On-going malaria transmission in The Gambia despite high coverage of control interventions: a nationwide cross-sectional survey

Background: As indicators of burden of malaria have substantially decreased in The Gambia, reaching a pre-elimination status may be attainable. Achieving this goal requires in-depth understanding of the current burden of Plasmodium falciparum infection. Methods: A nationwide cross-sectional survey was conducted in 2012 to determine the prevalence of P. falciparum infection, and to describe its heterogeneity and associated risk factors. Finger-prick blood samples were collected for microscopy, species-specific PCR and haemoglobin measurement. Results: A total of 9,094 participants were included and median age was 11.9 years (IQR 5, 28). Overall prevalence of P. falciparum was 16.01 % with marked heterogeneity between sites (4.32-36.75 %) and within villages in each site (1.63-49.13 %). Across all sites, 51.17 % (745/1,456) of infections were asymptomatic and 35.61 % (448/1,258) were sub-microscopic. The odds of P. falciparum infection were higher in older children; 5-15 years (OR = 1.90; 95 % CI 1.60-2.26), adults (OR = 1.48; 95 % CI 1.24-1.78) and participants with moderate anaemia (OR = 1.62; 95 % CI 1.32-1.99). Conclusions: The current malaria control interventions are not sufficient to interrupt transmission in The Gambia as malaria prevalence is still relatively high in the eastern part of the country. New interventions aiming at interrupting transmission are needed and should be urgently evaluated

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

Development of Potent and Selective PfCLK3 Inhibitors Based on GSK-TCMDC151 as a New Class of Antimalarials

The kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug target in malaria that offers prophylactic, transmission blocking and curative potential. Herein we describe the synthesis of our initial hit TCMDC-135051 1and efforts to establish a SAR with a 7-azaindole-based series. A total of 14 analogues were assessed in a TR-FRET assay against the full recombinant protein kinase PfCLK3 and 10 were further assessed in parasites 3D7 (chloroquine sensitive) strains of P. falciparum. SAR relating to rings A and B was established. These data suggest that TCMDC-135051 1is a promising lead compound for the development of new antimalarials with a novel mechanism of action targeting PfCLK3.</p

Development of Potent PfCLK3 Inhibitors Based on TCMDC-135051 as a New Class of Antimalarials.

The protein kinase PfCLK3 plays a critical role in the regulation of malarial parasite RNA splicing and is essential for the survival of blood stage Plasmodium falciparum. We recently validated PfCLK3 as a drug target in malaria that offers prophylactic, transmission blocking, and curative potential. Herein, we describe the synthesis of our initial hit TCMDC-135051 (1) and efforts to establish a structure-activity relationship with a 7-azaindole-based series. A total of 14 analogues were assessed in a time-resolved fluorescence energy transfer assay against the full-length recombinant protein kinase PfCLK3, and 11 analogues were further assessed in asexual 3D7 (chloroquine-sensitive) strains of P. falciparum parasites. SAR relating to rings A and B was established. These data together with analysis of activity against parasites collected from patients in the field suggest that TCMDC-135051 (1) is a promising lead compound for the development of new antimalarials with a novel mechanism of action targeting PfCLK3

Peptides derived from the SARS-CoV-2 receptor binding motif bind to ACE2 but do not block ACE2-mediated host cell entry or pro-inflammatory cytokine induction

SARS-CoV-2 viral attachment and entry into host cells is mediated by a direct interaction between viral spike glycoproteins and membrane bound angiotensin-converting enzyme 2 (ACE2). The receptor binding motif (RBM), located within the S1 subunit of the spike protein, incorporates the majority of known ACE2 contact residues responsible for high affinity binding and associated virulence. Observation of existing crystal structures of the SARS-CoV-2 receptor binding domain (SRBD)–ACE2 interface, combined with peptide array screening, allowed us to define a series of linear native RBM-derived peptides that were selected as potential antiviral decoy sequences with the aim of directly binding ACE2 and attenuating viral cell entry. RBM1 (16mer): S443KVGGNYNYLYRLFRK458, RBM2A (25mer): E484GFNCYFPLQSYGFQPTNGVGYQPY508, RBM2B (20mer): F456NCYFPLQSYGFQPTNGVGY505 and RBM2A-Sc (25mer): NYGLQGSPFGYQETPYPFCNFVQYG. Data from fluorescence polarisation experiments suggested direct binding between RBM peptides and ACE2, with binding affinities ranging from the high nM to low μM range (Kd = 0.207–1.206 μM). However, the RBM peptides demonstrated only modest effects in preventing SRBD internalisation and showed no antiviral activity in a spike protein trimer neutralisation assay. The RBM peptides also failed to suppress S1-protein mediated inflammation in an endogenously expressing ACE2 human cell line. We conclude that linear native RBM-derived peptides are unable to outcompete viral spike protein for binding to ACE2 and therefore represent a suboptimal approach to inhibiting SARS-CoV-2 viral cell entry. These findings reinforce the notion that larger biologics (such as soluble ACE2, ‘miniproteins’, nanobodies and antibodies) are likely better suited as SARS-CoV-2 cell-entry inhibitors than short-sequence linear peptides

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

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