Laboratory of Microbiology, Parasitology and Hygiene (LMPH), University of Antwerp, Antwerp, Belgium.
Centre de Recherche en Infectiologie du Centre de Recherche du Centre Hospitalier Universitaire de Québec, Université Laval, Québec, Québec, Canada.
Antimicrob Agents Chemother. 2020 Aug 20;64(9). doi: 10.1128/AAC.00152-20.
Current antileishmanial treatment is hampered by limitations, such as drug toxicity and the risk of treatment failure, which may be related to parasitic drug resistance. Given the urgent need for novel drugs, the Drugs for Neglected Diseases (DND) has undertaken a drug discovery program, which has resulted in the identification of aminopyrazoles, a highly promising antileishmanial chemical series. Multiple experiments have been performed to anticipate the propensity for resistance development. Resistance selection was performed by successive exposure of promastigotes () and intracellular amastigotes (both and in golden Syrian hamsters). The stability of the resistant phenotypes was assessed after passage in mice and sandflies. Whole-genome sequencing (WGS) was performed to identify mutated genes, copy number variations (CNVs), and somy changes. The potential role of efflux pumps (the MDR and MRP efflux pumps) in the development of resistance was assessed by coincubation of aminopyrazoles with specific efflux pump inhibitors (verapamil, cyclosporine, and probenecid). Repeated drug exposure of amastigotes did not result in the emergence of drug resistance either or Selection at the promastigote stage, however, was able to select for parasites with reduced susceptibility (resistance index, 5.8 to 24.5). This phenotype proved to be unstable after passage in mice and sandflies, suggesting that nonfixed alterations are responsible for the elevated resistance. In line with this, single nucleotide polymorphisms and indels identified by whole-genome sequencing could not be directly linked to the decreased drug susceptibility. Copy number variations were absent, whereas somy changes were detected, which may have accounted for the transient acquisition of resistance. Finally, aminopyrazole activity was not influenced by the MDR and MRP efflux pump inhibitors tested. The selection performed does not suggest the rapid development of resistance against aminopyrazoles in the field. Karyotype changes may confer elevated levels of resistance, but these do not seem to be stable in the vertebrate and invertebrate hosts. MDR/MRP efflux pumps are not likely to significantly impact the activity of the aminopyrazole leads.
当前的抗利什曼病治疗受到限制，例如药物毒性和治疗失败的风险，这可能与寄生虫的耐药性有关。鉴于对新型药物的迫切需求，被忽视疾病药物研发基金会（DND）开展了一项药物发现计划，由此确定了氨基吡唑这一极具前景的抗利什曼病化学系列。已经进行了多项实验来预测耐药性发展的倾向。通过连续暴露于前鞭毛体（）和细胞内无鞭毛体（两者在金黄地鼠中为 和 ）来进行抗性选择。在小鼠和沙蝇中转代后评估抗性表型的稳定性。进行全基因组测序（WGS）以鉴定突变基因、拷贝数变异（CNV）和染色体变化。通过与特定外排泵抑制剂（维拉帕米、环孢素和丙磺舒）共孵育来评估外排泵（MDR 和 MRP 外排泵）在耐药性发展中的潜在作用。无鞭毛体的重复药物暴露既没有导致药物耐药性（ 或 ）的出现，也没有导致药物耐药性的出现 在前鞭毛体阶段的选择，然而，能够选择对药物敏感性降低的寄生虫（耐药指数为 5.8 至 24.5）。这种表型在小鼠和沙蝇中转代后变得不稳定，表明非固定改变是导致耐药性升高的原因。与此一致，通过全基因组测序鉴定的单核苷酸多态性和插入缺失不能直接与降低的药物敏感性相关。不存在拷贝数变异，而检测到染色体变化，这可能导致耐药性的短暂获得。最后，氨基吡唑的活性不受测试的 MDR 和 MRP 外排泵抑制剂的影响。进行的选择并不表明在野外对抗氨基吡唑的耐药性迅速发展。核型变化可能赋予更高水平的耐药性，但在脊椎动物和无脊椎动物宿主中似乎不稳定。MDR/MRP 外排泵不太可能对氨基吡唑先导物的活性产生重大影响。
