Discovery and optimization of antifungal acetyl CoA synthetase inhibitors

抗真菌乙酰辅酶A合成酶抑制剂的发现和优化

基本信息

批准号：
10448463
负责人：
Damian J Krysan
金额：
$ 59.36万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-09 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10448463
关键词：
ATP Citrate (pro-S)-Lyase Acetate-CoA Ligase Acetyl Coenzyme A Active Sites Address Adenosine Monophosphate Affect Anti-Infective Agents Antifungal Agents Aspergillosis Azoles Back Bacterial Meningitis Binding Biochemical Biological Assay Candida Candida albicans Case Series Cellular Assay Chemistry Clinical Complement Complex Cryptococcal Meningitis Cryptococcus Cryptococcus neoformans Crystallization Development Enzymes Esters Fusarium Genetic study Goals Human In Vitro Industrial fungicide Infection Lead Libraries Life Link Mammals Molds Molecular Morbidity - disease rate Mycoses Organ Patients Persons Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacopoeias Plants Polyenes Roentgen Rays Saccharomyces cerevisiae Series Source Structure Testing Time Toxic effect anti-cancer base cancer cell design drug candidate fungus high throughput screening in vitro activity in vivo inhibitor interdisciplinary approach mortality nanomolar novel pathogen pathogenic fungus preclinical development scaffold screening small molecule small molecule inhibitor

项目摘要

PROJECT SUMMARY Recently, we discovered that a small molecule inhibitor of acetyl CoA synthetase (ACS), AR-12, has broad spectrum fungicidal activity in vitro and promising activity in vivo. Consistent with this broad spectrum of activity, genetic studies indicate that ACS is essential for viability in multiple fungi (C. albicans, Fusarium, S. cerevisiae). In contrast, ACS is not essential in mammals. This is likely because, in mammals and plants, the vast majority of acetyl CoA is derived from ATP-citrate lyase (ACL) and not ACS. The most important exception to this rule is the cancer cell where ACS is the predominant source of acetyl CoA. Consequently, ACS has emerged as an anti-cancer target. Although the development of AR-12 stalled, we propose that its target, ACS, remains worthy of further exploration as the basis for a new class of antifungal drugs.To identify novel inhibitors of fungal ACSs, we have developed a multi-disciplinary approach based on: 1) two complementary small molecule screening strategies; 2) the structural characterization ACS-inhibitor complexes from multiple pathogenic fungi: 3) whole cell assays of ACS function and inhibition, and 4) medicinal chemistry strategies that have already yielded micromolar inhibitors of ACS. An STD-NMR screen with C. neoformans Acs1 and identified 492 ACS interacting molecular fragments, of which the vast majority also interacted with multiple fungal ACS enzymes. In Aim 1, we will further characterize these hits. As a parallel strategy, we adapted our ACS activity assay for high throughput screening (HTS) with the goal of directly identifying small molecule ACS inhibitors. Our chemistry plan (Aim 2) is guided, in part, by the hypothesis that molecules mimicking the acetyl adenosine-monophosphate ester (AcAMP) intermediate are likely to be effective inhibitors. In Aim 2A, we will characterize the acetyl-PO3 binding pocket by a structure-activity study of AcAMP mimics derived from molecules already crystallized in the active site of fungal ACSs. Biochemically stable, potent acetyl-PO3 isosteres emerging from this analysis will then be linked with putative ATP/AMP-binding pocket-targeted fragments to assemble candidate non-nucleoside, bi- substrate ACS inhibitors. To complement this hypothesis-based strategy, candidate inhibitors will also be assembled from other strongly interacting fragments and we will optimize inhibitors directly identified in the ACS activity-based HTS screen (Aims 2B&C). New molecules will be evaluated (Aim 3) with a testing funnel that includes biochemical characterization of ACS inhibition, antifungal activity against a range of pathogenic fungi, whole cell assays of on-target activity against ACS, and initial in vitro toxicity/ADME characterization. Our goal is to identify a lead ACS inhibitor scaffold along with a back-up series for further pre-clinical development as broad-spectrum antifungal drug candidates.

项目概要最近，我们发现乙酰辅酶A合成酶（ACS）的小分子抑制剂AR-12具有广泛的应用前景。具有广谱的体外杀菌活性和有前景的体内活性。与这种广泛的活动相一致，遗传学研究表明 ACS 对于多种真菌（白色念珠菌、镰刀菌、酿酒酵母）的生存至关重要。相比之下，ACS 在哺乳动物中并不是必需的。这可能是因为，在哺乳动物和植物中，绝大多数乙酰 CoA 来源于 ATP-柠檬酸裂解酶 (ACL)，而不是 ACS。该规则最重要的例外是癌细胞中 ACS 是乙酰辅酶 A 的主要来源。因此，ACS 已成为一种抗癌目标。尽管AR-12的开发陷入停滞，但我们认为其目标ACS仍然值得进一步探索作为新型抗真菌药物的基础。为了鉴定新型真菌 ACS 抑制剂，我们开发了一种多学科方法，基于：1）两种互补的小分子筛选策略； 2) 来自多种病原真菌的 ACS 抑制剂复合物的结构表征：3) 完整 ACS 功能和抑制的细胞测定，以及 4) 已经产生的药物化学策略 ACS 的微摩尔抑制剂。对新型隐球菌 Acs1 进行 STD-NMR 筛选并鉴定出 492 个 ACS 相互作用分子片段，其中绝大多数还与多种真菌 ACS 酶相互作用。在目标 1 中，我们将进一步描述这些热门作品的特征。作为并行策略，我们调整了 ACS 活性测定以实现高通量筛选（HTS），目的是直接鉴定小分子 ACS 抑制剂。我们的化学计划（目标 2）部分是由模仿乙酰腺苷一磷酸酯的分子这一假设引导的 (AcAMP) 中间体可能是有效的抑制剂。在目标 2A 中，我们将表征乙酰基-PO3 结合通过对 AcAMP 模拟物进行结构活性研究，该模拟物衍生自已在活性物质中结晶的分子真菌 ACS 的位点。从该分析中产生的生化稳定、有效的乙酰基-PO3等排物将被与假定的 ATP/AMP 结合袋靶向片段连接以组装候选非核苷、双底物 ACS 抑制剂。为了补充这种基于假设的策略，候选抑制剂也将被由其他强相互作用片段组装而成，我们将优化 ACS 中直接识别的抑制剂基于活动的 HTS 筛选（目标 2B&C）。将使用测试漏斗评估新分子（目标 3）包括 ACS 抑制的生化特征、针对一系列病原真菌的抗真菌活性、针对 ACS 的靶向活性的全细胞测定，以及初始体外毒性/ADME 表征。我们的目标的目的是确定一个主要的 ACS 抑制剂支架以及用于进一步临床前开发的备用系列广谱抗真菌候选药物。