Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.
Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan, USA.
Antimicrob Agents Chemother. 2019 Sep 23;63(10). doi: 10.1128/AAC.00547-19. Print 2019 Oct.
The mycolate flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole-genome sequencing of isolated resistant mutants. Here, we describe the identification of four new inhibitors that select for resistance mutations in Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel growth inhibitors for differential growth inhibition of wild-type compared to the growth of a pool of 24 unique mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both and against intracellular cells treated with these compounds were shown to accumulate trehalose monomycolates, have reduced levels of trehalose dimycolate, and displace an MmpL3-specific probe, supporting MmpL3 as the target. The inhibitors were mycobacterium specific, with several also showing activity against the nontuberculous mycobacterial species Cluster analysis of cross-resistance profiles generated by dose-response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mutants defined two clades of inhibitors and two clades of mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance-conferring substitutions to the MmpL3 crystal structure revealed clade-specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small-molecule inhibitors.
Mycolate flippase MmpL3 一直是多种具有不同化学结构骨架的抑制剂的潜在靶标。这些不同的化学结构骨架使得很难在没有对分离的耐药突变体进行全基因组测序的情况下预测抑制 MmpL3 的化合物。在这里，我们描述了使用这些耐药突变体，对 163 种新型 生长抑制剂进行了靶向全细胞表型筛选，以确定它们对野生型 与 24 种独特 突变体的生长相比，是否有差异抑制作用。筛选成功地鉴定了另外六种潜在的 MmpL3 抑制剂。这些化合物对 和 具有杀菌活性，用这些化合物处理的细胞内 细胞积累了单分枝菌酸海藻糖酯，二分枝菌酸海藻糖酯水平降低，并且置换了 MmpL3 特异性探针，支持 MmpL3 作为靶标。抑制剂具有分枝杆菌特异性，其中几种抑制剂也对非结核分枝杆菌物种 具有活性。对 13 种 MmpL3 抑制剂对 24 种 突变体中的每一种的剂量反应实验的交叉耐药性分析生成的聚类分析定义了两个抑制剂簇和两个 突变体簇。对抑制剂的两两组合研究揭示了相互作用是特定于交叉耐药性分析中确定的簇的。此外，对 MmpL3 晶体结构的耐药性赋予取代的建模揭示了簇特异性定位的残基到 MmpL3 的特定结构域，并且簇显示出不同的耐药性。几种化合物在微粒体中表现出高溶解度和稳定性，在巨噬细胞中表现出低细胞毒性，支持进一步开发。对多种突变体和新型化合物的综合研究为 MmpL3 和小分子抑制剂的结构-功能相互作用提供了新的见解。
