Biology subjects

Luth, M. R.

Publications and source records attributed to Luth, M. R..

6 recordsLinked to original sources

bioRxiv2025

Mechanistic Insights into Dual-Active Liver and Blood-Stage Antiplasmodials

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.

microbiology↗

bioRxiv2024

A novel chemogenomic screening platform for scalable antimalarial drug target identification

Large-scale chemical-genetic screening, or chemogenomics, can faciliate rapid and scalable drug target identification. To establish a chemogenomic screen for antimalarial drug target identification, we leveraged [~]600 Plasmodium berghei artificial chromosomes (PbACs) encoding potential drug targets to generate a systematic overexpression library. PbACs were engineered with DNA barcodes, enabling their quantification within mixed pools using next generation sequencing (barcode sequencing or BarSeq). Pooled transfection of PbACs into the highly genetically tractable Plasmodium knowlesi demonstrated efficient vector uptake and transcription of encoded P. berghei genes. Parasite pools were exposed to antimalarial candidates, with pilot screens probing for known gene-compound associations identifying their targets with high sensitivity. Screening antimalarial inhibitors with unknown mechanisms of action successfully identified pi4k as the target for one novel compound, which was subsequently validated using in vitro evolution in Plasmodium falciparum parasites. This sensitive and scalable chemogenomics platform therefore represents a valuable early-stage tool for antimalarial target identification.

microbiology↗

bioRxiv2022

Collateral sensitivity as a strategy to suppress resistance emergence: the challenge of diverse evolutionary pathways

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.

microbiology↗

bioRxiv2021

Defining the Yeast Resistome through in vitro Evolution and Whole Genome Sequencing

In 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.

genetics↗

bioRxiv2019

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii.

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 T. gondii, a related apicomplexan parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing drug concentrations and whole genome sequencing identified several non-conservative 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 acids 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 mitochondrial genome, including genes encoding cytochrome b and cytochrome oxidase I. Consistent with prior studies in yeast and mammalian tumor cells that implicate the mitochondrion as a target of artemisinins, treatment of wild type parasites with artemisinin decreased mitochondrial membrane potential, and resistant parasites showed altered morphology and decreased membrane potential. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition in T. gondii.\n\nSignificanceArtemisinins provide important therapeutic agents for treatment of malaria and have potential for use in other infections and in cancer. Their use is threatened by the potential for resistance development, so understanding their mechanism of action and identifying genetic changes that alter sensitivity are important for improving clinical outcomes. Our findings suggest that mutations in novel targets can contribute to the emergence of parasites with increased tolerance to artemisinin treatment and that such mutations can confer a fitness advantage even in the absence of a notable shift in EC50. Our findings also support the idea that inhibition of mitochondrial function may be an important target in T. gondii, as previously suggested by studies in yeast and human cancer cells.

microbiology↗

bioRxiv2019

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

AO_SCPLOWBSTRACTC_SCPLOWOne of the most promising new compound classes in clinical development for the treatment of malaria is the imidazolopiperazines (IZPs) class. Human trials have demonstrated that members of the IZP series, which includes KAF156 (Ganaplacide) and GNF179, are potent and effective against Plasmodium symptomatic asexual blood-stage infections. Unlike other commonly used antimalarials, they also prevent transmission and block future infection in animal models. Despite the identification of several Plasmodium falciparum resistance mechanisms including mutations in ER-localized PfCARL (PfEMP65), Acetyl-coA transporter, and PfUGT transporter, IZPs mechanism of action remains unknown.\n\nTo investigate, we combined in vitro evolution and whole-genome analysis in the model organism Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods, in P. falciparum. S. cerevisiae clones that resist IZP activity carry multiple mutations in genes that encode endoplasmic reticulum(ER)-based lipid homeostasis and autophagy including elo2, elo3, sur2, atg15 and lcb4, as well as ER-based sec66. In Plasmodium, IZPs cause inhibition of protein trafficking, block the establishment of new permeation pathways and result in ER expansion. We also observe sensitization with other secretion inhibitors such as brefeldin A and golgicidin as well as synthetic lethality with PfSEC62. Our data show that IZPs target the secretory pathway and highlight a novel mechanism for blocking parasite growth and development that is distinct from those of standard compounds used to treat malaria. In addition, we provide physiological signatures and hallmarks for inhibitors that work through this mechanism of action and show that IZPs are tool compounds for studying ER-dependent protein processing in different species.

microbiology↗