Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents

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 <five-fold decreases in parasite susceptibility. This result compares favorably against previously published non-covalent inhibitors of the Plasmodium proteasome that can select for resistant parasites with >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

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

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

PfMFR3: A multidrug-resistant modulator in Plasmodium falciparum

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-stag

Elucidating the path to Plasmodium prolyl-tRNA synthetase inhibitors that overcome halofuginone resistance

© 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).info:eu-repo/semantics/publishedVersio

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum

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

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression

The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite

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

Advances in Malaria Pharmacology and the online Guide to MALARIA PHARMACOLOGY: IUPHAR Review X

Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

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 (PfAsnRS) 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 PfAsnRS 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 PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism