Rosa-Teijeiro Chloé, Wagner Victoria, Corbeil Audrey, d'Annessa Ilda, Leprohon Philippe, do Monte-Neto Rubens L, Fernandez-Prada Christopher
Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada.
The Research Group on Infectious Diseases in Production Animals (GREMIP), Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC, Canada.
Parasit Vectors. 2021 Aug 28;14(1):438. doi: 10.1186/s13071-021-04947-4.
The 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.
We 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.
We 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 (Top1B, Top1B, and Top1B). 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.
This 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.
耐药性的演变是利什曼病面临的最大挑战之一，这促使人们需要新的抗利什曼药物。重新利用已批准的药物是一种更快且极具吸引力的策略，正在全球范围内获得支持。不同的抗癌拓扑异构酶1B（TOP1B）抑制剂已显示出强大的抗利什曼活性和有前景的选择性指数，支持了这些药物重新利用的潜力。然而，癌细胞和利什曼原虫都有迅速产生耐药性的能力。本研究的目的是完成对拓扑替康暴露所引起影响的全基因组探索，以突出利什曼原虫在TOP1B抑制剂存在下有利于其存活所采用的潜在机制。
我们采用逐步耐药性筛选、全基因组测序、功能验证和理论方法相结合的方式，来探索婴儿利什曼原虫的三个独立克隆抵抗TOP1B抑制剂拓扑替康作用的倾向和潜在机制。
我们证明婴儿利什曼原虫能够对高浓度的拓扑替康产生耐药性，且生长能力不受损害。在三个耐药株系中，下一代测序数据均未发现基因缺失或扩增，排除了外排泵过度表达作为拓扑替康耐药性的首选机制。我们在利什曼原虫TOP1B的大亚基中鉴定出三种不同的突变（Top1B、Top1B和Top1B）。在野生型背景中这些突变等位基因的过表达导致对拓扑替康的高耐药水平。共价和非共价复合物中的计算分子动力学模拟表明，这些突变对催化五元组的排列以及这些残基与周围氨基酸和DNA的相互作用有影响。结合口袋结构的这种改变导致拓扑替康在三元复合物中的持久性降低。
这项工作有助于阐明此前尚不清楚的利什曼原虫中拓扑替康耐药性的潜在机制，即TOP1B大亚基中的突变，并为设计改进的抑制剂以对抗利什曼病和癌症中的耐药性提供了有价值的线索。我们的数据突出了在药物发现过程中纳入耐药性评估的重要性。
