A Fluorescence Displacement Assay for the Biotin Biosynthetic Enzyme BioA

生物素生物合成酶 BioA 的荧光置换测定

基本信息

批准号：
8403185
负责人：
Courtney C Aldrich
金额：
$ 3.66万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8403185
关键词：
AIDS-Related Opportunistic Infections Acyl Coenzyme A Anabolism Antitubercular Agents Ants Atypical Mycobacteria Bacillus (bacterium)Biochemical Biological Biological Assay Biotin Biotin Metabolism Pathway Carbon Dioxide Cell Death Cells Cessation of life Chemicals Chronic Communicable Diseases Coupled Coupling Custom Data Detection Development Drug Targeting Drug resistance Enzymes Extreme drug resistant tuberculosis Fluorescence Fluorescent Probes Genetic Gluconeogenesis Housing In Vitro Infection Label Laboratories Lead Libraries Measures Metabolic Microbiology Modeling Molecular Bank Multi-Drug Resistance Multidrug-Resistant Tuberculosis Mus Mycobacterium smegmatis Mycobacterium tuberculosis Mycobacterium tuberculosis H37Rv Nature Nutrient Pathway interactions Patients Persons Phase Phenotype Plague Production Pyruvate Carboxylase Reaction Reagent Recombinant Proteins Relative (related person)Research Series Source Specificity Staining method Stains Starvation Streptavidin System Tuberculosis Validation Virulence Virulent Vitamins base cofactor design desthiobiotin drug development drug discovery enzyme substrate fatty acid metabolism genome sequencing high throughput screening in vivo medical schools mortality mutant novel overexpression pathogen resistant strain response screening small molecule tuberculosis drugs

项目摘要

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.

描述（由申请人提供）：结核分枝杆菌是全世界细菌传染病死亡的主要原因。耐药菌株（MDR 和 XDR-TB）是一个新出现的问题，可能会在美国引发重大问题，因为超过一半的病例发生在外国出生的人身上。生物素生物合成最近被确定为开发新型蚂蚁结核病药物的一个有吸引力的目标。在本提案中，我们描述了一种新型 BioA 高温超导检测的设计、开发和优化，该检测可催化结核分枝杆菌中生物素生物合成的第二步。该应用的目的是使用我们的新型 HTS 测定法筛选 BioA，然后对已识别的命中进行二级和三级筛选。在第一个具体目标中，我们将为初级和次级测定准备化学和生化试剂。在第二个具体目标中，我们将使用基于正交 LC-MS 的测定来开发和筛选命中。在第三个具体目标中，我们将使用过度表达和不足表达 BioA 的耻垢分枝杆菌突变株进行三级筛选，以获取生物活性并表征目标特异性。