Poster - BMG Labtech

A Screen For Inhibitors of PfPK7 – A Malarial Kinase with Intrinsic ATPase Activity
David Whalley1, Keith Ansell1, Catherine Kettleborough1, Debbie Taylor1, Livia Vivas2, Natalie Bouloc1, John Large1, Ela Smiljanic1, Chris Edlin1, Justin Bryans1
1. Drug Discovery Group, MRC Technology, 1-3 Burtonhole Lane, London NW7 1AD
2. Department of Infectious and Tropical Diseases, London School of Hygeine and Tropical Medicine, London WC1E 7HT
Acknowledgements: Jane Endicott and Martin Nobel, Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Park Road, Oxford, OX1 3QU
PfPK7 and Malaria
Each year, several hundred million clinical cases of malaria are
reported, and the mortality reaches 1-3 million, the vast majority
of victims being children in sub-Saharan Africa. Plasmodium
falciparum, the most virulent species of human malaria has
become resistant to most anti-malarial drugs such as Chloroquine,
highlighting an urgent need for new treatments.
Following the publication of the complete genomic sequence of
P. falciparum genome in 2002, many potential drug targets have
been identified including the Plasmodium protein kinases. Many
of these kinases have been classified into eukaryotic kinase (ePK)
families; however several, including PfPK7, have been described
as “orphan” kinases that do not readily cluster with any predefined group.
We set out to develop a high throughput in vitro screening assay
for identification of PfPK7 inhibitors. During assay development
we observed that ATP turnover was dominated by ATP hydrolysis.
We used this to our advantage and ran a high throughput screen
which identified 4 series of ATP-competitive inhibitors.
Target Validation
ATPase Activity
1. ATP depletion measured
using luciferase and
luminescent substrate
luciferin (Promega Kinase
Glo Plus®). Results
show near complete ATP
turnover(200pmol) by both
4pmol and 20pmol PfPK7 and
indicate that ATP turnover
can not be attributed to
PfPK7 autophosphorylation
activity alone
The “promiscuous” kinase substrate,
Myelin Basic Protein (MBP) was
indentified as a substrate for PfPK7
using a gel-based 32P incorporation
assay2. In the ATP depletion assay,
signal generation was only dependent
on [PfPK7]. Clearly the rate of ATP
hydrolysis was faster than the rate of
phospho-transfer. This was also the
case for other PfPK7 substrates
2
PfPK7 is a ‘composite’ kinase
N-terminal lobe
homologous to fungal PKA
C-terminal lobe
homologous to the
MAPKKs MKK3/6
Although PfPK7 has C-terminal homolgy with MAPKKs (MEKs), the
two known PfMAPKs, Pfmap1 and Pfmap2, are not phosphorylated
by PfPK7. Interestingly, PfPK7 does not possess any MEK-like
activation loop at the expected location, and the upstream
activator is unknown2.
Specific inhibition of PfPK7 may be an attainable goal
due to the unique structure
Kinase Focussed Library
2. ATPase activity was confirmed
using a rhodamine-labelled
Phosphate Binding Protein
(PBP) Pi Biosensor, developed at the
MRC National Institute for Medical
Research by M. Webb3.
The RHPBP is labelled with 2
rhodamine moeities which are
stacked in the normal conformation.
On binding Pi the moeities move
apart, giving an increase in
fluorescence at 575nm.
The observed increase in fluorescence
over time was dependent on the
presence of both ATP and kinase
PfPK7 knockout parasites displayed two particular phenotypes:
PfPK7 could therefore represent a suitable “dual” target for
anti-malarial chemotherapy as inhibitors could be used in a
schizonticidal (“curative”) or transmission-blocking manner
Imidazopyridazines
Optimisation
MRT00064480
ATPase Activity
Exceeds Phospho
Transfer
1. Reduced asexual growth (schizogony) rate (human)
2. Impaired oocyst formation (mosquito vector)2
Primary HTS
Assay Development
Assay Validation
An ATP depletion assay was developed (Promega Kinase Glo Plus®)
using full-length recombinant PfPK7 alone to exploit the ATPase
activity of the kinase. The assay was optimised for single point
screening in 384-well format, and automated using the Biomek FX
(Beckman Coulter) and the Flexdrop (Perkin Elmer)
Novel PfPK7 Inhibitor
A novel family of inhibitors of PfPK7
(imidazopyridines) had been identified
initially through stabilization of the
protein to thermal denaturation.
The 3D structures of several PfPK7imidazopyridazine complexes have
recently been disclosed4, and have been
shown to bind to the PfPK7 active site.
One of these imidazopyridines, “K510”
(MRT00064480) displayed strong activity
in the thermal shift assay (ΔTm 7.3ºK).
The activity of this compound was
comfirmed in the ATP depletion assay.
IC50 = 300nM
N o E n zym e C o n tro ls
(R L U )
7 1 0 5 2 ±1 2 7 5
In te rn a l C o n tro ls (R L U )
(6 4 4 8 0 @ IC 50 )
5 8 5 7 6 ±2 1 6 4
E n zym e C o n tro ls (R L U )
3 9 1 4 1 ±2 8 3 5
S ig n a l:B a c k g ro u n d
M ean Z’
Anti-Parasitic Assay
0 .8 0 ±0 .0 5
H it ra te (< 8 0 % c o n tro l
a c tivity )
R e c o n firm e d h its @ 1 0 u M
457 cm pds = 5%
4 1 7 c m p d s = 9 1 .2 %
re c o n ra te
As expected for a kinase focussed library, a high hit rate (5%)
was obtained. Four compound series of ATP-competitive inhibitors,
including further examples of imidazopyridazines were identified
giving IC50s in the 0.3μM-96μM range. A chemistry programme
was subsequently initiated, focussing on the imidazopyridazine
series and one other.
These results suggest that despite structural divergence from
mammalian kinases, PfPK7 is not atypical in respect of its
susceptibility to inhibition by known kinase templates, modelled on
the active site of mammalian kinases.
1. Jones, M. K.; Good, M. F. Nature Medicine 2006, 12, 170-171.
2. Dorin, D; Semblat, J. P.; Poullet, P.; Alano, P.; Goldring, J.
P.; Whittle, C.; Patterson, S.; Chakrabarti, D.; Doerig, C. Mol
Microbiol. 2005, 55, 184-196.
3. Okoh, M. P.; Hunter, J. L.; Corrie, J. E. T.; Webb, M. R.
Biochemistry 2006, 45, 14764-14771.
4. Merckx, A.; Echalier, A.; Langford, K.; Sicard, A.; Langsley, G.;
Joore, J.; Doerig, C.; Noble, M.; Endicott, J. Structure 2008, 16, 228.
IC50 = 130nM
Synthetic and SAR investigations were undertaken which improved
the in vitro potency of 64480 in the ATP depletion assay, and gave
analogues with a good range of physicochemical parameters. These
compounds also inhibited incorporation of 32P into both PfPK7 and an
exogenous substrate in a gel-based assay, showing that inhibition of
ATPase activity is an indictor of inhibition of kinase activity.
1 .8
References
K510 docked in PfPK7 active site
MRT00066022
Com pound
No.
3 D 7 IC 50
K 1 IC 50
K B IC 50
S Id
C h lo ro q uine
0 .0 4
0 .8 0
9 7.4 8
2 4 37
10
7 .1 5
1 2.7 3
2 1.2 0
2 .9 6
11
2 .6 0
6 .8 6
5 2.1 7
2 0.1 0
13
2 .4 6
3 .8 9
1 2.9 8
5 .2 7
29
5 .2 6
9 .9 3
2 1.2 1
4 .0 3
33
6 .0 6
1 0.4 7
2 5.7 8
4 .2 5
34
1 .0 3
2 .6 5
2 4.0 2
2 3.3 2
(μ M ) a
(μ M ) b
(μ M ) c
IC50 assessed via 3H hypoxanthine incorporation - drug sensitive strain
IC50 assessed via 3H hypoxanthine incorporation – K1 multidrug resistant strain
c
Cytotoxicity against human KB cells
d
Selectivity index IC50 = KB/3D7 IC50
a
b
Some PfPK7 inhibitors
displayed modest activity
against a drug-sensitive
and a drug-resistant strain
of P. falciparum in a 3H
hypoxanthine incorporation
assay. However, the
selectivity of these
compounds is questionable
and further work will
be required in order to
determine the mechanism
of action.
Conclusions
•We found the malarial kinase PfPK7 to have intrinsic ATPase
activity which could be screened using a simple, cost-effective ATP
depletion assay.
•A novel family of PfPK7 ATP-competitive inhibitors
(imidazopyridazines) were identified through stabilisation of PfPK7
to thermal denaturation. A high throughput screening campaign
identified further imidazopyridazine examples and three other novel
series.
•Synthetic chemistry efforts enabled the preparation of a
number of analogues with promising in vitro potencies and good
physicochemical parameters
•We demonstrated that inhibitors of ATP hydrolysis also block the
productive phospho-transfer event.