Chemical biology studies of MmpL3 inhibition and resistance in mycobacteria

MmpL 3（分枝杆菌膜蛋白Large 3）是分枝杆菌生长抑制剂的常见靶标 通过全细胞、表型、高通量筛选鉴定。MmpL 3是一种移动海藻糖的真菌酸翻转酶 将单霉菌酸盐（TMM）转移至假周质间隙，TMM从假周质间隙被修饰为海藻糖二霉菌酸盐 (TDM)并被整合到菌膜中。结核分枝杆菌（Mtb）和M.耻垢病mmpL 3 敲低菌株显示mmpL 3对于体外和小鼠中的存活都是必需的。这种表型使 MmpL 3是一个有吸引力的治疗靶点，并支持表征靶向MmpL 3的分子的努力。多 MmpL 3抑制剂表现出与TB药物的协同相互作用，进一步支持了对该靶点的兴趣。 使用非靶向和靶向突变体筛选的创新组合，我们已经确定了十个新的， 抑制MmpL 3功能的不同支架。这些化合物在体外和抗 原代鼠巨噬细胞中细胞内Mtb。抑制剂是分枝杆菌特异性的， 抗非结核分枝杆菌（NTM）种M的活性。Mab，包括HC 2099 HC 2091系列中试构效关系（SAR）研究，涉及100多种类似物的合成 的HC 2099和HC 2091已经鉴定出具有约80 nM和约280 nM的全细胞Mtb半数最大功效的类似物。 nM，分别。几种类似物在微粒体中表现出高溶解度、稳定性，并且在细胞内没有细胞毒性。 巨噬细胞，支持其进一步发展。例如，HC 2099的类似物MSU-43085被口服给药。 生物可利用的，在急性鼠感染模型中对Mtb有活性，且活性与标准品相当 在体外和巨噬细胞中抗Mab的护理药物。因此，这些系列将是有价值的工具， 了解大肠杆菌-MmpL 3结构-功能相互作用，并作为新的结核病药物开发的领导者。 我们的MmpL 3抑制剂和突变体库也将使我们的团队能够确定耐药机制。 MmpL3.交叉耐药谱的聚类分析，由剂量反应实验产生， 针对24种不同mmpL 3突变体的13种MmpL 3抑制剂的组合，定义了两个抑制剂进化枝， 两个进化枝的抗性突变体。抑制剂的成对组合研究显示拮抗、协同 和特定于所鉴定的进化枝的加性相互作用。模拟的阻力替代， MmpL 3晶体结构揭示了MmpL 3的特定结构域的残基的进化枝特异性定位。这些 研究结果支持了我们的假设，即MmpL 3抑制剂或合理设计的分子的组合可以 以减少抵抗的频率。 本研究的总体目标是：1）优化新的MmpL 3抑制剂以确定抑制剂MmpL 3相互作用 并生成显示体内疗效的概念验证数据（目标1）;以及2）定义 MmpL 3设计策略以减少耐药性的演变并设计更持久的药物（目标2）。

MmpL3 (Mycobacterium membrane protein Large 3) is a common target of inhibitors of mycobacterial growth identified by whole cell, phenotypic, high throughput screens. MmpL3 is a mycolate flippase that moves trehalose monomycolate (TMM) to the pseudoperiplasmic space, from where TMM is modified to trehalose dimycolate (TDM) and incorporated into the mycomembrane. Mycobacterium tuberculosis (Mtb) and M. smegmatis mmpL3 knockdown strains show that mmpL3 is essential for survival both in vitro and in mice. This phenotype makes MmpL3 an attractive therapeutic target and supports efforts to characterize molecules targeting MmpL3. Multiple MmpL3 inhibitors exhibit synergistic interactions with TB drugs, further supporting interest in this target. Using an innovative combination of untargeted and targeted mutant screens, we have identified ten new and distinct scaffolds that inhibit MmpL3 function. These compounds are bactericidal both in vitro and against intracellular Mtb in primary murine macrophages. The inhibitors are mycobacteria specific with several showing activity against the non-tuberculous mycobacterial (NTM) species M. abscessus (Mab), including the HC2099 and HC2091 series. Pilot structure activity relationship (SAR) studies involving the synthesis of over 100 analogs of HC2099 and HC2091 have identified analogs with whole cell Mtb half-maximal efficacies of ~80 nM and ~280 nM, respectively. Several analogs exhibited high solubility, stability in microsomes and no cytotoxicity in macrophages, supporting their further development. For example, MSU-43085, an analog of HC2099, is orally bioavailable, active against Mtb in an acute murine model of infection and has activity comparable to standard of care drugs against Mab in vitro and in macrophages. Therefore, these series will be valuable tools to understand inhibitor-MmpL3 structure-function interactions and as leads for new TB drug development. Our library of MmpL3 inhibitors and mutants also will enable our team to define mechanisms of resistance in MmpL3. Cluster analysis of cross resistance profiles, generated by dose response experiments for each combination of 13 MmpL3 inhibitors against 24 different mmpL3 mutants, defined two clades of inhibitors and two clades of resistant mutants. Pairwise combination studies of the inhibitors revealed antagonistic, synergistic and additive interactions that were specific to the identified clades. Modeling of resistance substitutions to the MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3. These findings support our hypothesis that combinations of MmpL3 inhibitors or rationally designed molecules can be employed to reduce the frequency of resistance. The overall goals of this study are to: 1) optimize new MmpL3 inhibitors to define inhibitor MmpL3 interactions and generate proof-of-concept data showing efficacy in vivo (Aim 1); and 2) define mechanisms of resistance in MmpL3 to devise strategies to reduce the evolution of resistance and design more durable drugs (Aim 2).