Polymorphism in dhfr/dhps genes, parasite density and ex vivo response to pyrimethamine in Plasmodium falciparum malaria parasites in Thies, Senegal☆

Resistance to sulfadoxine–pyrimethamine (SP) in Plasmodium falciparum malaria parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes, and these mutations have spread resistance worldwide. SP, used for several years in Senegal, has been recommended for intermittent preventive treatment for malaria in pregnancy (IPTp) and has been widely implemented since 2003 in this country. There is currently limited data on SP resistance from molecular marker genotyping, and no data on pyrimethamine ex vivo sensitivity in Senegal. Molecular markers of SP resistance and pyrimethamine ex vivo sensitivity were investigated in 416 parasite samples collected from the general population, from the Thies region between 2003 and 2011. The prevalence of the N51I/C59R/S108N triple mutation in dhfr increased from 40% in 2003 to 93% in 2011. Furthermore, the prevalence of the dhfr N51I/C59R/S108N and dhps A437G quadruple mutation increased, from 20% to 66% over the same time frame, then down to 44% by 2011. There was a significant increase in the prevalence of the dhfr triple mutation, as well as an association between dhfr genotypes and pyrimethamine response. Conversely, dhps mutations in codons 436 and 437 did not show consistent variation between 2003 and 2011. These findings suggest that regular screening for molecular markers of antifolate resistance and ex vivo drug response monitoring should be incorporated with ongoing in vivo efficacy monitoring in areas where IPTp-SP is implemented and where pyrimethamine and sulfa drugs are still widely administered in the general population

Changes in drug sensitivity and anti-malarial drug resistance mutations over time among Plasmodium falciparum parasites in Senegal

Background: Malaria treatment efforts are hindered by the rapid emergence and spread of drug resistant parasites. Simple assays to monitor parasite drug response in direct patient samples (ex vivo) can detect drug resistance before it becomes clinically apparent, and can inform changes in treatment policy to prevent the spread of resistance. Methods: Parasite drug responses to amodiaquine, artemisinin, chloroquine and mefloquine were tested in approximately 400 Plasmodium falciparum malaria infections in Thiès, Senegal between 2008 and 2011 using a DAPI-based ex vivo drug resistance assay. Drug resistance-associated mutations were also genotyped in pfcrt and pfmdr1. Results: Parasite drug responses changed between 2008 and 2011, as parasites became less sensitive to amodiaquine, artemisinin and chloroquine over time. The prevalence of known resistance-associated mutations also changed over time. Decreased amodiaquine sensitivity was associated with sustained, highly prevalent mutations in pfcrt, and one mutation in pfmdr1 – Y184F – was associated with decreased parasite sensitivity to artemisinin. Conclusions: Directly measuring ex vivo parasite drug response and resistance mutation genotyping over time are useful tools for monitoring parasite drug responses in field samples. Furthermore, these data suggest that the use of amodiaquine and artemisinin derivatives in combination therapies is selecting for increased drug tolerance within this population

Identification of Collateral Sensitivity to Dihydroorotate Dehydrogenase Inhibitors in Plasmodium falciparum

Drug resistance has been reported for every antimalarial in use highlighting the need for new strategies to protect the efficacy of therapeutics in development. We have previously shown that resistance can be suppressed with a population biology trap: by identifying situations where resistance to one compound confers hypersensitivity to another (collateral sensitivity), we can design combination therapies that not only kill the parasite but also guide its evolution away from resistance. We applied this concept to the Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) enzyme, a well validated antimalarial target with inhibitors in the development pipeline. Here, we report a high-throughput screen to identify compounds specifically active against PfDHODH resistant mutants. We additionally perform extensive cross-resistance profiling allowing us to identify compound pairs demonstrating the potential for mutually incompatible resistance. These combinations represent promising starting points for exploiting collateral sensitivity to extend the useful lifespan of new antimalarial therapeutics

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

Sequence-Based Association and Selection Scans Identify Drug Resistance Loci in the Plasmodium Falciparum Malaria Parasite

Through rapid genetic adaptation and natural selection, the Plasmodium falciparum parasite—the deadliest of those that cause malaria—is able to develop resistance to antimalarial drugs, thwarting present efforts to control it. Genome-wide association studies (GWAS) provide a critical hypothesis-generating tool for understanding how this occurs. However, in P. falciparum, the limited amount of linkage disequilibrium hinders the power of traditional array-based GWAS. Here, we demonstrate the feasibility and power improvements gained by using whole-genome sequencing for association studies. We analyzed data from 45 Senegalese parasites and identified genetic changes associated with the parasites’ in vitro response to 12 different antimalarials. To further increase statistical power, we adapted a common test for natural selection, XP-EHH (cross-population extended haplotype homozygosity), and used it to identify genomic regions associated with resistance to drugs. Using this sequence-based approach and the combination of association and selection-based tests, we detected several loci associated with drug resistance. These loci included the previously known signals at pfcrt, dhfr, and pfmdr1, as well as many genes not previously implicated in drug-resistance roles, including genes in the ubiquitination pathway. Based on the success of the analysis presented in this study, and on the demonstrated shortcomings of array-based approaches, we argue for a complete transition to sequence-based GWAS for small, low linkage-disequilibrium genomes like that of P. falciparum.Molecular and Cellular BiologyOrganismic and Evolutionary Biolog

Prioritization of Molecular Targets for Antimalarial Drug Discovery

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

Potent acyl-CoA synthetase 10 inhibitors kill <i>Plasmodium falciparum</i> by disrupting triglyceride formation

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&nbsp;stability

Identification and Functional Validation of the Novel Antimalarial Resistance Locus PF10_0355 in Plasmodium falciparum

The Plasmodium falciparum parasite's ability to adapt to environmental pressures, such as the human immune system and antimalarial drugs, makes malaria an enduring burden to public health. Understanding the genetic basis of these adaptations is critical to intervening successfully against malaria. To that end, we created a high-density genotyping array that assays over 17,000 single nucleotide polymorphisms (~1 SNP/kb), and applied it to 57 culture-adapted parasites from three continents. We characterized genome-wide genetic diversity within and between populations and identified numerous loci with signals of natural selection, suggesting their role in recent adaptation. In addition, we performed a genome-wide association study (GWAS), searching for loci correlated with resistance to thirteen antimalarials; we detected both known and novel resistance loci, including a new halofantrine resistance locus, PF10_0355. Through functional testing we demonstrated that PF10_0355 overexpression decreases sensitivity to halofantrine, mefloquine, and lumefantrine, but not to structurally unrelated antimalarials, and that increased gene copy number mediates resistance. Our GWAS and follow-on functional validation demonstrate the potential of genome-wide studies to elucidate functionally important loci in the malaria parasite genome.Bill & Melinda Gates FoundationEllison Medical FoundationExxon Mobil FoundationFogarty International CenterNational Institute of Allergy and Infectious Diseases (U.S.)Burroughs Wellcome FundDavid & Lucile Packard FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra

Diversity-Oriented Synthesis Yields a Novel Lead for the Treatment of Malaria

Here, we describe the discovery of a novel antimalarial agent using phenotypic screening of Plasmodium falciparum asexual blood-stage parasites. Screening a novel compound collection created using diversity-oriented synthesis (DOS) led to the initial hit. Structure–activity relationships guided the synthesis of compounds having improved potency and water solubility, yielding a subnanomolar inhibitor of parasite asexual blood-stage growth. Optimized compound 27 has an excellent off-target activity profile in erythrocyte lysis and HepG2 assays and is stable in human plasma. This compound is available via the molecular libraries probe production centers network (MLPCN) and is designated ML238.Chemistry and Chemical Biolog