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

• 批准号: 10646327
• 负责人: Damian J Krysan
• 金额: $ 59.15万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 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/10646327
• 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; 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; Infection; Lead; Libraries; Life; Link; Mammals; Molds; Morbidity - disease rate; Mycoses; Organ; Patients; Persons; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacopoeias; Plants; Polyenes; Roentgen Rays; Saccharomyces cerevisiae; Series; Source; Structure; Testing; Toxic effect; X-Ray Crystallography; anti-cancer; cancer cell; design; drug candidate; fungicide; fungus; high throughput screening; in vitro activity; in vivo; inhibitor; interdisciplinary approach; meter; mortality; nanomolar; novel; pathogen; pathogenic fungus; preclinical development; scaffold; screening; small molecule; small molecule inhibitor

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.

项目摘要 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 在体外具有广谱杀菌活性，在体内具有良好的活性。与这种广泛的活动相一致， 遗传学研究表明ACS对于多种真菌的生存力是必需的（C. albicans、镰刀菌（Fusarium）、S. cerevisiae）。 相反，ACS在哺乳动物中不是必需的。这可能是因为，在哺乳动物和植物中， 乙酰辅酶A是从ATP-柠檬酸裂解酶（ACL），而不是ACS。这条规则最重要的例外是 ACS是乙酰辅酶A的主要来源的癌细胞。因此，ACS已经成为一个 抗癌靶点虽然AR-12的发展停滞不前，但我们认为其目标ACS仍然值得 作为一类新的抗真菌药物的基础进行进一步的探索。为了鉴定真菌ACS的新抑制剂， 我们已经开发了一种多学科方法，其基于：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）整个 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。用C.新形式Acs 1，并确定了492 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。目标1： 将进一步描述这些命中。作为一个平行的策略，我们调整我们的ACS活性测定高通量 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） 在某种程度上是由这样一种假设引导的，即模拟乙酰腺苷单磷酸酯的分子 （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-PO 3结合 通过对AcAMP模拟物的结构-活性研究， 真菌ACS的位置。然后，从该分析中出现的生物化学稳定的、有效的乙酰-PO 3电子等排体将被 与推定的ATP/AMP结合口袋靶向片段连接，以组装候选非核苷、双核苷、 底物ACS抑制剂。为了补充这一基于假设的策略，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屏幕（目标2B和C）。新分子将使用测试漏斗进行评估（目标3）， 包括ACS抑制的生化表征，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 是确定一种领先的ACS抑制剂支架沿着和一个备用系列，用于进一步的临床前开发， 广谱抗真菌候选药物。