Glucosamine-1-phosphate and serine acetylases: HTS assays and configurations

1-磷酸氨基葡萄糖和丝氨酸乙酰酶：HTS 测定和配置

• 批准号: 7678708
• 负责人: Michael R McNeil
• 金额: $ 3.68万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678708
• 关键词: Acetyl Coenzyme A; Acetylesterase; Antibiotics; Automation; Bacteria; Biological Assay; Cells; Chemicals; Coenzyme A; Compatible; Cysteine; Cytolysis; Data; Detection; Development; Disease; Drug resistance; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Future; Growth; Human; Liquid substance; Mycobacterium tuberculosis; Numbers; Parasites; Peptidoglycan; Pharmaceutical Preparations; Proteins; Public Health; Research; Robotics; Role; Screening procedure; Serine; Signal Transduction; Testing; Transferase; United States National Institutes of Health; Work; adduct; cost; drug development; extracellular; glucosamine 1-phosphate; human NAT2 protein; inhibitor/antagonist; novel strategies; pathogen; pre-clinical; serine O-acetyltransferase

DESCRIPTION (provided by applicant): We propose to develop and configure assays for high through put screening (HTS) for two acetyl transferases. The first, glucosamine-1-phosphate acetyl transferase (GAT) is an undeveloped target in peptidoglycan synthesis. It is essential for eubacterial growth, and its lack of function results in cell lysis. We propose to develop this assay using M. tuberculosis GAT enzyme (which is part of the bi-functional GlmU protein). Inhibitors of TB GAT are especially needed because there are no good peptidoglycan targeting antibiotics available for TB. Also new drugs against TB are needed because of the magnitude of the disease world wide and the emergence of drug resistant strains. We also propose to develop and configure assays for HTS of serine acetyl transferase (SAT) again using TB SAT. SAT is required for the synthesis of cysteine and has a role in bacterial extracellular signaling. It is not found in humans but is found in bacteria and several lower eukaryotic pathogens. Thus targeting SAT takes a novel approach to new drug development by hypothesizing that many pathogens, if unable to make cysteine, will not get adequate supplies from the host. Inhibitors of this enzyme, as can be found by HTS, are needed to validate this hypothesis. Both enzymes use acetyl-CoA to acetylate their substrate and the assay for both enzymes relies on the detection of free CoA produced by them. CoA is detected via its free SH group by forming a fluorescent adduct. We have preliminary data that this approach works for GAT and propose to develop it for GAT and extend it to SAT. After each assay is developed we propose to configure them for HTS by minimization of background, minimization of costs, demonstration of acceptable statistical parameters, and automation via a robotic liquid handler. We then propose to screen between 5 and 20 thousand compounds to test each assay. Future research will include submission of the assays to the NIH roadmap initiative for screening of large numbers of compounds to allow refinement of hits to candidates for preclinical development of new drugs. Relevance to Public Health: New drugs against bacteria and parasites are needed because drug resistance is resulting in current drugs not working. The work proposed herein will aid in the development of such new drugs by working with NIH to find chemical candidates that can be developed into new drugs.

描述（由申请人提供）：我们建议通过对两个乙酰基转移酶进行PUT筛选（HTS）开发和配置高分测定法。 第一个，葡萄糖胺1-磷酸乙酰基转移酶（GAT）是肽聚糖合成中未开发的靶标。 它对于细菌的生长至关重要，其缺乏功能会导致细胞裂解。 我们建议使用结核分枝杆菌GAT酶（这是双功能GLMU蛋白的一部分）开发该测定法。 特别需要TB GAT的抑制剂，因为没有可用于结核病的肽聚糖靶向抗生素。 由于全球疾病的大小和耐药菌株的出现，因此还需要针对结核病的新药。 我们还建议使用TB SAT再次开发和配置丝氨酸乙酰转移酶（SAT）HTS的测定法。 SAT是合成半胱氨酸所必需的，并且在细菌外信号传导中起作用。 它在人类中找不到，而是在细菌和几种较低的真核病原体中发现的。 因此，靶向SAT采取了一种新颖的方法来实现新药物开发，假设许多病原体，即使无法制作半胱氨酸，也不会从宿主那里获得足够的供应。 正如HTS所发现的那样，该酶的抑制剂是为了验证该假设的。 这两种酶都使用乙酰辅酶A乙酰化其底物，两种酶的测定都取决于检测它们产生的游离COA。 通过形成荧光加合物，通过其自由SH组检测到COA。 我们有初步的数据，该方法适用于GAT，并建议将其开发为GAT并将其扩展到SAT。 开发每个测定后，我们建议通过最小化背景，成本最小化，可接受的统计参数的证明以及通过机器人液体处理程序自动化来为HT配置它们。 然后，我们建议筛选5至20,000种化合物以测试每个测定法。 未来的研究将包括将测定法提交给NIH路线图计划，以筛选大量化合物，以使命中率改进候选人，以促进新药的临床前开发。 与公共卫生的相关性：需要针对细菌和寄生虫的新药，因为耐药性导致目前的药物不起作用。 本文提出的工作将通过与NIH合作寻找可以发展为新药的化学候选物来帮助开发此类新药。