Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential anti-malarials we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound towards clinical candidate status.

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication.

Ideally antimalarial drugs can be developed which target multiple life cycle stages, thus impacting prevention, treatment and transmission of disease. Here we introduce 4-(1H)-quinolone-3-diarylethers that are selectively potent inhibitors of the parasite’s mitochondrial cytochrome bc1 complex. These compounds are highly active against the primary human malarias (falciparum and vivax), targeting the parasite at both the liver and blood stages as well as the forms that are crucial to disease transmission: gametocytes ⇒ zygotes ⇒ ookinetes ⇒ oocysts. Chosen as the preclinical candidate, ELQ-300 has good oral bioavailability at efficacious dosages in mice, is metabolically stable, and is highly active in rodent malaria models. Given a low predicted dose in patients and a long predicted half-life, ELQ-300 offers the hope of a new molecule for the treatment, prevention and, ultimately, eradication of malaria.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapsein a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.