RESUMEN
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 < 1 × 10-100). 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.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Xenobióticos/metabolismo , Xenobióticos/farmacologíaRESUMEN
Recent advances in cell-based, high-throughput phenotypic screening have identified new chemical compounds that are active against eukaryotic pathogens. A challenge to their future development lies in identifying these compounds' molecular targets and binding modes. In particular, subsequent structure-based chemical optimization and target-based screening require a detailed understanding of the binding event. Here, we use directed evolution and whole-genome sequencing of a drug-sensitive S. cerevisiae strain to identify the yeast ortholog of TcCyp51, lanosterol-14-alpha-demethylase (TcCyp51), as the target of MMV001239, a benzamide compound with activity against Trypanosoma cruzi, the etiological agent of Chagas disease. We show that parasites treated with MMV0001239 phenocopy parasites treated with another TcCyp51 inhibitor, posaconazole, accumulating both lanosterol and eburicol. Direct drug-protein binding of MMV0001239 was confirmed through spectrophotometric binding assays and X-ray crystallography, revealing a binding site shared with other antitrypanosomal compounds that target Cyp51. These studies provide a new probe chemotype for TcCyp51 inhibition.
Asunto(s)
Inhibidores de 14 alfa Desmetilasa/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Evolución Molecular Dirigida , Tripanocidas/uso terapéutico , Inhibidores de 14 alfa Desmetilasa/química , Inhibidores de 14 alfa Desmetilasa/farmacología , Secuencia de Aminoácidos , Enfermedad de Chagas/parasitología , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Cromatografía de Gases y Espectrometría de Masas , Simulación del Acoplamiento Molecular , Plasmodium falciparum/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Espectrofotometría Ultravioleta , Esterol 14-Desmetilasa/efectos de los fármacos , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimologíaRESUMEN
The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.