Developing High-Throughput Assays for M. tuberculosis Tat Pathway Inhibitors

描述(申请人提供)：结核分枝杆菌(结核分枝杆菌)，结核病的病原体，是一个主要的健康问题，每年导致170万人死亡。结核分枝杆菌多重和广泛耐药菌株的出现使这种情况更加令人担忧，因为它引发了人们对无法治疗的结核病的担忧。迫切需要开发治疗结核病的新药。许多细菌病原体具有双精氨酸转位(TAT)途径，该途径将蛋白质输出到细菌的细胞膜或宿主环境。哺乳动物细胞没有TAT途径，这使得该途径成为新的抗菌药的一个有吸引力的候选者。结核分枝杆菌的Tat途径是一个特别引人注目的药物靶点：它是结核杆菌的基本途径，它输出结核分枝杆菌的毒力因子，它输出结核分枝杆菌 ？-对内酰胺类抗生素产生耐药性的内酰胺酶。在这个项目中，我们将开发强大的高通量筛选(HTS)方法来识别Mtb Tat途径的抑制剂。将使用快速生长的非致病模式污垢分枝杆菌(Msmeg)开发全细胞表型分析。Msmeg的Tat途径不是必需的，但对β-内酰胺类耐药是必要的。在目标1中，我们将建立一种基于缺乏TAT途径的Msmeg突变体的β-内酰胺敏感性的TAT抑制剂的初步筛选方法。在目标2中，我们将开发二次检测，以区分通过抑制TAT而不是其他机制而引起内酰胺类药物敏感性的化合物。还将开发建立靶标特异性和检测毒力结核分枝杆菌的检测方法。在目标3中，我们将进行试点筛查，以评估我们的检测和筛查级联的有效性，HITS进入结核分枝杆菌的测试阶段。通过本研究鉴定的化合物将有助于验证Mtb TAT途径作为新的药物靶点的有效性，它们也将作为研究细菌TAT途径的第一个化学探针具有价值。在这项工作完成后，我们打算申请我们的筛选活动的快速通道进入NIH分子库探针生产中心网络。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major health problem responsible for 1.7 million deaths each year. The emergence of multiply and extensively drug resistant strains of Mtb makes the situation even more alarming as it raises concern about untreatable tuberculosis. There is an urgent need to develop new drugs to treat tuberculosis. Many bacterial pathogens possess a twin arginine translocation (Tat) pathway that exports proteins to the bacterial cell envelope or host environment. Mammalian cells do not have a Tat pathway, making this pathway an attractive candidate for novel antimicrobials. The Tat pathway of Mtb is a particularly compelling drug target: it is an essential pathway in Mtb, it exports Mtb virulence factors, and it exports the Mtb ?-lactamase that imparts resistance to ?-lactam antibiotics. In this project we will develop robust high-throughput screening (HTS) assays for identifying inhibitors of the Mtb Tat pathway. Whole-cell phenotypic assays will be developed using the fast-growing and nonpathogenic model mycobacteria M. smegmatis (Msmeg). The Tat pathway of Msmeg is not essential but it is necessary for ?-lactam resistance. In Aim 1, we will develop a primary screening assay for Tat inhibitors that is based on the ?-lactam sensitivity of Msmeg mutants lacking the Tat pathway. In Aim 2, we will develop secondary assays to distinguish compounds that elicit ?-lactam sensitivity through Tat inhibition as opposed to other mechanisms. Assays for establishing target-specificity and for testing on virulent Mtb will also be developed. In Aim 3, w will perform pilot screening to assess the effectiveness of our assays and the screening cascade with hits progressing to testing on Mtb. The compounds identified through this research will help to validate the Mtb Tat pathway as a novel drug target and they will also be valuable as the first chemical probes for studying the Tat pathway of bacteria. Upon completion of this work, we intend to apply for Fast Track entry of our screening campaign into the NIH Molecular Libraries Probe Production Centers Network.