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

描述（由申请人提供）：结核分枝杆菌（Mtb），结核病的病原体，是一个主要的健康问题，每年造成170万人死亡。多重和广泛耐药结核菌株的出现使情况更加令人震惊，因为它引起了对无法治愈的结核病的关注。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易位（达特）途径，其将蛋白质输出到细菌细胞包膜或宿主环境。哺乳动物细胞不具有达特途径，使得该途径成为新型抗微生物剂的有吸引力的候选者。结核分枝杆菌的达特途径是一个特别引人注目的药物靶点：它是结核分枝杆菌的一个重要途径，它输出结核分枝杆菌毒力因子，并且它输出结核分枝杆菌毒素。 ?-内酰胺酶，赋予耐药？-内酰胺类抗生素在这个项目中，我们将开发强大的高通量筛选（HTS）检测用于识别Mtb达特途径的抑制剂。全细胞表型分析将使用快速生长和非致病性模型分枝杆菌M。斯美格（Msmeg）。Msmeg的达特途径不是必需的，但它是必需的？内酰胺抗性。在目标1中，我们将开发一种达特抑制剂的初步筛选方法，该方法基于？缺乏达特途径的Msmeg突变体的内酰胺敏感性。在目标2中，我们将开发二次测定来区分引起？通过达特抑制内酰胺敏感性，而不是其他机制。还将开发用于建立目标特异性和检测毒性结核分枝杆菌的测定法。在目标3中，我们将进行试点筛选，以评估我们的检测方法和筛选级联的有效性，并进行结核分枝杆菌检测。通过本研究鉴定的化合物将有助于验证Mtb达特途径作为一种新的药物靶点，它们也将作为研究细菌达特途径的第一个化学探针。完成这项工作后，我们打算申请快速通道进入我们的筛选活动到NIH分子图书馆探针生产中心网络。

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.