Calcium dependent protein kinases and Malaria:
Identification of chemical start point for drug discovery
Oliver Billker & Katie Chapman
Symposium: Academic Drug Discovery:
Challenges and perspectives
Imperial College London, March 2011
Antimalarial drugs
Chloroquine
Artemisinin
•
No longer useful against
P. falciparum
•
First-line treatment of P. falciparum
•
Component of a combination.
Wide spread resistance
•
Reduced efficacy observed in SE
Asia.
•
Important to develop a well supplied pipeline of new antimalarials.
Antimalarial drug development
•
Antimalarials are not economically attractive to the pharmaceutical industry.
•
Public-private partnerships play a key role.
E.g. Medicines for Malaria Venture, Wellcome Trust, Bill & Melinda Gates Foundation
Opportunity and need for academic institutions to make an impact.
A paradigm shift in antimalarial screening
Prioritise by chemistry,
IP, etc.
Target
based
screening
Cell based
screens
Libraries of
>20.000
starting
points for
drug
development
Identify mechanism of
induced resistance.
Screen against validated
targets
Genetic tools for target
identification at scale
(Sanger team)
https://www.ebi.ac.uk/chembl/
Nature, March 2010
13533 inhibitors of
P. falciparum growth
IC50 < 1 µM
2 million GSK compounds
242 inhibitors P. falciparum
CDPK 1, 4 or 5
Systematic prioritisation of parasite kinase
targets in a malaria parasite of rodents
•
Gene deletion analysis of 65 protein kinases.
•
Kinomes are highly conserved across Plasmodium species.
•
>1/3 of parasite protein kinases are redundant in blood stages.
Comparative analysis of Plasmodium kinomes
Redundant in P. berghei
Different in P. falciparum
Rita Tewari
Calcium dependent protein kinases (CDPKs)
•
Plant-like kinases with a unique activation mechanism.
calmodulin-like
domain
kinase
domain
Billker & Doerig 2010
Target selection
Plant like  Opportunity for selectivity
Family  Opportunity for multi-target inhibitor.
CamK
CDPK1 and 5:
Essential in blood stages.
CDPK1 and 4
Essential for transmission.
Redundant in P. berghei
Predicted CDPK functions
CDPK4
CDPK1
CDPK1
CDPK5
CDPK1
CDPK4
CDPK1
CDPK4 is required for male gamete formation
OH
N
X a n th u re n ic a c id
Te m p e ra tu re
C O OH
OH
R e c e p to r
P LC
Guanlyl
cyclase
Phosphodiesterases
cGMP
PKG
P IP 2
PKG
IP 3
ER
C a2+
SRPK
Male gamete
formation
Map-2
C a2+
CDPK4
C a2+
Differential gene expression
Host cell lysis
Katie Chapman, Imperial
College Drug Discovery
M e xile tin e H C l
PH A 665752
3 -1 B P P 1
3 -M B P P 1
1 -N A P P 1
C1 selective
3 -B R P P 1
1 -N M P P 1
..
S ta u ro s p o rin e
R O 318820
.
unselective
P u rfa lc a m in e
Q u e rc e tin
PP1
PP2
R e s v e ra to l
P ic e a ta n n o l
1 /p IC 5 0
Comparative profiling of recombinant PfCDPK1 and 4
C4 selective
10
9
8
CDPK1
CDPK4
7
6
5
4
3
2
CDPK4 is required for male gamete formation
cdpk4
A ve ra g e n u m b e r o f o o cysts
The CDPK4 inhibitor 1NM-PP1 blocks parasite
transmission to mosquitoes
50
40
30
20
10
0
D M S O 1NM-PP1
R m -1 -1 3 2 1 N M -P P 1
DMSO
DMSO
1NM-PP1
Lotta Burström
P. berghei CDPK1-GFP is expressed
throughout life cycle
schizont
ookinete
gametocytes
Sarah Sebastian
oocyst
sporozoites
cdpk1 knock down blocks development…
zygote
retort
WT ookinete
Sarah Sebastian
ookinete
…and prevents transmission of P. berghei to
mosquitoes
average oocysts/midgut
feed
n
WT
CDPK1
fold change
1
20
120
0.05
2,400
2
35
275
0.09
3,000
Sarah Sebastian
Target prioritisation: CDPK5 is also
essential for blood stage development.
Science 238 (2010)
Target summary
• CDPK1, 4 & 5 are individually essential for parasite
development at different life cycle stages.
• Absence of redundancy due to different subcellular
localisations (lipid modification), expression patterns,
substrate preferences.
• Pan-CDPK inhibitor could exploit synergies and delay
emergence of resistance.
Aim: Screen P. falciparum versions of all three targets
to explore feasibility of pan-CDPK inhibition