Three Different Mutations in the DNA Topoisomerase 1B in Leishmania Infantum Contribute to Resistance to Antitumor Drug Topotecan

Abstract

BackgroundThe evolution of drug resistance is one of the biggest challenges in leishmaniasis and has prompted the need for new antileishmanial drugs. Repurposing of approved drugs is a faster and very attractive strategy that is gaining supporters worldwide. Different anticancer topoisomerase 1B (TOP1B) inhibitors have shown strong antileishmanial activity and promising selective indices, supporting the potential repurposing of these drugs. However, cancer cells and Leishmania share the ability to become rapidly resistant. The aim of this study was to complete a whole-genome exploration of the effects caused by exposure to topotecan in order to highlight the potential mechanisms deployed by Leishmania to favor its survival in the presence of a TOP1B inhibitor. MethodsWe used a combination of stepwise drug-resistance selection, whole-genome sequencing, functional validation, and theoretical approaches to explore the propensity of and potential mechanisms deployed by three independent clones of L. infantum to resist the action of TOP1B-inhibitor topotecan. ResultsWe demonstrated that L. infantum is capable of becoming resistant to high concentrations of topotecan without impaired growth ability. No gene deletions or amplifications were identified from the next-generation sequencing data in any of the three resistant lines, ruling out the overexpression of efflux pumps as the preferred mechanism of topotecan resistance. We identified three different mutations in the large subunit of the leishmanial TOP1B (Top1BF187Y, Top1BG191A and Top1BW232R). Overexpression of these mutated alleles in the wild-type background led to high levels of resistance to topotecan. Computational molecular dynamics simulations, in both covalent and non-covalent complexes, showed that these mutations have an effect on the arrangement of the catalytic pentad and on the interaction of these residues with surrounding amino acids and DNA. This altered architecture of the binding pocket results in decreased persistence of topotecan in the ternary complex. ConclusionsThis work helps elucidate the previously-unclear potential mechanisms of topotecan resistance in Leishmania by mutations in the large subunit of TOP1B and provides a valuable clue for the design of improved inhibitors to combat resistance in both leishmaniasis and cancer. Our data highlights the importance of including drug-resistance evaluation in drug-discovery cascades.

Novel Pyrido[2′,1′:2,3]imidazo[4,5-c]quinoline Derivative Selectively Poisons Leishmania donovani Bisubunit Topoisomerase 1 to Inhibit the Antimony-Resistant Leishmania Infection in Vivo

“…Further studies are warranted to explore whether C17 may drug develop resistance to antileishmanial therapy by generating point mutations in LdTop1. 48 Moreover, additional steps are required for testing this drug resistance potential prior to clinical trials and further validation will confirm the lab results to be used and practiced in the clinics.…”

Antileishmanial metallodrugs and the elucidation of new drug targets linked to post-translational modifications machinery: pitfalls and progress

“…Stepwise drug resistance selection can provide clues on predicting parasite's adaptations towards drug tolerance or developing specific drug resistance mechanisms. (33) Drug combination -Is also encouraged and justified by reducing the chances of selecting resistant parasites and to improve therapeutical schemes, reducing dose and treatment time. (17) It could be tested in vivo, guided or not by previous in vitro isobologram tests.…”