Exploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome b.

Drug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype.

Lactam Truncation Yields a Dihydroquinazolinone Scaffold with Potent Antimalarial Activity that Targets PfATP4.

The emergence of resistance against current antimalarial treatments has necessitated the need for the development of novel antimalarial chemotypes. Toward this goal, we recently optimised the antimalarial activity of the dihydroquinazolinone scaffold and showed it targeted PfATP4. Here, we deconstruct the lactam moiety of the tricyclic dihydroquinazolinone scaffold and investigate the structure-activity relationship of the truncated scaffold. It was shown that SAR between scaffolds was largely transferrable and generated analogues with potent asexual stage activity. Evaluation of the truncated analogues against PfATP4 mutant drug resistant parasite strains and in assays measuring PfATP4-associated ATPase activity demonstrated retention of PfATP4 as the molecular target. Analogues exhibited activity against both male and female gametes and multidrug resistant parasites. Limited efficacy of analogues in a P. berghei asexual stage mouse model was attributed to their moderate metabolic stability and low aqueous stability. Further development is required to address these attributes toward the potential use of the dihydroquinazolinone class in a curative and transmission blocking combination antimalarial therapy.

Property and Activity Refinement of Dihydroquinazolinone-3-carboxamides as Orally Efficacious Antimalarials that Target PfATP4.

To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na+-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.

Optimization of pyrazolopyridine 4-carboxamides with potent antimalarial activity for which resistance is associated with the P. falciparum transporter ABCI3.

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class.

Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.