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Infections caused by malaria parasites place an enormous burden on the world's poorest communities. Breakthrough drugs with novel mechanisms of action are urgently needed. As an organism that undergoes rapid growth and division, the malaria parasite Plasmodium falciparum is highly reliant on protein synthesis, which in turn requires aminoacyl-tRNA synthetases (aaRSs) to charge tRNAs with their corresponding amino acid. Protein translation is required at all stages of the parasite life cycle; thus, aaRS inhibitors have the potential for whole-of-life-cycle antimalarial activity. This review focuses on efforts to identify potent plasmodium-specific aaRS inhibitors using phenotypic screening, target validation, and structure-guided drug design. Recent work reveals that aaRSs are susceptible targets for a class of AMP-mimicking nucleoside sulfamates that target the enzymes via a novel reaction hijacking mechanism. This finding opens up the possibility of generating bespoke inhibitors of different aaRSs, providing new drug leads. Expected final online publication date for the Annual Review of Microbiology, Volume 77 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5′-monophosphate–mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid–sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5′-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum , namely tyrosine RS ( Pf YRS). ML901 exerts whole-life-cycle–killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn2C6 transcription factors YRR1 and YRM1 (p −100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.

Identifying how small molecules act to kill malaria parasites can lead to new “chemically validated” targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

Development of antimalarial compounds into clinical candidates remains costly and arduous without detailed knowledge of the target. As resistance increases and treatment options at various stages of disease are limited, it is critical to identify multistage drug targets that are readily interrogated in biochemical assays. Whole-genome sequencing of 18 parasite clones evolved using thienopyrimidine compounds with submicromolar, rapid-killing, pan–life cycle antiparasitic activity showed that all had acquired mutations in the P. falciparum cytoplasmic isoleucyl tRNA synthetase (cIRS). Engineering two of the mutations into drug-naïve parasites recapitulated the resistance phenotype, and parasites with conditional knockdowns of cIRS became hypersensitive to two thienopyrimidines. Purified recombinant P. vivax cIRS inhibition, cross-resistance, and biochemical assays indicated a noncompetitive, allosteric binding site that is distinct from that of known cIRS inhibitors mupirocin and reveromycin A. Our data show that Plasmodium cIRS is an important chemically and genetically validated target for next-generation medicines for malaria.

Here we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

Here we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the β5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

Changes in host cell morphology and transcription after apicomplexan parasite infection have long been noted, but there have been few studies of the functional consequences of host cell remodeling. Here we show, using time-dependent immunofluorescence microscopy of multiple human cell lines (HepG2, HC-04, Huh7.5.1 and primary human hepatocytes), infected with multiplePlasmodiumspecies (Plasmodium berghei, P. falciparumandP. vivax(hypnozoites and schizonts)), and antibodies to multiple human proteins (HsNR4A3, HsMUC13, HsGOLGA8A, HsCGA, HsBiP, HsCXCL2), that human protein trafficking is extensively modified inPlasmodiuminfected cells. Using conventional as well as ultrastructure expansion microscopy we show that newly-synthesized human proteins are trafficked to the parasitophorous vacuole instead of the infected-cell plasma membrane, nucleus or extracellular space. Universal redirection of human signaling proteins cells the parasitophorous vacuole may provide a mechanistic explanation for how apicomplexan parasites can block host cells response to infection.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human “mammalian target of rapamycin” (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kβ) and cyclic guanosine monophosphate–dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kβ in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kβ. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kβ and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite

Current malaria treatments are threatened by drug resistance and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations inPlasmodium falciparumcyclic amine resistance locus (PfCARL) andP. falciparumacetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their target(s) are unknown. The tyramine of (S)-SW703 is shared with some reportedPfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for activity of (S)-SW703, suggesting differences in mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity on multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

Malaria is a devastating disease that resulted in an estimated 627,000 deaths in 2020. About 80% of those deaths were among children under the age of five. Our approach is to develop small molecule inhibitors against cytoskeletal targets that are vital components of parasite function, essential at multiple stages of parasite infection, can be targeted with high specificity, and are highly druggable. Here we describe KNX-115, which inhibits purified Plasmodium falciparum myosin A (PfMyoA) actin-activated ATPase with a potency in the 10s of nanomolar range and >50-fold selectivity against cardiac, skeletal, and smooth muscle myosins. KNX-115 inhibits the blood and liver stages of Plasmodium with an EC50 of about 100 nanomolar, with negligible liver cell toxicity. In addition, KNX-115 inhibits sporozoite cell traversal and blocks the gametocyte to oocyst conversion in the mosquito. KNX-115 displays a similar killing profile to pyrimethamine and parasites are totally killed after 96 hours of treatment. In line with its novel mechanism of action, KNX-115 is equally effective at inhibiting a panel of Plasmodium strains resistant to experimental and marketed antimalarials. In vitro evolution data likely suggests a refractory potential of KNX-115 in developing parasite resistance.

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 revealed a ∼5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. When challenged with KAE609, high-level resistance was obtained more rapidly and at lower inoculum than wild-type parasites. Selections were also successful with an “irresistible” compound, MMV665794 that failed to yield resistance with other strains. Mutations in a previously uncharacterized gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), were validated as causal for resistance to MMV665794 and an analog, MMV007224. The increased genetic repertoire available to this “mutator” parasite can be leveraged to drive P. falciparum resistome discovery.

Here we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,600-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 cells, which is largely driven by the accommodation of the parasite proteasome for a d-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits th...

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 (pfmdr1). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

A novel diazaspiro[3.4]octane series was identified from a Plasmodium falciparum whole-cell high-throughput screening campaign. Hits displayed activity against multiple stages of the parasite lifecycle, which together with a novel sp3-rich scaffold provided an attractive starting point for a hit-to-lead medicinal chemistry optimization and biological profiling program. Structure-activity-relationship studies led to the identification of compounds that showed low nanomolar asexual blood-stage activity (

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

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. 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.

Systems glycobiology aims to provide models and analysis tools that account for the biosynthesis, regulation, and interactions with glycoconjugates. To facilitate these methods, there is a need for a clear glycan representation accessible to both computers and humans. Linear Code, a linearized and readily parsable glycan structure representation, is such a language. For this reason, Linear Code was adapted to represent reaction rules, but the syntax has drifted from its original description to accommodate new and originally unforeseen challenges. Here, we delineate the consensuses and inconsistencies that have arisen through this adaptation. We recommend options for a consensus-based extension of Linear Code that can be used for reaction rule specification going forward. Through this extension and specification of Linear Code to reaction rules, we aim to minimize inconsistent symbology thereby making glycan database queries easier. With a clear guide for generating reaction rule descriptions, glycan synthesis models will be more interoperable and reproducible thereby moving glycoinformatics closer to compliance with FAIR standards. Here, we present Linear Code for Reaction Rules (LiCoRR), version 1.0, an unambiguous representation for describing glycosylation reactions in both literature and code.

Highlights • In-vitro evolution and whole genome sequencing elucidates gene–drug interactions. • Cellular thermal shift assay and mass spectrometry directly reveals small molecule compound–protein interactions. • Metabolomic profiling complements target identification with broad mode of action., To this day, malaria remains a global burden, affecting millions of people, especially those in sub-Saharan Africa and Asia. The rise of drug resistance to current antimalarial treatments, including artemisinin-based combination therapies, has made discovering new small molecule compounds with novel modes of action an urgent matter. The concerted effort to construct enormous compound libraries and develop high-throughput phenotypic screening assays to find compounds effective at specifically clearing malaria-causing Plasmodium parasites at any stage of the life cycle has provided many antimalarial prospects, but does not indicate their target or mode of action. Here, we review recent advances in antimalarial drug discovery efforts, focusing on the following ‘omics’ approaches in mode of action studies: IVIEWGA, CETSA, metabolomic profiling.

Malaria remains one of the most deadly infectious diseases, causing hundreds of thousands of deaths each year, primarily in young children and pregnant mothers. Here, we report the discovery and derivatization of a series of pyrazolo[3,4-b]pyridines targeting Plasmodium falciparum, the deadliest species of the malaria parasite. Hit compounds in this series display sub-micromolar in vitro activity against the intraerythrocytic stage of the parasite as well as little to no toxicity against the human fibroblast BJ and liver HepG2 cell lines. In addition, our hit compounds show good activity against the liver stage of the parasite but little activity against the gametocyte stage. Parasitological profiles, including rate of killing, docking, and molecular dynamics studies, suggest that our compounds may target the Qo binding site of cytochrome bc1.

Genetic screens in Saccharomyces cerevisiae have allowed for the identification of many genes as sensors or effectors of DNA damage, typically by comparing the fitness of genetic mutants in the presence or absence of DNA-damaging treatments. However, these static screens overlook the dynamic nature of DNA damage response pathways, missing time-dependent or transient effects. Here, we examine gene dependencies in the dynamic response to ultraviolet radiation-induced DNA damage by integrating ultra-high-density arrays of 6144 diploid gene deletion mutants with high-frequency time-lapse imaging. We identify 494 ultraviolet radiation response genes which, in addition to recovering molecular pathways and protein complexes previously annotated to DNA damage repair, include components of the CCR4-NOT complex, tRNA wobble modification, autophagy, and, most unexpectedly, 153 nuclear-encoded mitochondrial genes. Notably, mitochondria-deficient strains present time-dependent insensitivity to ultraviolet radiation, posing impaired mitochondrial function as a protective factor in the ultraviolet radiation response.

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.

Most phenotypic screens aiming to discover new antimalarial chemotypes begin with low cost, high-throughput tests against the asexual blood stage (ABS) of the malaria parasite life cycle. Compounds active against the ABS are then sequentially tested in more difficult assays that predict whether a compound has other beneficial attributes. Although applying this strategy to new chemical libraries may yield new leads, repeated iterations may lead to diminishing returns and the rediscovery of chemotypes hitting well-known targets. Here, we adopted a different strategy to find starting points, testing ∼70,000 open source small molecules from the Global Health Chemical Diversity Library for activity against the liver stage, mature sexual stage, and asexual blood stage malaria parasites in parallel. In addition, instead of using an asexual assay that measures accumulated parasite DNA in the presence of compound (SYBR green), a real time luciferase-dependent parasite viability assay was used that distinguishes slow-acting (delayed death) from fast-acting compounds. Among 382 scaffolds with the activity confirmed by dose response (

© The Author(s) 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/., 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., This work was supported by NIH R01AI143723 (R.M. and D.F.W.), NIH R01AI152533 (M.R.L. and E.A.W.), 5F31AI129412 (L.F.), and the Bill & Melinda Gates Foundation (OPP1054480, E.A.W. and D.F.W.), LEAN program of the Leducq Foundation (U.O.), Arthritis Research UK 20522 (U.O.), Cancer Research UK A23900 (U.O.). N.C.P. was supported by a National Science Foundation Graduate Research Fellowship (DGE1745303). M.R.L. was supported in part by a Ruth L. Kirschstein Institutional National Research Award from the National Institute for General Medical Sciences (T32 GM008666). This publication includes data generated at the University of California, San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929).

Phenotypic screening methods have placed numerous preclinical candidates into the antimalarial drug-discovery pipeline. As more chemically validated targets become available, efforts are shifting to target-based drug discovery. Here, we briefly review some of the most attractive targets that have been identified in recent years.

There is a shift in antimalarial drug discovery from phenotypic screening toward target-based approaches, as more potential drug targets are being validated in Plasmodium species. Given the high attrition rate and high cost of drug discovery, it is important to select the targets most likely to deliver progressible drug candidates. In this paper, we describe the criteria that we consider important for selecting targets for antimalarial drug discovery. We describe the analysis of a number of drug targets in the Malaria Drug Accelerator (MalDA) pipeline, which has allowed us to prioritize targets that are ready to enter the drug discovery process. This selection process has also highlighted where additional data are required to inform target progression or deprioritization of other targets. Finally, we comment on how additional drug targets may be identified.

Novel bis-1,2,4-triazine compounds with potent in vitro activity against Plasmodium falciparum parasites were recently identified. The bis-1,2,4-triazines represent a unique antimalarial pharmacophore and are proposed to act by a novel but as-yet-unknown mechanism of action. This study investigated the activity of the bis-1,2,4-triazine MIPS-0004373 across the mammalian life cycle stages of the parasite and profiled the kinetics of activity against blood and transmission stage parasites in vitro and in vivo. MIPS-0004373 demonstrated rapid and potent activity against P. falciparum, with excellent in vitro activity against all asexual blood stages. Prolonged in vitro drug exposure failed to generate stable resistance de novo, suggesting a low propensity for the emergence of resistance. Excellent activity was observed against sexually committed ring stage parasites, but activity against mature gametocytes was limited to inhibiting male gametogenesis. Assessment of liver stage activity demonstrated good activity in an in vitro P. berghei model but no activity against Plasmodium cynomolgi hypnozoites or liver schizonts. The bis-1,2,4-triazine MIPS-0004373 efficiently cleared an established P. berghei infection in vivo, with efficacy similar to that of artesunate and chloroquine and a recrudescence profile comparable to that of chloroquine. This study demonstrates the suitability of bis-1,2,4-triazines for further development toward a novel treatment for acute malaria.

Significance Here, we describe inhibitors of the Plasmodium proteasome, an enzymatic complex that malaria parasites rely on to degrade proteins. Starting from inhibitors developed to treat cancer, derivatives were designed and synthesized with the aim of increasing potency against the Plasmodium proteasome and decreasing activity against the human enzyme. Biochemical and cellular assays identified compounds that exhibit selectivity and potency, both in vitro and in vivo, at different stages of the parasite’s lifecycle. Cryo-electron microscopy revealed that the inhibitors bind in a hydrophobic pocket that is structurally different in the human proteasome—underpinning their selectivity. The work will help develop antimalarial therapeutics, which are desperately needed to treat a disease that kills nearly half a million people annually., The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) β5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax. They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

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 EC 50 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 .

Spreading antimalarial resistance threatens effective treatment of malaria, an infectious disease caused by Plasmodium parasites. We identified a compound, BCH070, that inhibits asexual growth of multiple antimalarial-resistant strains of Plasmodium falciparum (half maximal inhibitory concentration [IC50] = 1–2 µM), suggesting that BCH070 acts via a novel mechanism of action. BCH070 preferentially kills early ring-form trophozoites, and, importantly, equally inhibits ring-stage survival of wild-type and artemisinin-resistant parasites harboring the PfKelch13:C580Y mutation. Metabolomic analysis demonstrates that BCH070 likely targets multiple pathways in the parasite. BCH070 is a promising lead compound for development of new antimalarial combination therapy that retains activity against artemisinin-resistant parasites.

A major advance in antimalarial drug discovery has been the shift towards cell-based phenotypic screening, with notable progress in the screening of compounds against the asexual blood stage, liver stage, and gametocytes. A primary method for drug target deconvolution in Plasmodium falciparum is in vitro evolution of compound-resistant parasites followed by whole-genome scans. Several of the most promising antimalarial drug targets, such as translation elongation factor 2 (eEF2) and phenylalanine tRNA synthetase (PheRS), have been identified or confirmed using this method. One drawback of this method is that if a mutated gene is uncharacterized, a substantial effort may be required to determine whether it is a drug target, a drug resistance gene, or if the mutation is merely a background mutation. Thus, the availability of high-throughput, functional genomic datasets can greatly assist with target deconvolution. Studies mapping genome-wide essentiality in P. falciparum or performing transcriptional profiling of the host and parasite during liver-stage infection with P. berghei have identified potentially druggable pathways. Advances in mapping the epigenomic regulation of the malaria parasite genome have also enabled the identification of key processes involved in parasite development. In addition, the examination of the host genome during infection has identified novel gene candidates associated with susceptibility to severe malaria. Here, we review recent studies that have used omics-based methods to identify novel targets for interventions against protozoan parasites, focusing on malaria, and we highlight the advantages and limitations of the approaches used. These approaches have also been extended to other protozoan pathogens, including Toxoplasma, Trypanosoma, and Leishmania spp., and these studies highlight how drug discovery efforts against these pathogens benefit from the utilization of diverse omics-based methods to identify promising drug targets.

Herein we describe the optimization of a phenotypic hit against Plasmodium falciparum based on an aminoacetamide scaffold. This led to N‐(3‐chloro‐4‐fluorophenyl)‐2‐methyl‐2‐{[4‐methyl‐3‐(morpholinosulfonyl)phenyl]amino}propanamide (compound 28) with low‐nanomolar activity against the intraerythrocytic stages of the malaria parasite, and which was found to be inactive in a mammalian cell counter‐screen up to 25 μm. Inhibition of gametes in the dual gamete activation assay suggests that this family of compounds may also have transmission blocking capabilities. Whilst we were unable to optimize the aqueous solubility and microsomal stability to a point at which the aminoacetamides would be suitable for in vivo pharmacokinetic and efficacy studies, compound 28 displayed excellent antimalarial potency and selectivity; it could therefore serve as a suitable chemical tool for drug target identification., New roads to novel chemotypes: Compound selection from the TCAMS library (GSK), followed by iterative rounds of inhibitor design and synthesis afforded a single‐digit nanomolar inhibitor of P. falciparum (3D7), based on an aminoacetamide core. Compound 28 is a potent antimalarial and displayed excellent selectivity in a mammalian counter‐screen. This compound could be used as a suitable chemical tool for drug target identification or as a lead compound for further optimization.

Plasmodium falciparum and Plasmodium vivax, the two protozoan parasite species that cause the majority of cases of human malaria, have developed resistance to nearly all known antimalarials. The ability of malaria parasites to develop resistance is primarily due to the high numbers of parasites in the infected person’s bloodstream during the asexual blood stage of infection in conjunction with the mutability of their genomes. Identifying the genetic mutations that mediate antimalarial resistance has deepened our understanding of how the parasites evade our treatments and reveals molecular markers that can be used to track the emergence of resistance in clinical samples. In this review, we examine known genetic mutations that lead to resistance to the major classes of antimalarial medications: the 4-aminoquinolines (chloroquine, amodiaquine and piperaquine), antifolate drugs, aryl amino-alcohols (quinine, lumefantrine and mefloquine), artemisinin compounds, antibiotics (clindamycin and doxycycline) and a napthoquinone (atovaquone). We discuss how the evolution of antimalarial resistance informs strategies to design the next generation of antimalarial therapies.

A series of novel 8-aminoquinolines (8-AQs) with an aminoxyalkyl side chain were synthesized and evaluated for in vitro antiplasmodial properties against asexual blood stages, liver stages, and sexual stages of Plasmodium falciparum. 8-AQs bearing 2-alkoxy and 5-phenoxy substituents on the quinoline ring system were found to be the most promising compounds under study, exhibiting potent blood schizontocidal and moderate tissue schizontocidal in vitro activity.

The exoerythrocytic stage of Plasmodium infection is a critical window for prophylactic intervention. Using genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we show that the human mucosal immunity gene, mucin-13 (MUC13), is strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm MUC13 transcript increases in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, marking both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes. This data provides insights into host-parasite interactions in Plasmodium infection, and demonstrates that a component of host mucosal immunity is reprogrammed during the progression of infection., Host-parasite interactions during the exoerythrocytic stage of Plasmodium infection remains poorly understood. Using dual RNA-Seq, the authors show that human mucosal immunity protein mucin-13 is upregulated during Plasmodium hepatic-stage infection and marks infected cells independent of tested Plasmodium species.

Summary Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stage P. falciparum parasites in the low nanomolar to low micromolar range. In order to understand their mechanism of action, parasites from two different genetic backgrounds were exposed to sublethal concentrations of MMV085203 and GNF-Pf-3600 until resistance emerged. Whole genome sequencing revealed all 17 resistant clones acquired nonsynonymous mutations in the gene encoding the orphan apicomplexan transporter PF3D7_0312500 (pfmfr3) predicted to encode a member of the major facilitator superfamily (MFS). Disruption of pfmfr3 and testing against a panel of antimalarial compounds showed decreased sensitivity to MMV085203 and GNF-Pf-3600 as well as other compounds that have a mitochondrial mechanism of action. In contrast, mutations in pfmfr3 provided no protection against compounds that act in the food vacuole or the cytosol. A dihydroorotate dehydrogenase rescue assay using transgenic parasite lines, however, indicated a different mechanism of action for both MMV085203 and GNF-Pf-3600 than the direct inhibition of cytochrome bc1. Green fluorescent protein (GFP) tagging of PfMFR3 revealed that it localizes to the parasite mitochondrion. Our data are consistent with PfMFR3 playing roles in mitochondrial transport as well as drug resistance for clinically relevant antimalarials that target the mitochondria. Furthermore, given that pfmfr3 is naturally polymorphic, naturally occurring mutations may lead to differential sensitivity to clinically relevant compounds such as atovaquone.

SummaryIn vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in the model microbe, Saccharomyces cerevisiae. Analysis of 355 curated, laboratory-evolved clones, resistant to 80 different compounds, demonstrates differences in the types of mutations that are identified in selected versus neutral evolution and reveals numerous new, compound-target interactions. Through enrichment analysis we further identify a set of 137 genes strongly associated with or conferring drug resistance as indicated by CRISPR-Cas9 engineering. The set of 25 most frequently mutated genes was enriched for transcription factors and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function, single nucleotide variants found in 170-amino-acid domains in two Zn2C6 transcription factors, YRR1 and YRM1 (p < 1x 10 −100). This remarkable enrichment for transcription factors as drug resistance genes may explain why it is challenging to develop effective antifungal killing agents and highlights their important role in evolution.

A novel diazaspiro[3.4]octane series was identified from a

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages., Here, Reader et al. screen the Medicines for Malaria Venture Pandemic Response Box in parallel against Plasmodiumasexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. They identify two potent transmission-blocking drugs: a histone demethylase inhibitor ML324 and the antitubercular SQ109.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) beta5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Tres Cantos Open Lab is a collaborative initiative that integrates teams from academia and GlaxoSmithKline to enable rapid testing of innovative therapeutic hypotheses for endemic infectious diseases. Here, we provide an overview of the key scientific achievements in its first decade. Tres Cantos Open Lab is a collaborative initiative that integrates teams from academia and GlaxoSmithKline to enable rapid testing of innovative therapeutic hypotheses for endemic infectious diseases. Here, we provide an overview of the key scientific achievements in its first decade.

Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway., Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparum.