Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

Article abstract of DOI:10.1016/j.bmcl.2013.03.017

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.

Full text of DOI:10.1016/j.bmcl.2013.03.017

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3064–3069
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted imidazopyridazines are potent and selective
inhibitors of Plasmodium falciparum calcium-dependent
protein kinase 1 (PfCDPK1)
a
a
a
a
Timothy M. Chapman a, , Simon A. Osborne , Nathalie Bouloc , Jonathan M. Large , Claire Wallace ,
Kristian Birchall a, Keith H. Ansell a, Hayley M. Jones a, Debra Taylor a, Barbara Clough b, Judith L. Green b,
Anthony A. Holder b
⇑
a Centre for Therapeutics Discovery, MRC Technology, Mill Hill, London NW7 1AD, UK
b Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent
protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme.
Subsequent exploration of the SAR and optimisation has yielded leading members which show promising
in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial
in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a
Plasmodium berghei mouse model of malaria.
Received 12 December 2012
Revised 28 February 2013
Accepted 4 March 2013
Available online 21 March 2013
Keywords:
Plasmodium falciparum
Ó 2013 Elsevier Ltd. All rights reserved.
Calcium-dependent protein kinase 1
Malaria
Imidazopyridazine
SAR
Malaria is one of the most prevalent infectious diseases of the
developing world. In excess of 3 billion people are at risk, and it
currently leads to the deaths of almost 1 million people each year,
with the majority of these occurring in sub-Saharan Africa among
children under 5 years of age.1 Resistance to existing anti-malarial
drugs is widespread2 and therefore new therapeutic approaches
are urgently needed. Calcium-dependent protein kinases (CDPKs)
are directly regulated by Ca2+ and are found in plants and organ-
isms in the alveolate lineage,3 but are absent in humans. They
are present in Apicomplexan parasites including Plasmodium falci-
parum, the causative agent of the most severe form of malaria.
CDPKs in Plasmodium are present as a multigene family containing
at least five members,4 and different CDPKs are proposed to be
functional at different stages of the parasite life cycle. P. falciparum
calcium-dependent protein kinase 1 (PfCDPK1), first identified by
Zhao et al.,5 is expressed in the asexual blood stages of the parasite
responsible for disease pathology. It has been shown to be encoded
by an essential gene6 and it is implicated in parasite motility and
host cell invasion, where it is able to phosphorylate components
of the molecular motor that drive parasite invasion of red blood
cells.7 If this invasion process can be prevented the parasite
lifecycle would be broken, leading the parasites to die and the
infection to be cleared. PfCDPK1 therefore represents a novel target
for the potential treatment of malaria and offers promise for
achieving selectivity over the kinases of the human host. More re-
cently its role in translational regulation of motor complex tran-
scripts has been reported8 but hitherto few inhibitors of PfCDPK1
have been described in the literature.9
A high throughput screen of our compound collection against
the isolated recombinant PfCDPK1 enzyme was performed10 and
a series containing a 3,6-disubstituted imidazopyridazine core
template was identified as the primary series of interest (Fig. 1).
Early examples with R1 as a 2- or 3-aminoethylpyridyl group and
R2 as a phenyl ring carrying an appended amide, cyano or fluoro
group all showed sub-100 nanomolar IC50 values against the en-
zyme (Table 1). Initial screening of these compounds against the
P. falciparum parasite in vitro showed strong inhibition of parasite
growth in a number of cases. However, despite the promising po-
tency of these early compounds, they typically showed high logD
values and low stability in microsomes. Furthermore, they exhib-
ited poor selectivity for PfCDPK1 over a panel of human kinases,
and their anti-parasite effect may be driven by significant
off-target activity. Related imidazopyridazines have been de-
scribed in the literature as effective inhibitors of other kinases such
as human PIM kinase,11 IKKb12 and malarial PfPK7.13
⇑
Corresponding author. Tel.: +44 02089067100.
E-mail address: tim.chapman@tech.mrc.ac.uk (T.M. Chapman).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.017

T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3064–3069
3065
N
N
R1
N
R2
Figure 1. 3,6-Imidazopyridazine hit core template.
Table 1
Examples from hit series
Compound
R1
R2
PfCDPK1 IC50
(lM)
P. falciparum growth inhibitiona (%)
N
N
H
1
0.059
99
O
O
NH2
NH2
N
N
2
0.061
0.066
99
35
H
N
H
N
3
4
N
H
O
N
N
N
0.071
0.080
98
6
H
CN
5
N
H
F
a
At 1 lM inhibitor concentration.
The aim at this point was to explore SAR towards improving the
potency alongside the selectivity, ADME and physical properties of
the series. In order to assist compound design, a homology model
of PfCDPK1 was built based on the published crystal structure of
TgCDPK114 and docking studies using Glide (Schrödinger Inc.) sug-
gested that the imidazopyridazine core could form a key H-bond
interaction between the nitrogen at the 1-position and the back-
bone N-H of Tyr-148 at the kinase hinge region (Fig. 2).
range of different alkyl groups were investigated and the isopentyl
group was found to be optimal for enzyme affinity (examples 9 and
10) with sub-micromolar anti-parasite EC50. Compounds were pre-
pared following the synthetic route shown in Scheme 1: installa-
tion of the basic amine side chain was achieved by nucleophilic
substitution at the 6-chloro substituent of 11 to afford the interme-
diates 12 and 14. The 3-position N-linked amides or carbamate 6–8
were accessed by Suzuki coupling either directly or through the
intermediate aniline 15 with subsequent functionalisation. The 4-
position C-linked amides were accessed by Suzuki coupling fol-
lowed by hydrolysis to give the carboxylic acids 13 and 16 then
amide coupling with isopentyl amine.
The aminoethylpyridine group at R1 could form an interaction
with the Lys-85 but was also directed towards the Glu-152 residue
at the entrance to the pocket, leading out towards solvent. The R2
portion was proposed to occupy a pocket where the model sug-
gested there was sufficient space to append larger groups onto the
phenyl ring. This offered a potential opportunity to gain improved
potency and selectivity in comparison to compounds such as 1
and 2.
In order to try to further improve the physical properties of the
compounds, decrease the logD and improve anti-parasite potency,
replacement of the phenyl ring attached to the imidazopyridazine
core with a heteroaryl ring was investigated.
Synthesis of a range of analogues with variation of the groups at
both R1 and R2 was undertaken in order to build the SAR, and
examples given in Table 2 illustrate the results from assays against
both the PfCDPK1 enzyme and P. falciparum parasite.15 It was rap-
idly found that the pyridyl group at the R1 position of the molecule
was less important in contributing to the binding affinity than the
core and R2 groups, so this R1 could be replaced with a more basic
amine group with the aim of lowering the logD and improving the
ADME and physical properties of the compounds. Exploration of a
range of different basic amine side chains at R1 revealed that N-
methyl piperidine and 1,4-diaminocyclohexane in particular gave
good enzyme affinity. At the R2 position, N-linked phenyl amides
and carbamates showed good enzyme affinity and sub-micromolar
EC50 values against the P. falciparum parasite (Table 2, examples 6–
8). C-linked phenyl amides also showed good enzyme affinity: a
The replacement of the phenyl ring by pyridyl and directly
linking the alkylamine to the pyridyl ring resulted in a compound
with good enzyme affinity and sub-500 nanomolar cell potency
(Table 3, example 17), which also displayed a good in vitro ADME
profile (see Table 6). A range of alternative alkyl groups was ex-
plored and while changes could be accommodated (e.g., 18 and
19), none were superior to the isopentyl group for potency.
The introduction of polarity led to a small loss in potency (20)
and the alternative pyridine isomer carrying the isopentylamine
substituent (21) displayed a sevenfold loss in potency against
the enzyme in comparison to 17. The compounds were obtained
through the synthetic route shown in Scheme 2: Suzuki cou-
pling gave the chloropyridine intermediates 22 and 23 and
the alkylamines were subsequently introduced by nucleophilic
displacement.

3066
T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3064–3069
Figure 2. Proposed binding orientation from docking of compound 1 in homology model of PfCDPK1.
Table 2
SAR with basic amine groups at R1 and substituted phenyl groups at R2
Compound
R1
R2
PfCDPK1 IC50
(l
M)
P. falciparum EC50 (lM)
H2N
H
N
6
0.011
0.32
N
H
O
N
O
7
8
0.022
0.018
0.56
0.75
N
H
N
H
N
O
OMe
N
H
N
H
H2N
N
H
9
0.016
0.023
0.46
0.78
N
O
H
N
N
H
10
N
H
O
Variation in the basic side-chain at R1 with constant R2 was then
explored (Table 4). This showed that reducing the size of the ring to
the pyrrolidine was well tolerated (24), however the azetidine (25)
lost significant potency against both the enzyme and parasite, and
this was also observed for the N-methyl piperazine (26). As pre-
dicted by the homology model the presence of the NH was not
found to be essential: although the 4-dimethylaminopiperidine
(27) was less potent than compound 17, its desmethyl analogue
(28) showed good potency against both the enzyme and parasite.
Similarly, the compound 17 analogue without the N-methyl group
(29) was well accommodated, with no loss in enzyme binding
affinity and slightly improved anti-parasite activity.
Returning to the R2 position, further changes in the heteroaryl
ring and the appended groups were investigated (Table 5). The nitro-
gen linker atom between the heteroaryl ring and the alkyl chain was
replaced with an oxygen atom, by performing a nucleophilic substi-
tution with isopentyl alcohol deprotonated with sodium hydride in
place of the amine. However, the product (30) showed a significant
loss in potency, indicating the importance of this N–H donor.
Replacement of the pyridine ring with pyrimidine was investigated,
and this revealed that compounds containing the pyrimidine at-
tached to the core through the 5-position (31 and 32) showed good
inhibitory activity whereas attachment at the 4-position (33) re-
sulted in a significant loss of potency against the enzyme.

T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3064–3069
3067
O
O
N
N
N
(b), (c)
(a)
(i) or (j)
HN
N
N
N
(6)
(9)
N
H
N
Cl
N
(7) or (8)
Br
N
N
N
H
N
(11)
(15)
NH2
(12)
Br
(d), (e)
(g)
(h), (c)
O
O
HN
N
N
N
(f)
N
(d), (e)
(f)
N
N
N
N
N
H
N
(10)
N
H
N
H
N
Br
(16)
(13)
(14)
CO2H
CO2H
Scheme 1. Reagents and conditions: (a) 1,4-cyclohexanediamine, dioxane/NMP, microwave, 180 °C then di-tert-butyldicarbonate, DMAP, CH3CN, 50 °C; (b) 3-
acetamidophenylboronic acid pinacol ester, Pd(dppf)Cl2, aq Na2CO3, dioxane, reflux; (c) 4 M HCl/dioxane; (d) 4-ethoxycarbonylphenylboronic acid, Pd(dppf)Cl2, aq Na2CO3,
dioxane, reflux; (e) LiOH, THF/MeOH/H2O; (f) isopentylamine, TBTU, DIPEA, and DMF; (g) 1-methyl-4-aminopiperidine, NMP, microwave, 180 °C; (h) 3-N-tert-
butoxycarbonylaminophenylboronic acid, Pd(dppf)Cl2, aq Na2CO3, dioxane, reflux; (i) cyclopropanecarbonyl chloride, DIPEA, and CH2Cl2; (j) methyl chloroformate, DIPEA,
CH2Cl2.
Table 3
SAR with heteroaryl R2 (nt = not tested)
N
N
N
N
H
N
R2
Compound
R2
PfCDPK1 IC50
(l
M)
P. falciparum EC50 (lM)
N
17
0.013
0.014
0.036
0.40
N
H
N
18
19
0.43
0.45
N
H
N
N
H
N
20
21
0.025
0.088
0.67
nt
OMe
N
H
N
H
N
The synthetic routes used to access these analogues are detailed
in Scheme 3: installation of the S-methyl pyrimidine through Suzu-
ki coupling on the BOC-protected compounds 34 and 12 gave the
intermediates 35–37. These were functionalised through oxidation
of the S-methyl group using mCPBA and subsequent introduction
of the alkylamine by nucleophilic substitution. The sequence was
completed by removal of the BOC protecting group and then in
the case of the piperidine by the introduction of the N-methyl
group through reductive amination.
microsomes alongside significant improvements in kinase selectiv-
ity against a human kinase panel.
Compounds possessing the best profiles with respect to po-
tency, in vitro ADME and selectivity were advanced to testing for
in vivo efficacy in a P. berghei mouse model of malaria. In advance
of in vivo testing, it was shown that the inhibitors retained potency
against the isolated P. berghei CDPK1 enzyme.16 Compounds were
dosed with an oral, once daily 50 mg/kg regime over 4 days in
the standard Peters test, and their in vitro ADME and in vivo effi-
cacy data is shown in Table 6. The best efficacy was displayed by
compound 17, with a 46% reduction in the level of parasitaemia
relative to vehicle. This offers promise at this stage considering
the relatively modest cellular potency of these compounds and
17 represents an interesting early lead. PK profiling of compound
Overall, the introduction of a basic side chain at the R1 position
and a heteroaryl ring with an appended aminoalkyl group at R2 led
to improved potency, physical properties and in vitro ADME char-
acteristics compared with the initial hits. These compounds dis-
played lower logD and higher stability in both mouse and human

3068
T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3064–3069
Table 4
Table 6
SAR with alternative basic amine groups
In vitro ADME and in vivo efficacy data for selected compounds
N
Compound
N
17
24
27
28
R1
N
PfCDPK1 IC50
(
l
M)
(lM)
0.013
0.40
63
85
3.4
81
0.023
0.17
74
63
3.5
0.044
0.57
84
0.013
0.14
90
90
2.5
55
N
P. falciparum EC50
MLMa (% rem)
HLMa (% rem)
mlogD
N
80
H
3.2
137
7
PAMPA Papp (nm sÀ1
)
114
30
Compound
R1
PfCDPK1 IC50
(l
M)
P. falciparum EC50 (lM)
Reduction in parasitaemia in vivob (%)
46
11
a
% Remaining at 30 min.
4-Day Peters test in P. berghei mouse model, with oral dosing once daily at
N
24
0.023
0.17
b
N
H
50 mg/kg; compounds were dissolved or suspended in 70/30 Tween-80/ethanol
and diluted 10-fold with water before dosing.
N
25
26
27
0.089
0.175
0.044
0.93
2.40
0.57
N
H
N
N
(a)
N
N
N
(14)
(c)
N
N
N
H
N
(22)
(b)
N
N
Cl
N
28
29
0.013
0.015
0.14
0.24
N
H2N
HN
N
N
H
N
N
N
(23)
N
H
Cl
N
N
N
N
H
(b)
N
N
N
N
H
17 revealed that it possessed a half-life of 2 h and good oral bio-
availability in mouse (Fig. 4), although it displayed moderate to
high clearance.
R
N
N
N
i-pentyl (17)
n-butyl (18)
H
R=
H
(21)
Compounds 17, 24 and 28 exhibited good selectivity profiles
N
N
(19)
when screened against a panel of human kinases at 1 lM inhibitor
concentration (Fig. 3).17 Pleasingly, screening against the isolated
CDPK1 enzyme of the related malarial parasite Plasmodium vivax
revealed that these compounds were highly potent against this
species,16 which is also an important human pathogen causing
considerable morbidity.
(20)
O
Scheme 2. Reagents and conditions: (a) 2-chloro-5-pyridine boronic acid,
Pd(dppf)Cl2, aq Cs2CO3, THF, reflux; (b) RNH2, NMP, microwave, 190 °C; (c) 2-
chloro-4-pyridine boronic acid, Pd(dppf)Cl2, aq Cs2CO3, THF, reflux.
Table 5
SAR profiles with alternative R2 heteroaryl groups or heteroatom linkers (nt = not tested)
N
N
R1
N
R2
Compound
R1
R2
PfCDPK1 IC50
(
lM)
P. falciparum EC50 (lM)
H2N
N
30
0.170
0.54
N
H
O
N
N
H
N
31
0.014
0.47
N
N
N
H
H2N
N
N
H
32
33
0.016
0.424
0.17
nt
N
H
N
N
N
H
N
N
H

T. M. Chapman et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3064–3069
3069
O
O
(a) or (b)
(c), (d), (e), (f)
N
N
N
N
O
N
O
N
N
N
N
N
H
N
N
H
N
N
H
N
R2
R2
Br
(34)
R2 =
R2 =
or
(31)
or
N
N
N
N
N
N
N
S
(36)
N
N
N
(33)
H
S
(35)
H
O
O
H2N
N
HN
N
N
(a)
(c), (d), (e)
N
N
H
N
(12)
N
H
N
(32)
N
(37)
N
N
N
N
S
H
Scheme 3. Reagents and conditions: (a) 2-(methylthio)pyrimidine-5-boronic acid pinacol ester, Pd(dppf)Cl2, aq Cs2CO3, dioxane, reflux; (b) 2-(methylthio)pyrimidine-4-
boronic acid pinacol ester, Pd(dppf)Cl2, aq Cs2CO3, dioxane, reflux; (c) m-chloroperoxybenzoic acid, CH2Cl2; (d) isopentylamine, dioxane, 65 °C; (e) 4 M HCl/dioxane; (f)
formaldehyde, AcOH, Na(OAc)3BH, THF.
References and notes
Kinase
Compound
1. World Health Organisation. World Malaria Report, 2010.
2. Petersen, I.; Eastman, R.; Lanzer, M. FEBS Lett. 2011, 1551.
3. Harper, J. F.; Harmon, A. Nat. Rev. Mol. Cell Biol. 2005, 6, 555.
17
24
28
4. Ward, P.; Equinet, L.; Packer, J.; Doerig, C. BMC Genomics 2004, 5, 79.
5. Zhao, Y.; Kappes, B.; Franklin, R. M. J. Biol. Chem. 1993, 268, 4347.
6. (a) Tewari, R.; Straschil, U.; Bateman, A.; Bohme, U.; Cherevach, I.; Gong, P.;
Pain, A.; Billker, O. Cell Host Microbe 2010, 8, 377; (b) Kato, N.; Sakata, T.;
Breton, G.; Le Roch, K. G.; Nagle, A., et al Nat. Chem. Biol. 2008, 4, 347.
7. Green, J. L.; Rees-Channer, R. R.; Howell, S. A.; Martin, S. R.; Knuepfer, E.; Taylor,
H. M.; Grainger, M.; Holder, A. A. J. Biol. Chem. 2008, 283, 30980.
8. Sebastian, S.; Brochet, M.; Collins, M. O.; Schwach, F.; Jones, M. L.; Goulding, D.;
Rayner, J. C.; Choudhary, J. S.; Billker, O. Cell Host Microbe 2012, 12, 9.
9. (a) Lemercier, G.; Fernandez-Montalvan, A.; Shaw, J. P.; Kugelstadt, D.; Bomke,
J.; Domostoj, M.; Schwarz, M. K.; Scheer, A.; Kappes, B.; Leroy, D. Biochemistry
2009, 48, 6379; For a review of CDPKs as drug targets see: (b) Kugelstadt, D.;
Derrer, B.; Kappes, B. Drug Discov. Infect. Dis. 2011, 2, 319 (Apicomplexan
parasites).
10. A high throughput screen of 35,422 compounds comprising a diverse set of
small molecules from a variety of commercial suppliers and 8100 kinase-
focused compounds (BioFocus) was performed using Kinase-GloÒ (Promega) to
measure ATP depletion resulting from the kinase reaction. Full length
recombinant PfCDPK1 was incubated with MyoA-Tail domain Interacting
Protein (MTIP) substrate and the amount of ATP remaining after 1 h was
measured by luminescence using a Pherastar plate reader (BMG Labtech).
11. Bullock, A. N.; Debreczeni, J. E.; Fedorov, O. Y.; Nelson, A.; Marsden, B. D.;
Knapp, S. J. Med. Chem. 2005, 48, 7604.
Figure 3. Kinase selectivity data on selected compounds screened at 1 lM inhibitor
concentration against a 73-member human kinase panel; green: <50% inhibition;
amber: 50–80% inhibition; red: >80% inhibition. Kinases hit by compound 17 are:
MKK1, RSK1, PKD1, CHK2, Aurora B, NUAK1, GCK, MLK1, Src, Lck, YES1, and VEGFR;
compound 24: RSK1 and HER4; compound 28: RSK1, CAMK1, PHK, NUAK1, and
HER4.
i.v. t1/2
Clint
2.0 h
2984 mL/h/kg
84%
Oral %F
ppb
86%
Figure 4. Mouse pharmacokinetic and plasma-protein binding data for compound
17.
12. Shimizu, H.; Yamasaki, T.; Yoneda, Y.; Muro, F.; Hamada, T.; Yasukochi, T.;
Tanaka, S.; Toki, T.; Yokoyama, M.; Morishita, K.; Iimura, S. Bioorg. Med. Chem.
Lett. 2011, 21, 4550.
13. Bouloc, N.; Large, J. M.; Smiljanic, E.; Whalley, D.; Ansell, K. H.; Edlin, C. E.;
Bryans, J. S. Bioorg. Med. Chem. Lett. 2008, 18, 5294.
14. Ojo, K. K.; Larson, E. T.; Keyloun, K. R.; Castaneda, L. J.; DeRocher, A. E.;
Inampudi, K. K., et al Nat. Struct. Mol. Biol. 2010, 17, 602 (PDB ref: 3I7C).
15. P. falciparum EC50 values were measured using an in vitro model of malaria
parasite growth. Compounds were diluted into 2% DMSO and added to
parasites 24 h post-invasion in a 96-well plate and incubated under static
conditions. Cells were recovered 48 h later and processed for FACS analysis
using hydroethidine to stain parasite DNA. Data was acquired using CellQuest
Pro software on a FACSCalibur (Becton Dickinson). Growth inhibition was
In summary, a series of imidazopyridazines which are potent
inhibitors of PfCDPK1 has been identified. Leading compounds
have shown promising in vitro anti-parasite activity, in vitro ADME
and kinase selectivity profiles and in vivo pharmacokinetics in
mouse, with modest in vivo efficacy in a P. berghei mouse model
of malaria. Improving the in vitro anti-parasite activity, in vivo effi-
cacy and PK profile of this series is the subject of further work and
will be described in a future publication.
calculated
using
the
following
formula:
%
growth
Acknowledgments
inhibition = (1 À [parasitaemia of culture/parasitaemia of control culture])
Â100.
We thank David Tickle and Sadhia Mahmood at MRCT for
in vitro ADME, David Whalley for testing against PbCDPK1 and
PvCDPK1 and Munira Grainger at NIMR for provision of parasites.
We are grateful to the Medicines for Malaria Venture for providing
support for this project, including Paul Willis, Didier Leroy and Si-
mon Campbell for their input and Sergio Wittlin at the Swiss Trop-
ical and Public Health Institute for conducting P. berghei in vivo
efficacy studies. Mouse pharmacokinetic studies were performed
by Pharmidex. A.A.H. is funded by the MRC (U117532067) and
the EU FP7 Grant agreement 242095 (EviMalar).
16. To establish activity of the compounds against recombinant P. berghei CDPK1
and P. vivax CDPK1 enzymes, ATPase activity was measured using a biosensor
sensitive to ADP (rhodamine-labeled ParM, gift of M. Webb, NIMR). The
progress of the reactions was monitored by an increase in fluorescence
corresponding to accumulation of ADP using a Pherastar plate reader (BMG
Labtech).
17. Kinase selectivity profiling was carried out at the National Centre for Protein
Kinase Profiling in the MRC Protein Phosphorylation Unit at the University of
Dundee.