Biology subjects

Ottilie, S.

Publications and source records attributed to Ottilie, S..

6 recordsLinked to original sources

bioRxiv2025

Shared Binding Site but Divergent Resistance Profiles Uncover Novel Resistance Mechanisms in Plasmodium HSP90 Inhibitors

Drug resistance is a widespread problem across therapeutic areas including malaria, but what accounts for resistance propensity remains poorly understood. Here, we reveal that two HSP90 inhibitors targeting the identical ATP-binding site exhibit dramatically different resistance profiles in P. falciparum. Geldanamycin readily selected 10 distinct resistance mutations conferring up to 22-fold resistance, while AUY-922 required 44 weeks to yield a single A41S mutation with only 2-fold resistance to AUY-922 but not to geldanamycin. Resistance mapping in parasites and yeast revealed geldanamycin resistance mutations distributed throughout the binding pocket whereas AUY-922 resistance mutations localized close to the ATP-binding site. Unexpectedly, the A41S mutation enhanced AUY-922 binding affinity without changing geldanamycin binding. In silico analysis suggested this enhancement occurs through additional hydrogen bonding, yet stronger binding correlated with resistance. In yeast, A41S had opposite effects, hypersensitizing cells to all HSP90 inhibitors tested. Additionally, conditional HSP90 knockdown increased geldanamycin sensitivity but left AUY-922 activity unaffected, indicating different target dependencies despite shared binding sites. Based on these data, we propose a multi-target hypothesis where AUY-922s lower resistance risk stems from engaging multiple HSP90 family members. Our findings reveal how enhanced drug-target binding can paradoxically correlate with resistance and demonstrate that resistance risk cannot be predicted from binding site identity alone, providing insights for developing more durable drugs across therapeutic areas.

microbiology↗

bioRxiv2022

Development of Highly Selective Epoxyketone-based Plasmodium Proteasome Inhibitors with Negligible Cytotoxicity

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 O_SCPLOWDC_SCPLOW-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.

biochemistry↗

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↗

bioRxiv2020

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

New chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel screening of the Medicines for Malaria Venture Pandemic Response Box to identify multistage-active and stage-specific compounds against various life cycle stages of Plasmodium parasites (asexual parasites, stage IV/V gametocytes, gametes, oocysts and liver stages) and for endectocidal activity. Hits displayed unique chemotypes and included two multistage-active compounds, 16 asexual-targeted, six with prophylactic potential and ten gametocyte-targeted compounds. Notably, four structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity were identified: the JmjC inhibitor ML324, two azole antifungals including eberconazole, and the antitubercular clinical candidate SQ109. Besides ML324, none of these have previously attributed antiplasmodial activity, emphasizing the success of de novo parallel screening against different Plasmodium stages to deliver leads with novel modes-of-action. Importantly, the discovery of such transmission-blocking targeted compounds covers a previously unexplored base for delivery of compounds required for malaria elimination strategies.

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↗