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)的分析方法。第一，氨基葡萄糖-1-磷酸乙酰转移酶(GAT)是合成肽聚糖的一个未开发的靶标。它对真细菌的生长是必不可少的，而它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)来开发这种检测方法。尤其需要结核病GAT的抑制剂，因为目前还没有针对结核病的好的针对抗生素的肽聚糖。此外，由于世界范围内结核病的严重程度和抗药性菌株的出现，因此需要治疗结核病的新药。我们还建议再次使用TB SAT来开发和配置丝氨酸乙酰转移酶(SAT)的HTS分析。Sat是合成半胱氨酸所必需的，并在细菌细胞外信号转导中发挥作用。它不在人类身上发现，但在细菌和几种低等真核病原体中发现。因此，靶向SAT为新药开发提供了一种新的方法，它假设许多病原体如果不能产生半胱氨酸，就不能从宿主那里获得足够的供应。这种酶的抑制剂，就像HTS所发现的那样，需要用来验证这一假设。这两种酶都使用乙酰辅酶A来乙酰化它们的底物，两种酶的检测都依赖于它们产生的游离辅酶A的检测。辅酶A通过其游离的SH基团通过形成荧光加合物来检测。我们有初步数据表明，这种方法适用于GAT，并建议为GAT开发它，并将其扩展到SAT。在开发每一种分析方法后，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机实现自动化来配置它们以用于HTS。然后，我们建议筛选5000到20000种化合物来测试每种检测方法。未来的研究将包括向NIH路线图倡议提交分析，以筛选大量化合物，以便改进对新药临床前开发候选药物的命中。与公共卫生相关：需要针对细菌和寄生虫的新药，因为抗药性导致现有药物不起作用。这里提出的工作将通过与美国国立卫生研究院合作寻找可以开发成新药的候选化学物质来帮助开发这种新药。

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.