PfCLK3 as a Therapeutic Antimalarial Drug Target

Artemisinin resistance, the current frontline antimalarial, is threatening to significantly increase the global incidence of malaria. Hence, novel targets for development of next generation antimalarials is urgently required. Here I focus on the essential malaria protein kinase PfCLK3, involved in RNA processing, to investigate if it is a suitable pharmacological target for malaria treatment. The molecule TCMDC-135051, identified from a screen at GlaxoSmithKline, was used as a probe inhibitor of PfCLK3. The evidence presented here shows that TCMDC-135051 had parasiticidal activity at multiple stages of P. falciparum growth at low nanomolar potency, with an IC50 of 180 nM in asexual ring stage parasites. TCMCD-135051 is also potent against asexual P. knowlesi and P. berghei parasites, meeting cross species requirements for new antimalarial agents. Against recombinant CLK3 kinases, TCMDC-135051 demonstrated a high potency, with an IC50 value of ~40 nM towards PfCLK3, PvCLK3 and PbCLK3 kinases. Additionally, a mode of action behaviour suggestive of non-ATP competitive inhibition was observed. For target validation, a mutant PfCLK3 parasite line (PfCLK3_G449P) reduced TCMDC-135051 potency by ~1.5 fold log units compared to wild type. Long-term exposure of Dd2 parasites to TCMDC-135051 showed two single point mutations on the PfCLK3 gene, indicating TCMDC-135051 selectivity towards PfCLK3. Using the parasite reduction rate to investigate the speed of action, TCMDC-135051 demonstrate activity levels similar to dihydroartemisinin, a standard antimalarial. Furthermore, inhibition of PfCLK3 resulted in impaired splicing in wild type 3D7 parasites compared to mutant PfCLK3_G449P, demonstrating the role of PfCLK3 in regulating RNA splicing. In conclusion, inhibition of PfCLK3 activity results in rapid killing of asexual P. falciparum parasites and other Plasmodium species at multiple stages through inhibition of RNA splicing. Therefore, the data presented here revealed PfCLK3 as a suitable target for treatment of symptomatic malaria and a potential transmissionblocking target

Tajima's and Fu & Li's summary indices of nucleotide site frequency spectrum for each of 2,853 <i>P. falciparum</i> genes with 3 or more SNPs in the Gambian population.

<p>A. Frequency distribution histograms for the individual gene values for Tajima's D, Fu & Li's F* and Fu & Li's D* respectively. B. Two-dimensional plot of Tajima's D and Fu & Li's F* values for each of the 2853 genes (<i>r</i> = 0.67; correlation between Fu & Li's F* and D* indices is stronger, <i>r</i> = 0.96; correlation between Tajima's D and Fu & Li's D* is less, <i>r</i> = 0.50; P<0.001 for all correlations). Those in the top right tail of the distribution with high indices of both are considered further as genes with candidate signatures of balancing selection.</p

Distribution of Tajima's D values in members of different gene families and groups of genes defined by expression location.

<p>For each, plots show the mean (mid-line), one standard deviation (boxes), and 2 standard deviations (whiskers) with any individual outlier genes as points. The proportions of genes with values above zero and the numbers of genes analysed in each gene family are shown above the plot.</p

Mapping signatures to particular regions within genes.

<p>A. Plots of linkage disequilibrium (<i>r</i>²) with distance between polymorphic nucleotides within genes each containing 10 or more SNPs. Nine genes are illustrated: left hand column shows genes with data on SNPs covering <500 bp, middle column 500–1000 bp, and right hand column >1000 bp, each column plotted with a different x-axis scale. Decline of LD with distance is evident in most genes, although the bottom plots show examples with some extended LD over most of the sequence analysed. B. Sliding window analysis identifies regions of genes with candidate signatures of balancing selection: top plot shows a <i>PHISTa</i> gene (PFL2555w) with high Tajima's D values in the 5′-region; middle plot shows the strongest signature on a <i>clag</i>-like gene (MAL7P1.229) is in the 3′-region; bottom plot for PF10_0355 shows the signature in the middle of the sequence. Window size of 100 bp was applied with step size of 25 bp.</p

Genes with estimated peak expression at the merozoite stage have highest Tajima's D values overall.

<p>Assignment of peak stage transcript expression for 2710 genes in data from microarray studies <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002992#pgen.1002992-LeRoch1" target="_blank">[37]</a> used an expression time series query implemented by PlasmoDB <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002992#pgen.1002992-Aurrecoechea1" target="_blank">[36]</a>, and are plotted against the polymorphism data from the present study. The points show the values for individual genes (and horizontal bars the medians of all genes) with estimated peak expression at each stage (ER, early ring; LR, late ring; ET, early trophozoite; LT, late trophozoite; ES, early schizont; LS, late schizont; M, merozoite; G, gametocyte). The proportions of genes with values above zero are shown at the top (this is highest for merozoite-stage genes, with 72/404 or 17.8%, p<0.0001 compared with all other genes). Asterisks indicate p values for Mann-Whitney tests on the comparisons of distributions between pairs of stages (* p<0.01, *** p<0.0001).</p

25 genes with ≥10 SNPs showing highest values of Tajima's D index in a genome-wide analysis of sequences of 65 Gambian <i>P. falciparum</i> isolates.

<p>N, number of aligned nucleotide positions analysed; % CDS, percentage of the complete gene coding sequence analysed; S, number of polymorphic sites analysed per gene; π, pairwise nucleotide diversity index; peak expression, as determined by previous microarray transcriptome analyses; dN/dS, Nei & Gojobori ratio of pairwise nucleotide diversity at nonsynonymous sites compared with synonymous sites. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002992#s2" target="_blank">Results</a> for all 2853 genes with 3 or more SNPs are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002992#pgen.1002992.s005" target="_blank">Table S2</a>.</p

Distribution of numbers of SNPs per gene for 5,056 <i>P. falciparum</i> genes analyzed with a population sample of 65 Gambian clinical isolates.

<p>Distribution of numbers of SNPs per gene for 5,056 <i>P. falciparum</i> genes analyzed with a population sample of 65 Gambian clinical isolates.</p

Transcript profiles of the six <i>msp3</i>-like genes in <i>P. falciparum</i> clinical and laboratory isolates grown to schizont stages.

<p>(A) Genomic loci of the six <i>msp3</i>-like genes on parasite chromosome 10 (nomenclature and map is based on 3D7 genome sequence version 2.1). Quantitative RT-PCR was based on non-polymorphic sequences (oligonucleotide primers and probes are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002992#pgen.1002992.s007" target="_blank">Table S4</a>). (B) Variation in relative transcript levels for the six <i>msp3</i>-like genes among 45 Gambian clinical isolates. Relative transcript levels for each gene in each isolate are normalized as a proportion of the sum for all six genes within the isolate. (C) Variation in transcript levels of the genes among 11 diverse laboratory-adapted cultured isolates. (D) Cluster analysis of expression profiles in the 45 clinical isolates and 11 laboratory-adapted isolates. Laboratory isolates are interspersed with the clinical isolates throughout, except for a divergent cluster of only clinical isolates on the right of the figure expressing little or no transcript of the <i>msp6</i> gene PF10_0346 (including one isolate that abundantly expressed the <i>h103</i>/<i>msp11</i> gene PF10_0352).</p

Distribution of Tajima's D values across all 14 chromosomes for each of the 2,853 <i>P. falciparum</i> genes with 3 or more SNPs in the Gambian population.

<p>A. All values for individual genes are plotted as individual points positioned according to the order of the genes along each of the chromosomes. B. Chromosomal locations of each of the genes with positive Tajima's D values (genes with values between zero and 1.0 are shown in grey, those with values >1.0 in black).</p