Drug discovery targeting Malaria

1. 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 Dundee, imperial DDC and GSK, Sanger Centre Medicinal Chemistry Imperial-DDC and CRO-India Lead Optimisation DDC and Sanger Centre

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

3. Assay optimisation • Endpoint Assay • Measure in the initial rate • Optimal ATP and Peptide • Run at Km to prevent desensitisation to certain mode of action inhibitors • Km is the concentration of substrate that leads to half-maximal velocity. Km c. 25uM for CDPK5 Reagent optimisation • Ca ions, Mg ions, DMSO, Detergent • Majority of enzymes are saturated with Ca. Unlikely to identify compounds that compete at the Ca site

4. Quality Control • Robust signal • Identify the dynamic range • Z’= 1-(3*STDEV HC+ 3*STDEV LC) • (Mean HC-Mean LC) • Standard compounds • Measured by pIC50 • pIC50=-Log.IC50 • Staurosporine • broad spectrum kinaseinhibitor

5. Screening Compound Set Single shot Target assay N=1, N=2 single concentration 100 uM-1uM Mean Inhibition + 3SD Arbitrary cut off To get a certain number of compounds Hit compound pIC50 confirmation 11 point curve, serial dilution in target assay Biochemical assay usually pIC50 greater than 5 (10uM) Cut off selection Chemical re-synthesis/ purchase Selectivity Cut off selection Cell assay/ Phenotypic assay Look for correlation between cell and target assay Cut off selection Lead Optimization Target Validation

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 1500 Biologically active compounds SS against pfCDPK1FL SS against pfCDPK5FL SS against pfCDPK4FL Hit identification 5.6% hit rate Purchase Compounds pIC50 confirmation

8. Compound Identification Resveratrol- and analogues pIC50 CDPK1 <4 CDPK4 <4 CDPK5 4.59 CDPK5KD 4.63 Natural product- Shown to kill Parasite Quercetin pIC50 CDPK1 6.17 CDPK4 6.06 CDPK5 5.78 CDPK5KD 6.31 Natural product PHA 665752 pIC50 CDPK1 <4 CDPK4 8.31 CDPK5 <4 CDPK5KD <4 Role in inhibiting transmission? PP1 pIC50 CDPK1 7.34 CDPK4 7.49 CDPK5 <4 CDPK5KD <4 (Ojo et al., 2010) ~200 Quercetin and Resveratrol analogue compounds kindly donated by PrabhatArya (Hyderabad University) to be screened

9. Anti-malarial set, GSK- 2010

10. Anti-malarial set, GSK- 2010 GSK compound Library Parasitic proliferation assay Performed at GSK 13,000 Hits Hit identification SS against pfCDPK5FL SS against pfCDPK4FL SS against pfCDPK1FL

11. Single Shot Screening Hit rate at 75 % inhibition CDPK1 1.27% CDPK4 0.53% CDPK5 FL 0.16% • 242 Compounds tested for IC50 based on <80% inhibition for CDPK1 or 4, < 50% inhibition for CDPK5FL

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

13. Results ATP competitive Not ATP competitive

14. Published Data (Gamo et al., 2010) • 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 Parasitic proliferation assay 13000 Hits Hit identification SS against pfCDPK1FL SS against pfCDPK4FL SS against pfCDPK5FL Cut off selection pIC50 confirmation Run in ATP and ATP desensitised mode Cut off selection Target Validation Lead Optimization

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 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 Imperial, DDC Albert Jaxa- Chamiec Caroline Low Cathy Tralau Stewart Hayley Cordingley Michelle Heathcote Ojay Oka Imperial College funding University Of Hyderabad PrabhatArya

22. HTRF peptide assay Eu XL665 Biotin ADP ATP Biotin P • 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 Robust signal , Reproducible Pharmacology

23. Diverse biological active compounds, 2010 1500 Biologically active compounds SS against pfCDPK5FL SS against pfCDPK1FL SS against pfCDPK4FL Hit identification Purchase Compounds Cut off selection pIC50 confirmation Cut off selection Selectivity Published data Cut off selection Parasitic proliferation assay In progress Target Validation