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Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria.

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5–8 fold elevation in the mutation rate, with an increase of 13–28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an “irresistible” compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this “ mutator” parasite can be leveraged to drive P. falciparum resistome discovery. The ability to evolve Plasmodium drug resistance in vitro is challenging and time consuming. Here, Kümpornsin et al. generated a Plasmodium falciparum parasite line with an elevated mutation rate by impairing the proof-reading activity of DNA polymerase, which results in a higher mutation rate, quick resistance development, and a lower inoculum than wild type to support the identification of new antimalarial targets and understand drug resistance mechanisms.

The development of next-generation antimalarials that are efficacious against the human liver and asexual blood stages is recognized as one of the world’s most pressing public health challenges. In recent years, aminoacyl-tRNA synthetases, including prolyl-tRNA synthetase, have emerged as attractive targets for malaria chemotherapy. We describe the development of a single-step biochemical assay for Plasmodium and human prolyl-tRNA synthetases that overcomes critical limitations of existing technologies and enables quantitative inhibitor profiling with high sensitivity and flexibility. Supported by this assay platform and co-crystal structures of representative inhibitor-target complexes, we develop a set of high-affinity prolyl-tRNA synthetase inhibitors, including previously elusive aminoacyl-tRNA synthetase triple-site ligands that simultaneously engage all three substrate-binding pockets. Several compounds exhibit potent dual-stage activity against Plasmodium parasites and display good cellular host selectivity. Our data inform the inhibitor requirements to overcome existing resistance mechanisms and establish a path for rational development of prolyl-tRNA synthetase-targeted anti-malarial therapies. The development of antimalarials against the human liver and asexual blood stages is one of the top public health challenges. Here, the authors report a single-step biochemical assay for the characterization of prolyl-tRNA synthetase inhibitors, and develop high-affinity inhibitors for the enzyme, including elusive triple-site ligands.

Primary Amoebic Meningoencephalitis (PAM) is caused by Naegleria fowleri, a free-living amoeba that occasionally infects humans. While considered \"rare\" (but likely underreported) the high mortality rate and lack of established success in treatment makes PAM a particularly devastating infection. In the absence of economic inducements to invest in development of anti-PAM drugs by the pharmaceutical industry, anti-PAM drug discovery largely relies on drug 'repurposing'-a cost effective strategy to apply known drugs for treatment of rare or neglected diseases. Similar to fungi, N. fowleri has an essential requirement for ergosterol, a building block of plasma and cell membranes. Disruption of sterol biosynthesis by small-molecule inhibitors is a validated interventional strategy against fungal pathogens of medical and agricultural importance. The N. fowleri genome encodes the sterol 14-demethylase (CYP51) target sharing ~35% sequence identity to fungal orthologues. The similarity of targets raises the possibility of repurposing anti-mycotic drugs and optimization of their usage for the treatment of PAM. In this work, we (i) systematically assessed the impact of anti-fungal azole drugs, known as conazoles, on sterol biosynthesis and viability of cultured N. fowleri trophozotes, (ii) identified the endogenous CYP51 substrate by mass spectrometry analysis of N. fowleri lipids, and (iii) analyzed the interactions between the recombinant CYP51 target and conazoles by UV-vis spectroscopy and x-ray crystallography. Collectively, the target-based and parasite-based data obtained in these studies validated CYP51 as a potentially 'druggable' target in N. fowleri, and conazole drugs as the candidates for assessment in the animal model of PAM.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum , the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum . Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium , IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing. Imidazolopiperazines (IZPs) are a class of compounds under clinical development for malaria, but their mechanism of action is unclear. Here, the authors show that IZPs inhibit the parasite’s secretory pathway, affecting protein trafficking and export.

Despite recent progress in the development of the next generation of antimalarial drug candidates, the risk of resistance remains. Thus, the identification of new targets that are essential at multiple stages of the malaria parasite lifecycle is an important priority. Validation of targets essential for the liver-stage not only would be relevant for prophylaxis but may also limit the potential generation of resistance due to the lower parasite numbers encountered at this stage. Here, we have leveraged compounds with dual liver- and blood-stage activity, combined with evolution of resistance, to chemically validate targets with multi-stage essentiality. We successfully evolved resistance to four, which we further characterize by genome sequencing, CRISPR editing and biochemical assays. The remaining five resistance-refractory compounds showed no interactions when profiled against a barcoded parasite library representing >30 known modes of action, suggesting these may have novel targets and represent interesting starting points for further exploration.

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. In six selections with TCMDC-125334, the most common mechanism of resistance to this compound was copy number variation of the dhodh locus, although we did identify one mutation, DHODH I263S, which conferred resistance to TCMDC-125334 but not DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme. Malaria affects around 240 million people around the world every year. The microscopic parasite responsible for the disease are carried by certain mosquitoes and gets transmitted to humans through bites. These parasites are increasingly acquiring genetic mutations that make anti-malaria medication less effective, creating an urgent need for alternative treatment approaches. Several new malaria drugs being explored in preclinical research work by binding to an enzyme known as DHODH and preventing it from performing its usual role in the parasite. Previous work found that, in some cases, malaria parasites that evolved resistance to one type of DHODH inhibitor (by acquiring mutations in their DHODH enzyme) then became more vulnerable to another kind. It may be possible to leverage this ‘collateral sensitivity’ by designing treatments which combine two DHODH inhibitors and therefore make it harder for the parasites to evolve resistance. To investigate this possibility, Mandt et al. first tested several DHODH inhibitors to find the one that was most potent against drug-resistant parasites. In subsequent experiments, they combined TCMDC-125334, the best candidate that emerged from these tests, with a DHODH inhibitor that works well against vulnerable parasites. However, the parasites still rapidly evolved resistance. Further work identified a new DHODH mutation that allowed the parasites to evade both drugs simultaneously. Together, these findings suggest that the DHODH enzyme may not be the best target for new malaria drugs because many it can acquire many possible mutations that confer resistance. Such results may inform other studies that aim to harness collateral sensitivity to fight against a range of harmful agents.

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance in Plasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability of Toxoplasma gondii, a related parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing concentrations and whole-genome sequencing identified several nonconservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acid changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild-type parasites in the presence of drug, despite not changing EC50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondria genome, including genes encoding cytochrome b and cytochrome c oxidase I. Prior studies in yeast and mammalian tumor cells implicate the mitochondrion as a target of artemisinins, and treatment of wild-type parasites with high concentrations of drug decreased mitochondrial membrane potential, a phenotype that was stably altered in the resistant parasites. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition of resistance in T. gondii.

Malaria parasites are evolutionarily prepared to resist drug attack. Resistance is emerging to even the latest frontline combination therapies, which target the blood stages of the Plasmodium parasite. As an alternative strategy, Antonova-Koch et al. investigated the possibilities of drugs against liver-stage parasites (see the Perspective by Phillips and Goldberg). To do so, they devised a luciferase-reporter drug screen for the rodent parasite Plasmodium berghei. Three rounds of increasingly stringent screening were used. From this regime, several chemotypes that inhibit Plasmodium mitochondrial electron transport were identified. Excitingly, several new scaffolds, with as-yet-unknown modes of action but solely targeting the parasites' liver stages, emerged as promising drug leads for further development. Science , this issue p. eaat9446 ; see also p. 1112 Screening of more than half a million compounds for their ability to inhibit liver-stage Plasmodium development yields thousands of candidates. To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase ( Pf AsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of Pf AsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound Pf AsnRS provide insights into the structure-activity relationship and the selectivity mechanism. New antimalarials are urgently needed. Here, the authors identify Open Source Malaria compound, OSMS-106, as a reaction hijacking inhibitor of the malaria parasite protein synthesis machinery, with potential use for treatment and prophylaxis.