MmpL3 as a target for novel anti-TB agents

总结 分枝杆菌酸是所有分枝杆菌外膜的基本组成部分， 过去几种药物的治疗功效清楚地说明了针对它们的生物合成和输出的目标 目前的抗分枝杆菌剂包括异烟肼、乙硫异烟胺、硫醋腙和异舒利，以及许多 最近发现的小分子抑制剂据报道可抑制完整的膜分枝菌酸 转运蛋白MmpL3。我们的财团在上一个资助期之前和期间的共同努力， MmpL3的发现以及该转运蛋白作为治疗目标的验证。这些 进一步的研究使我们能够第一次深入了解它的结构，能量学和机制， 对抑制剂的敏感性和抗性。 尽管取得了这些进展，但在我们对分枝菌酸易位的理解方面仍然存在重要差距 从细胞质到周质和外膜，MmpL3在这一过程中发挥的确切作用， 以及相关的分子机制。一些证据表明，分枝菌酸的出口， 细胞表面涉及比唯一的MmpL3蛋白更多的组分，并且MmpL3可能作为一种免疫调节剂。 用于多蛋白复合物偶联合成和输出的支架。我们的初步证据进一步表明 MmpL3可能在质膜水平上协调细胞的局灶性沉积， 细胞伸长和细胞分裂过程中的壁核成分。 这项基础研究的目的1是确定MmpL3相关机制的性质， 用于结核分枝杆菌中分枝菌酸的有效合成和输出，并探测 MmpL3参与细胞伸长过程中新细胞壁沉积的时空协调， 师.根据目标2，将对MmpL3进行详细的生化检查，以描述MmpL3的精确表达。 在分枝杆菌细胞中MmpL3介导的分枝菌酸转运的程度， 涉及的机制。在此目标下开发的测定将进一步用于探测精确的 MmpL3抑制剂的作用机制和相关的耐药性机制。 除了提供关于所有分枝杆菌的基本生理过程的基础知识外， 这些研究的结果将促进目前针对MmpL3的药物开发工作， 进一步导致创新的治疗策略，靶向细胞交叉点的关键代谢过程， 被膜生物合成和细胞伸长和分裂。

Summary Mycolic acids are essential building blocks of the outer membrane of all mycobacteria and the interest in drugs targeting their biosynthesis and export is clearly illustrated by the therapeutic efficacy of several past and present anti-mycobacterial agents including isoniazid, ethionamide, thiacetazone and isoxyl, and a number of recently discovered small molecule inhibitors reported to inhibit the integral membrane mycolic acid transporter, MmpL3. Joint efforts by our Consortium prior to and during the previous funding period have led to the discovery of MmpL3 and to the validation of this transporter as a target of therapeutic interest. These studies further allowed us to gain the first insights into its structure, energetics and mechanisms of susceptibility and resistance to inhibitors. Despite these advances, important gaps still remain in our understanding of the translocation of mycolic acids from the cytoplasm to the periplasm and outer membrane, the precise role played by MmpL3 in this process, and the molecular mechanisms involved. Several lines of evidence indicate that the export of mycolic acids to the cell surface involves more components than the only MmpL3 protein, and that MmpL3 may act as a scaffold for a multiprotein complex coupling synthesis and export. Our preliminary evidence further indicates that MmpL3 may play a role in coordinating, at the level of the plasma membrane, the focal deposition of cell wall core constituents during cell elongation and cell division. Aim 1 of this basic research proposal is to define the nature of the MmpL3-associated machinery responsible for the efficient synthesis and export of mycolic acids in Mycobacterium tuberculosis, and to probe the involvement of MmpL3 in the spatiotemporal coordination of new cell wall deposition during cell elongation and division. Under Aim 2, a detailed biochemical examination of MmpL3 will be conducted to delineate the precise extent of mycolic acid transport mediated by MmpL3 in mycobacterial cells and to gain insight in the molecular mechanisms involved. The assays developed under this aim will further be used to probe the precise mechanism of action of MmpL3 inhibitors and associated mechanisms of resistance. In addition to providing fundamental knowledge about essential physiological processes of all mycobacteria, the results from these studies will facilitate current drug development efforts targeted to MmpL3 and could further lead to innovative therapeutic strategies targeting key metabolic processes at the intersection of cell envelope biosynthesis and cell elongation and division.