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