Targeting SecA1 of Mycobacterium tuberculosis for Novel Drug Development

描述（由申请人提供）：结核病（TB）是当今世界面临的最可怕的健康问题之一，由于TB-HIV综合征和越来越多的多重、广泛甚至完全耐药（MDR、XDR、TDR）结核分枝杆菌（Mtb）菌株的报告，TB危机的前景是不祥的。迫切需要靶向新的Mtb途径的新药。所有细菌都具有高度保守的Sec途径，该途径将蛋白质从细胞质输出到细菌细胞包膜或宿主环境。在Sec系统输出的蛋白质中有在毒力中具有重要功能或作用的蛋白质，使得Sec途径成为有吸引力的药物靶标。Sec途径的中心组分之一是SecA ATP酶，其提供来自ATP水解的能量以驱动蛋白质跨膜易位。SecA没有哺乳动物同源物，这使其成为一个有吸引力的靶标。与其他细菌一样，结核分枝杆菌也有一个功能性的Sec途径。在分枝杆菌中，典型的SecA被命名为SecA 1。该两阶段提案的R21阶段的目标是使用条件性Mtb secA 1突变体明确证明Mtb SecA 1对于体外和体内感染期间的Mtb生长和存活是必需的。此外，作为R21阶段的一部分，我们将开发可用于定量高通量筛选（qHTS）的全细胞测定法，以识别Mtb SecA 1的抑制剂。在该项目的R33阶段，我们将自动化和验证qHTS检测，并进行中试筛选，以评估检测性能，通过鉴定抑制Mtb SecA 1的化合物的能力来衡量。我们还将解决与Mtb SecA 1结合的一小组抑制剂的结构，以帮助理解抑制剂的作用模式。拟议的工作将验证Mtb SecA 1作为一种新的药物靶标，由于SecA在细菌中高度保守，因此这些结果也可能有助于未来针对其他耐药细菌病原体SecA靶标的药物设计。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is one of the most frightening health problems facing the world today and the outlook for the TB crisis is ominous as a result of the TB-HIV syndemic and increasing reports of multiply, extensively and even totally drug resistant (MDR, XDR, TDR) Mycobacterium tuberculosis (Mtb) strains. New drugs that target novel Mtb pathways are desperately needed. All bacteria possess a highly conserved Sec pathway that exports proteins from the cytoplasm to the bacterial cell envelope or host environment. Among the proteins exported by the Sec system are proteins with essential functions or roles in virulence, making the Sec pathway an attractive drug target. One of the central components of the Sec pathway is the SecA ATPase that provides energy from ATP hydrolysis to drive protein translocation across the membrane. There are no mammalian homologues of SecA, which makes it an appealing target. Like other bacteria, Mtb has a functional Sec pathway. In mycobacteria, the canonical SecA is named SecA1. The goals of the R21 phase of this two stage proposal are to definitively prove Mtb SecA1 is essential for Mtb growth and survival in vitro and during in vivo infection, using conditional Mtb secA1 mutants. Also as part of the R21 phase, we will develop whole-cell assays that can be used in quantitative high-throughput screening (qHTS) for identifying inhibitors of Mtb SecA1. In the R33 phase of the project, we will automate and validate the qHTS assays and undertake pilot screening to assess assay performance as measured by the ability to identify compounds that inhibit Mtb SecA1. We will additionally solve the structure of a small set of inhibitors bound to Mtb SecA1 as a way to help understand inhibitor mode of action. The proposed work will validate Mtb SecA1 as a new drug target and, because SecA is highly conserved among bacteria, the results may also aid future drug design directed at SecA targets of other drug-resistant bacterial pathogens.