Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.

Antimalarial drugs have thus far been chiefly derived from two sources—natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic-stage of the rodent malaria parasite, Plasmodium berghei, and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9,886 Diversity-Oriented Synthesis (DOS)-derived compounds. Of the compounds screened we obtain hit rates of 12–13% and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM, and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages.

Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites.