Chemical biology studies of MmpL3 inhibition and resistance in mycobacteria

分枝杆菌 MmpL3 抑制和耐药性的化学生物学研究

基本信息

批准号：
10734240
负责人：
Robert B Abramovitch
金额：
$ 75.9万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-13 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10734240
关键词：
Acute Air American Antibiotics Biological Availability Biology Cells Cessation of life Chemicals Chronic Obstructive Pulmonary Disease Cluster Analysis Collection Cord Factors Cystic Fibrosis Data Development Disease Dose Drug Kinetics Drug resistance Drug resistant Mycobacteria Tuberculosis Ethambutol Evolution Exhibits Frequencies Genus Mycobacterium Goals Growth Health In Vitro Individual Infection Libraries Macrophage Membrane Proteins Microsomes Mission Modeling Mus Mycobacterium abscessus Mycobacterium smegmatis Mycobacterium tuberculosis National Institute of Allergy and Infectious Disease Oral Pharmaceutical Preparations Phenotype Pyrazinamide Resistance Resistance profile Rifampin Series Solubility Structure Structure-Activity Relationship Trehalose Tuberculosis United States analog bactericide cytotoxicity design disease transmission drug development efficacy study experimental study fitness global health high throughput screening in vivo inhibitor innovation interest isoniazid knock-down mouse model mutant mycobacterial mycolate non-tuberculosis mycobacteria non-tuberculous mycobacterial infection novel rational design resistance frequency resistance mechanism resistance mutation resistant strain response scaffold standard of care therapeutic target tool tuberculosis drugs

项目摘要

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).

MmpL3（分枝杆菌膜蛋白大 3）是分枝杆菌生长抑制剂的常见靶点通过全细胞、表型、高通量筛选进行鉴定。 MmpL3 是一种移动海藻糖的霉菌酸翻转酶单菌酸盐 (TMM) 至假周质空间，TMM 在此被修饰为海藻糖二霉菌酸盐（TDM）并纳入菌膜中。结核分枝杆菌 (Mtb) 和耻垢分枝杆菌 mmpL3 敲除菌株表明 mmpL3 对于体外和小鼠的生存都是必需的。这种表型使得 MmpL3 是一个有吸引力的治疗靶点，并支持表征针对 MmpL3 的分子的努力。多种的 MmpL3 抑制剂与结核病药物表现出协同相互作用，进一步支持了人们对该靶点的兴趣。通过非靶向和靶向突变体筛选的创新组合，我们已经确定了十种新的和抑制 MmpL3 功能的独特支架。这些化合物在体外和对细菌都有杀菌作用原代小鼠巨噬细胞中的细胞内 Mtb。该抑制剂具有分枝杆菌特异性，有多种表现针对非结核分枝杆菌 (NTM) 物种脓肿分枝杆菌 (Mab) 的活性，包括 HC2099 和HC2091系列。涉及 100 多种类似物合成的中试结构活性关系 (SAR) 研究 HC2099 和 HC2091 已鉴定出全细胞 Mtb 半最大功效分别为 ~80 nM 和 ~280 nM 的类似物分别为nM。几种类似物在微粒体中表现出高溶解度、稳定性，并且在微粒体中没有细胞毒性。巨噬细胞，支持其进一步发育。例如，MSU-43085是HC2099的类似物，可口服具有生物利用度，在急性鼠感染模型中具有抗 Mtb 的活性，并且具有与标准相当的活性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抑制剂-MmpL3 结构-功能相互作用，并作为新结核病药物开发的先导。我们的 MmpL3 抑制剂和突变体库也将使我们的团队能够定义耐药机制 MmpL3。交叉耐药性概况的聚类分析，由每个药物的剂量反应实验生成 13 种 MmpL3 抑制剂针对 24 种不同的 mmpL3 突变体的组合，定义了两个抑制剂分支抗性突变体的两个分支。抑制剂的配对组合研究揭示了拮抗、协同作用以及特定于已识别进化枝的附加相互作用。耐药替代模型 MmpL3 晶体结构揭示了残基对 MmpL3 特定结构域的进化枝特异性定位。这些研究结果支持我们的假设，即 MmpL3 抑制剂或合理设计的分子的组合可以用于减少电阻频率。本研究的总体目标是：1）优化新的 MmpL3 抑制剂以定义抑制剂 MmpL3 相互作用并生成显示体内功效的概念验证数据（目标 1）； 2）定义抵抗机制 MmpL3 制定策略来减少耐药性的演变并设计更持久的药物（目标 2）。