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

描述（由申请人提供）：我们建议开发和配置用于两种乙酰转移酶的高通量筛选（HTS）的分析。首先，葡萄糖氨基-1-磷酸乙酰转移酶（GAT）是肽聚糖合成中尚未开发的靶标。它是真细菌生长所必需的，缺乏它的功能会导致细胞裂解。我们建议使用结核分枝杆菌GAT酶（它是双功能GlmU蛋白的一部分）开发这种检测方法。结核病GAT抑制剂是特别需要的，因为没有好的肽聚糖靶向抗生素可用于结核病。此外，由于结核病在世界范围内的严重程度以及耐药菌株的出现，需要新的抗结核病药物。我们还建议再次使用TB SAT开发和配置丝氨酸乙酰转移酶（SAT）的HTS检测方法。SAT是半胱氨酸合成所必需的，并且在细菌细胞外信号传导中起作用。它在人类中没有发现，但在细菌和几种低级真核病原体中发现。因此，通过假设许多病原体如果不能制造半胱氨酸，就不能从宿主获得足够的供应，靶向SAT为新药开发提供了一种新的途径。正如HTS所发现的那样，需要这种酶的抑制剂来验证这一假设。这两种酶都使用乙酰辅酶a来使底物乙酰化，这两种酶的测定都依赖于检测它们产生的游离辅酶a。CoA通过其游离SH基团形成荧光加合物来检测。我们有初步数据表明该方法适用于GAT，并建议将其开发用于GAT，并将其扩展到SAT。在开发每个分析后，我们建议通过最小化背景，最小化成本，演示可接受的统计参数以及通过机器人液体处理器自动化来配置它们用于HTS。然后，我们建议筛选5到2万种化合物来测试每个分析。未来的研究将包括将检测结果提交给NIH路线图倡议，以筛选大量化合物，以便对新药临床前开发的候选药物进行优化。与公共卫生的相关性：需要针对细菌和寄生虫的新药，因为耐药性正在导致现有药物不起作用。本文提出的工作将通过与NIH合作寻找可以开发成新药的候选化学物质来帮助开发此类新药。

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.