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Validation of The Protein Kinase PF CLK3 As A Multistage Cross-Species Malarial Drug Target
Validation of The Protein Kinase PF CLK3 As A Multistage Cross-Species Malarial Drug Target
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Validation of a ultra-sensitive qRT-PCR for the detection of P. vivax gametocytes in asymptomatic individuals from Panama. Validación de una prueba molecular ultra-
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PfCLK3 as a multistage cross-species tion that inhibition of PfCLK3 killed the malaria
parasite by preventing the splicing of essential
parasite genes. Because there is a high degree
malarial drug target of homology between orthologs of CLK3 in other
Plasmodium species, it might be expected that
Mahmood M. Alam*, Ana Sanchez-Azqueta*, Omar Janha*, Erika L. Flannery, our probe molecule would both inhibit CLK3
Amit Mahindra, Kopano Mapesa, Aditya B. Char, Dev Sriranganadane, contained in other malaria parasite species and
Nicolas M. B. Brancucci, Yevgeniya Antonova-Koch, Kathryn Crouch, have effective parasiticidal activity in these para-
Nelson Victor Simwela, Scott B. Millar, Jude Akinwale, Deborah Mitcheson, sites. This was indeed found to be the case, with
Lev Solyakov, Kate Dudek, Carolyn Jones, Cleofé Zapatero, Christian Doerig, our molecule showing potent inhibition of CLK3
Davis C. Nwakanma, Maria Jesús Vázquez, Gonzalo Colmenarejo, ◥
from P. vivax and P. berghei
Maria Jose Lafuente-Monasterio, Maria Luisa Leon, Paulo H. C. Godoi,
ON OUR WEBSITE as well as killing the blood
Gametocyte development
malarial drug target of the PfCLK family (17), plays an essential role in
parasite pre-mRNA processing (18).
Mahmood M. Alam1*, Ana Sanchez-Azqueta2*, Omar Janha2*, Erika L. Flannery3, High-throughput screen identifies
Amit Mahindra4, Kopano Mapesa4, Aditya B. Char5, Dev Sriranganadane6, selective P f CLK3 inhibitors
Nicolas M. B. Brancucci7, Yevgeniya Antonova-Koch8, Kathryn Crouch1, We established high-throughput inhibition assays
Nelson Victor Simwela1, Scott B. Millar1, Jude Akinwale9, Deborah Mitcheson10, for two essential members of the Pf CLK family,
Lev Solyakov9, Kate Dudek9, Carolyn Jones9, Cleofé Zapatero11, Christian Doerig12, PfCLK1 and PfCLK3 (fig. S1). Both of these pro-
Davis C. Nwakanma13, Maria Jesús Vázquez11, Gonzalo Colmenarejo14, tein kinases were purified as active recombinant
Maria Jose Lafuente-Monasterio11, Maria Luisa Leon11, Paulo H. C. Godoi6, proteins (fig. S2A) and were used in a high-
D
hibitor set” from within the Medicines for Malaria
espite artemisinin-based combination ther- than half have been reported to be essential for Venture (MMV) box, a collection of 400 anti-
apies offering effective frontline treatment blood-stage survival (8–12). These studies, together malarial compounds (24), were used to generate
for malaria, there are still more than 200 with the generally accepted potential of target- concentration inhibition curves (Fig. 1C and
million cases of malaria worldwide each ing protein kinases in the treatment of numerous table S1). On the basis of the selectivity criterion
year, resulting in an estimated 500,000 human diseases (13, 14), suggest that inhibition of a difference of more than 1.5 log units in the
deaths. This, combined with the fact that there is of parasite protein kinases might offer a viable negative logarithm of the half-maximal inhibition
now clear evidence for the emergence of resistance strategy for the treatment of malaria (6, 15) (pIC50), 28% of the hits showed specific inhibition
not only to artemisinin (1, 2) but also to partner To directly test this hypothesis, we focused on of PfCLK1 and 13% specifically inhibited PfCLK3,
drugs including piperaquine and mefloquine one of the four members of the P. falciparum whereas 23% of the compounds inhibited both
(3, 4), means that there is an urgent need for cyclin-dependent–like (CLK) protein kinase fam- PfCLK3 and PfCLK1; the remainder (36%) were
novel therapeutic strategies to cure malaria while ily, Pf CLK3 (PF3D7_1114700), a protein kinase inactive (Fig. 1, C and D, and table S1). Exemplar
also preventing transmission. Global phospho- essential for maintaining the asexual blood stage molecules from each of these three classes are
proteomic studies on the most virulent species of of both P. falciparum (8, 12) and P. berghei (10, 11). shown in fig. S3.
human malaria, Plasmodium falciparum, have In mammalian cells, the CLK protein kinase fam- Highlighted in Fig. 1C is TCMDC-135051, which
established protein phosphorylation as a key ily and the closely related SRPK family are crucial showed the highest selectivity and potency for
regulator of a wide range of essential parasite mediators of multiple phosphorylation events on inhibition of PfCLK3. TCMDC-135051 also showed
processes (5–8). Furthermore, of the 65 eukaryotic splicing factors, including serine-arginine–rich lower activity against the closely related human
protein kinases in the parasite kinome (9), more (SR) proteins, which are necessary for the correct kinase CLK2 (29% sequence identity with PfCLK3)
1
Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow G12 8QQ, UK. 2Centre for Translational Pharmacology, Institute of Molecular Cell and Systems Biology, University
of Glasgow, Glasgow G12 8QQ, UK. 3Novartis Institute for Biomedical Research, Emeryville, CA 94608, USA. 4School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK. 5Institute of
Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Science, University of Glasgow, Glasgow G12 8QQ, UK. 6Structural Genomics Consortium,
Universidade Estadual de Campinas, Campinas, São Paulo 13083-886, Brazil. 7Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, 4051 Basel,
Switzerland. 8Skaggs School of Pharmaceutical Sciences, UC Health Sciences Center for Immunology, Infection and Inflammation, University of California, San Diego, School of Medicine, La Jolla,
CA 92093, USA. 9Medical Research Council Toxicology Unit, University of Leicester, Leicester LE1 9HN, UK. 10Department of Molecular Cell Biology, University of Leicester, Leicester LE1 9HN,
UK. 11Diseases of the Developing World, GlaxoSmithKline, 28760 Tres Cantos, Madrid, Spain. 12Biomedical Science Cluster, School of Health and Biomedical Sciences, Royal Melbourne Institute
of Technology, Melbourne, VIC 3000, Australia. 13MRC Unit the Gambia, Fajara, Banjul, The Gambia. 14Biostatistics and Bioinformatics Unit, IMDEA Food Institute, 28049 Madrid, Spain.
15
Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX3 7DQ, UK.
*These authors contributed equally to this work.
†Corresponding author. Email: andrew.tobin@glasgow.ac.uk
by a factor of ~100 (table S1). Similarly, TCMDC- TCMDC-135051 is a member of a series of but no change in sensitivity to chloroquine or
135051 showed no evidence of interacting with molecules that were contained in the high- artemisinin (Fig. 2A and Table 1). Whole-genome
the human ortholog of PfCLK3, PRPF4B. This was throughput screen with the same chemical scaf- sequencing of the three resistant lines revealed
seen in thermostability assays, using differential fold. This series showed similar inhibitory activity mutations in PfCLK3 (lines TM051A and TM051C)
scanning fluorimetry, where staurosporine, acting against PfCLK3 (fig. S6). Note that the TCMDC- and a mutation in the putative RNA processing
as a positive control, increased the melting tem- 135051 is part of the TCAMS and has previously protein PfUSP39 (PF3D7_1317000) (line TM051B;
perature of PRPF4B by 2.40° ± 0.14°C. In contrast, been shown to have antiparasiticidal activity Fig. 2B and Table 1). The resistant clone TM051A,
TCMDC-135051 showed no change in PRPF4B (half-maximal response EC50 = 320 nM); the which contained the mutation Pro196 → Arg
thermostability (fig. S4A). Furthermore, in a mass structure has been published (21). However, (P196R) in the N-terminal region outside the
spectrometry–based PRPF4B activity assay, the resynthesis of TCMDC-135051, together with nu- Pf CLK3 kinase domain (Fig. 2B), showed the
published inhibitor Compound A (25) showed clear magnetic resonance (NMR) analysis, has deter- smallest change in sensitivity to TCMDC-135051
inhibition of PRPF4B, whereas TCMDC-135051 at mined the correct structure for TCMDC-135051 to (factor of 4.2 shift in EC50 relative to parental
concentrations up to 50 mM showed no inhibi- be the one shown in Fig. 1C and fig. S3. Dd2 parasites). Examination of the in vitro enzy-
tory activity (fig. S4B). In further counterscreens, matic properties of the P196R mutant found in
TCMDC-135051 showed no activity against the Parasite strains resistant to TM051A did not detect any changes in enzyme
P. falciparum protein kinases PfPKG and PfCDPK1 TCMDC-135051 show mutations in Pf CLK3 kinetics or sensitivity to inhibition by TCMDC-
(fig. S5, A to C). Thus, TCMDC-135051 showed We next sought to confirm that PfCLK3 was the 135051 relative to the wild-type kinase; this find-
selective inhibition of Pf CLK3 when compared target of TCMDC-135051 parasiticidal activity. ing suggests that this mutation could potentially
against the closely related human kinases PRPF4B Exposing P. falciparum Dd2 parasites to increas- stabilize the protein or be otherwise involved in
and CLK2, as well as the closest parasite kinase, ing concentrations of TCMDC-135051 resulted the interaction between Pf CLK3 and its sub-
Pf CLK1, and other parasite kinases (Pf PKG, in the emergence of three independent lines that strates or regulatory proteins.
Fig. 1. High-throughput screen identifies inhibitors of PfCLK1 and PfCLK3. (A) Percent Genetic target validation of Pf CLK3
inhibition distribution pattern of compounds screened against PfCLK3, binned in 5% intervals. To further confirm Pf CLK3 as the target of
Active “hit” compounds were defined as those that were positioned >3 SDs from the mean. TCMDC-135051 parasiticidal activity, we de-
(B) Pie chart summary of the primary single-dose screen. (C) Hit compounds were used signed a variant of Pf CLK3 that showed reduced
in concentration response curves. Shown is a comparison of pIC 50 values for inhibition of sensitivity to TCMDC-135051. To generate this
PfCLK3 versus PfCLK1. TCMDC-135051 (structure shown) is highlighted as the most potent variant, we took advantage of the highly selec-
and selective PfCLK3 hit. (D) The same data as shown in (C) but in pie chart format of tive inhibition of Pf CLK3 over Pf CLK1 shown
compounds designated as inactive, pan-active (active against both PfCLK1 and PfCLK3), or by TCMDC-135051. By exchanging amino acids
selective for either PfCLK1 or PfCLK3. within subdomain V of the PfCLK3 kinase domain
Table 1. Adaptive resistance to TCMDC-135051. Dd2 parasites were exposed to subthreshold concentrations of TMDC-135051, and three lines were
isolated that were less sensitive to TCMDC-135051 but had unchanged sensitivity to artemisinin and chloroquine. Shown are nucleotide changes and
associated amino acid changes in genes from the resistance lines as well as the identity and annotated function of the mutant genes. EC50 values associated
with each line for artemisinin, chloroquine, and TCMDC-135051 are shown, as well as the relative (fold) change in IC50 for TCMDC-135051 compared to Dd2
parent parasites. Data are means ± SEM of three experiments.
EC50
sensitivity to an inhibitor. By swapping residues continued to the 50-hour time point (Fig. 4A). changes in gene transcription in parent Dd2
between the kinases, we introduced inhibitor These data indicated that Pf CLK3 inhibition parasites and the drug-resistant stain TM051C
insensitivity into our target kinase (e.g., Pf CLK3) prevented the transition of the parasites at early in response to exposure to TCMDC-135051. RNA
in a strategy that could be applied to other pro- stages (ring to trophozoite) as well as late stages isolated from trophozoite-stage parasites was
tein kinases. (trophozoite to schizont) of development and did extracted after treatment with 1 mM TCMDC-
not allow parasites to reach the next invasion 135051 for 60 min, during which the Dd2 and
Inhibition of Pf CLK3 prevents cycle (Fig. 4A). These data further indicated that TM051C parasites maintained normal morphol-
trophozoite-to-schizont transition PfCLK3 inhibition resulted in rapid killing, with ogy. Genome-wide transcriptional patterns were
To characterize the phenotypic response to no evidence that the compound resulted in qui- determined using oligonucleotide microarray
Pf CLK3 inhibition and to understand Pf CLK3 escence from which the parasite could recover chips that probed 5752 P. falciparum genes (31).
function, we treated P. falciparum 3D7 parasites after drug withdrawal. These features were con- Under these conditions, 779 gene transcripts
synchronized at ring stage (time point zero) with firmed in parasite reduction rate assays, which were significantly down-regulated in response
1 mM TCMDC-135051. The parasites progressed showed that treatment of parasites with 10 × to Pf CLK3 inhibition in the Dd2 parasites and
to late ring stage (time point 20 hours) (Fig. 4A) EC50 of TCMDC-135051 completely killed the 155 genes were up-regulated (Fig. 4C and table
but did not progress further to trophozoite stage parasite in 48 hours; viable parasites could not S2). That the majority of these transcriptional
(time point 30 and 40 hours), arresting with a be observed despite maintaining the parasite changes were due to inhibition of Pf CLK3 and
condensed and shrunken appearance (Fig. 4A). culture for 28 days after withdrawal of TCMDC- not off-target events was supported by the fact
Similar effects were observed if the parasites were 135051 (Fig. 4B). that under the same conditions, only six genes
treated at mid-ring stage (time point 10 hours). were up-regulated and 88 down-regulated in the
Treatment of the parasite at later time points (20 Inhibition of Pf CLK3 resistant TM051C parasite strain (Fig. 4D and
or 30 hours) blocked development of the parasite disrupts transcription table S3). By subtracting the transcriptional
from the trophozoite to the schizont stage. The Because PfCLK3 has been proposed to regulate changes observed in the TM051C strain, defined
fact that the parasites at the schizont stage were RNA processing (20) and is closely related to the here as “off-target,” from those observed with the
not viable was further evidenced by the absence human kinases PRPF4B and CLK2 that are in- Dd2 parent, the transcriptional changes due to
of ring-stage parasites when the culture was volved in RNA splicing (19), we investigated “on-target” inhibition of Pf CLK3 were defined
(table S4). Among these “on-target” down- essential for asexual P. falciparum survival (12) 93% of the “on-target” down-regulated genes
regulated genes were those involved in key par- (table S4). contained introns (Fig. 4F and table S4), versus
asite processes, such as egress and invasion, Gene ontology enrichment analysis was used 52% of genes in the P. falciparum genome that
cytoadherence, parasite protein export, and in- to determine biological functions that were dis- are annotated as containing introns (32) (Fig. 4G).
volvement in sexual stages, as well as house- proportionally down-regulated by Pf CLK3 inhi- Hence, Pf CLK3 inhibition significantly affected
keeping functions including metabolism, RNA bition. In this analysis, genes associated with the transcription of genes that contained introns
processing, lipid modification, and mitochon- key biological functions, particularly protein (P < 0.0001, Pearson c2 test), further supporting
drial function (Fig. 4E and table S4). Of the 696 modification, phospholipid biosynthesis, and its role in splicing.
“on-target” genes identified as down-regulated lipid modification, were significantly overrep- In addition to the nearly 700 genes down-
by Pf CLK3 inhibition (table S4), 425 matched resented among those genes that were down- regulated in response to Pf CLK3 inhibition,
those that have recently been determined to be regulated (fig. S8 and table S5). We found that there were 154 genes that were significantly
up-regulated (table S4). Among these were genes dose-related reduction in parasitemia over a allowed to develop to stage V in the continued
associated with RNA processing, such as splicing 5-day infection period, where the maximal dose presence of drug. These experiments showed a
factor 1 (PF3D7_1321700) and pre-mRNA splic- (50 mg/kg) resulted in near-complete clearance concentration-dependent decrease in stage V
ing factor SYF1 (PF3D7_1235900), indicating that of parasites from peripheral blood (Fig. 5C). gametocyte number (Fig. 5D). When analyzed
at least some of the up-regulated genes may rep- using a generalized linear mixed model (GLMM),
resent compensatory mechanisms. In support of Activity of TCMDC-135051 at liver this effect had a potency of EC50 = 0.8 mM (Fig.
this notion was the finding that Pf CLK3 itself invasion and sporozoite development 5E). Furthermore, the inhibition of Pf CLK3
was within the up-regulated genes (table S4). TCMDC-135051 showed potent activity against showed a statistically significant decrease in
P. berghei sporozoites in a liver invasion and exflagellation (Fig. 5, F and G; EC50 = 0.2 mM),
Cross-species and in vivo activity of development assay (34) in which the compound which, combined with the effect on gametocyte
TCMDC-135051 showed a pEC50 value of 6.17 ± 0.10 (EC50 = number, contributed to a statistically significant
It might be predicted that the close similarity 0.40 mM) (fig. S11), although hepatocyte toxicity reduction in transmission, as measured by the
between orthologs of CLK3 in different malaria (fig. S11) was observed but only significantly at prevalence of oocysts in the gut of mosquitos in
parasite species would result in TCMDC-135051 10 mM (fig. S11). membrane feeding assays (Fig. 5, H and I).
showing similar activities against CLK3 from These studies were further extended to test the
different Plasmodium species. This indeed was Targeting Pf CLK3 reduces transmission effects of Pf CLK3 inhibition on stage V gameto-
the case, as in vitro kinase assays using recombi- to the mosquito vector cytes. Although exposure of stage V gametocytes
nant PvCLK3 (P. vivax) and PbCLK3 (P. berghei) The effects of Pf CLK3 inhibition on sexual-stage to TCMDC-135051 for 24 hours did not affect
(fig. S9, A and B) showed that TCMDC-135051 parasites were tested in an assay developed using gametocyte number (fig. S13, A and B), a small
had near-equipotent inhibition at these two the P. falciparum Pf 2004 parasite strain, which but significant reduction in exflagellation (~25%
orthologs, with pIC50 values of 7.47 ± 0.12 (IC50 = shows high levels of gametocyte production (35). reduction, P < 2 × 10−16 as determined by GLMM)
Fig. 5. Inhibition of PfCLK3 has parasiticidal activity at multiple ****P < 0.0001. (D to I) Concentration effect of exposure of stage II to V
parasite species, shows in vivo parasiticidal activity in P. berghei, blocks P. falciparum (clone 3D7) gametocytes to TCMDC-135051 on gametocyte
gametocyte development, and reduces transmission to the mosquito (GC) numbers in culture [(D) and (E)], exflagellation [(F) and (G)], and
vector. (A and B) Concentration effect curve of TCMDC-135051 on blood- prevalence (number of mosquitos with oocyst infection per number of
stage P. knowlesi (A) and P. berghei (B) parasites. (C) TCMDC-135051 mosquitos dissected) of infection of Anopheles coluzzii mosquitos [(H) and (I)].
P. berghei in vivo growth inhibition curves and day 4 percentage suppression (D), (F), and (H) show means ± SEM of four independent experiments;
plots (inset). Error bars are SD from n = 4 mice groups. Statistical (E), (G), and (I) show the predicted effects of drug concentrations according to
comparisons between mice treated with drug and vehicle are shown using the maximal GLMM, with the shaded area indicating 95% confidence intervals.
one-way analysis of variance and Dunnett multiple-comparisons test. From the GLMM analysis, the approximate EC50 values were calculated.
including the liver stage. This suggests that in malaria, and there is abundant evidence that vector using restriction sites HpaI and BglII. The
targeting Pf CLK3 might be a novel strategy for phosphorylation and phosphosignaling are cru- rest of the clk3 gene sequence downstream of
developing curative treatments for malaria by cial for the viability of both asexual and sexual CLK3-HR, corresponding to 1798 to 3152 bp of
clearance of asexual blood-stage parasites and stages of the malaria parasite (5, 8, 10, 11). Es- the Pf Clk3 genomic sequence, was modified by
as a potential prophylactic by targeting the liver sential parasite protein kinase targets have been removing introns and the stop codon, and the
stage; moreover, the parasiticidal activity afforded identified (8, 11), and academic and industrial coding sequence was optimized for E. coli codon
by PfCLK3 inhibition at gametocytes would indi- laboratories have gained much experience in the usage to make it dissimilar to Pf clk3 genomic
cate that through this mechanism, transmission design of protein kinase inhibitor drugs (14, 37). sequence. This fragment of gene, which we
to the insect vector could also be affected. Because But despite these developments, the targeting of named as Pf CLK3-codon optimized (CLK3-CO),
splicing of essential transcripts occurs at many parasite protein kinases in antimalarial drug de- was commercially synthesized and included BglII
stages of the parasite life cycle, it is attractive to velopment is only in its infancy (6, 39). By focus- recognition site at 5′ and XhoI recognition site at
hypothesize that inhibition of PfCLK3, which has ing on an essential parasite kinase and taking 3′. CLK3-CO was cloned downstream of CLK3-
been implicated in the phosphorylation of splic- advantage of high-throughput phenotypic screens HR region in the parent plasmid using BglII and
ing factors necessary for the assembly and activ- of commercial and academic libraries (21, 40, 41) XhoI restriction sites in such a way that the triple
ity of the spliceosome (17, 18, 20), would have a as a starting point to screen for inhibitors, we HA tag sequence in the parent plasmid remained
wide-ranging impact on parasite viability. In sup- have identified a probe molecule that has not in frame with the PfClk3 sequence. The BglII re-
port of this notion is the finding that Pf CLK3 only established the validity of PfCLK3 as a target striction site that was artificially introduced for
inhibition down-regulated more than 400 essen- in malaria but also determined that this protein cloning purpose was mutated back to original
tial parasite transcripts. Interestingly, the major- kinase is susceptible to selective pharmacological Pf CLK3 coding sequence by site-directed muta-
ity of down-regulated transcripts are from genes inhibition by small drug-like molecules. In this genesis using CLK3-BglII-KN1 and CLK3-BglII-
that contain introns (91%), providing further way, our study lends weight to the argument that KN2 primers. Site-directed mutagenesis was used
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