A Fluorescence Displacement Assay for the Biotin Biosynthetic Enzyme BioA

描述（由申请人提供）：结核分枝杆菌是全球细菌感染性疾病死亡的主要原因。耐药菌株（MDR和XDR-TB）是一个新出现的问题，可能在美国导致重大问题，因为超过一半的病例是在外国出生的人。生物素的生物合成最近已被确定为一个有吸引力的目标，为新的抗结核药物的发展。在该提案中，我们描述了一种新的HTS检测BioA的设计，开发和优化，该检测催化Mtb生物素生物合成的第二步。本申请的目的是使用我们的新型HTS测定筛选BioA，然后对鉴定的命中进行二级和三级筛选。在第一个具体目标中，我们将准备用于初级和次级检测的化学和生化试剂。在第二个具体目标中，我们将使用基于正交LC-MS的测定开发和筛选命中。在第三个具体目标中，我们将使用M的突变株进行三级筛选。过表达和低表达BioA以获得生物活性和表征靶特异性的smeglobulin。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is the leading cause of bacterial infectious disease deaths worldwide. Drug-resistant strains (MDR and XDR-TB) are an emerging problem and could lead to significant issues in the US because over half of the cases are in foreign born persons. Biotin biosynthesis has recently been identified as an attractive target for the development of new ant tubercular agents. In this proposal we describe the design, development, and optimization of a novel HTS-assay for BioA, which catalyzes the second step of biotin biosynthesis in Mtb. The objectives of this application are to screen BioA using our novel HTS assay and then perform secondary and tertiary screening of identified hits. In the first specific aim, we will prepare chemical and biochemical reagents for the primary and secondary assays. In the second specific aim, we will develop and screen hits using an orthogonal LC-MS based assay. In the third specific aim, we will perform tertiary screening using mutant strains of M. smegmatis that over- and under-express BioA to access biological activity and characterize on-target specificity.