Validation of drug targets in malaria parasites predicted by metabolic modelling using CRISPR-Cas9

In recent years, despite the significant decline in number of malaria infections, malaria continues to be a threatening disease to humans with the emergence of artemisinin-resistant parasites. During the symptomatic, infection stage, malaria treatments rely on antimalarial chemotherapy to control the progress of infection. Hence, new drugs that can control infection and prevent transmission are urgently needed. In the early phase of drug discovery, metabolic modeling has been conducted to model growth at a genomic level and predict drug targets against Plasmodium falciparum, leading to the uncovering of 18 new predicted drug targets. However, those targets need to be verified by genetic or chemical target validation. Herein, two candidate drug targets; UMP-CMP kinase and dicarboxylate/ tricarboxylate carrier (DTC) protein were examined for necessity using gene knockouts by clustered regularly interspaced short palindromic repeats and Cas9 endonuclease mediated genome editing (CRISPR-Cas9). The gene disruption of both target loci along with the non-essential KAHRP gene was validated through genotyping analysis and confirmed by DNA sequencing. Subsequently, phenotypic analysis by microscopy implicated the essentiality of DTC and UMP-CMP kinase as, there were no visible viable parasites by thin blood smearing from transgenic parasites, while KAHRP2 transgenic parasites were observed on day 20 post-transfection. However, quantitative RT-qPCR data is important to validate the visual observations; comparing with the KAHRP2 transgenic cultures. Demonstrating essentiality of UMP-CMP kinase and DTC is the first step in targeting these proteins for future drug development. Furthermore, setting up the gene disruption system here could be applied to other predicted malaria drug targets in the future

CRISPR-Cas9 based validation of potential antimalarial targets

With the ability of Plasmodium falciparum, the pathogen responsible for the most mortality from malaria, to evade the front-line drug treatment artemisinin and ongoing resistance to other antimalarial drugs. There is an urgent need for continued development of new therapeutics to treat malaria and to combat the resistant parasites. In this research project, the aim was to validate UMP-CMP kinase (UCK), pivotal in the exclusive pyrimidine biosynthesis for DNA and RNA in malaria parasites, as an essential enzyme and druggable target. Herein, the essentiality of UMP-CMP kinase (UCK) gene for parasite survival and reproduction during the blood stages was discovered by using CRISPR/Cas9-based gene replacement combined with dimerisable Cre-mediated recombination. Induced deletion of the sequence encoding a catalytic domain of UCK via the Di-Cre system resulted in defective asexual growth and parasite development failure. The phenotypic analysis showed truncated parasites were arrested in the trophozoite stage and failed to progress to schizonts. The presence of an antisense circular RNA of UCK (antisense circRNA-UCK) and its cellular functions were also investigated. We hypothesised that an antisense circRNA-UCK might regulate the parental gene (UCK) expression given the coincident expression of the UCK gene initiating during early nucleic acid synthesis stages and antisense circRNA-UCK expression near the end of this stage. Experimental detection of antisense circRNA-UCK was confirmed and the antisense circRNA-UCK knockout parasite generated using CRISPR-Cas9 recombination. An antisense circRNA-UCK knockout parasite line E4 showed a significant increase in the growth rate when compared to the control parasite with two other lines (B11 and D10) slightly increased in their growth rate. This finding suggests that antisense circRNA-UCK might play a role in blood stage parasite replication although further experimentation is needed to verify these observations. In addition, recombinant PfUCK was kinetically similar in substrate preference of CMP and UMP and was more active in reducing conditions as compared to human UCK. Screens of novel compounds from in silico prediction identified the first selective PfUCK inhibitors, active at single micromolar concentrations for future lead identification and optimisation. Altogether, these data suggest that PfUCK is a promising target for antimalarial drug development

Evaluation of the Activities of Pyrimethamine Analogs against Plasmodium vivax and Plasmodium falciparum Dihydrofolate Reductase-Thymidylate Synthase Using In Vitro Enzyme Inhibition and Bacterial Complementation Assays

Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system. The 50% inhibitory concentrations derived from PvDHFR-TS-dependent bacteria were correlated with their corresponding inhibition constants (K(i)) from an enzyme inhibition assay, pointing to the likelihood that the potent enzyme inhibitors will also have potent antimalarial activities. Active compounds against both wild-type and S58R S117N (SP21) double-mutant P. vivax include analogs with structures which can avert a steric clash with the asparagine (S117N) side chain of the mutant, similar to those found for homologous Plasmodium falciparum mutants, raising the possibility that the same compounds can be developed against both types of antifolate-resistant malaria. This rapid and convenient drug screening system should be useful for development of new antifolates against P. vivax, for which a continuous culture system is not yet available

Preclinical Evaluation of the Antifolate QN254, 5-Chloro- N′6′-(2,5-Dimethoxy-Benzyl)-Quinazoline-2,4,6-Triamine, as an Antimalarial Drug Candidate▿ †

Drug resistance against dihydrofolate reductase (DHFR) inhibitors—such as pyrimethamine (PM)—has now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N′6′-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparum V1S strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparum clinical isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S) forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The drug was remarkably efficacious against Plasmodium berghei and could fully cure infected mice with three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative in vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent