP Robust signal, Reproducible Pharmacology

23. 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