Discovery and optimization of antifungal acetyl CoA synthetase inhibitors

项目总结 最近，我们发现了一种乙酰辅酶A合成酶(ACS)的小分子抑制剂AR-12，它具有广泛的 体外抑菌活性和体内预期活性。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)的生存是必不可少的。 相比之下，ACS在哺乳动物中并不是必需的。这很可能是因为，在哺乳动物和植物中，绝大多数 乙酰辅酶A的活性来源于三磷酸腺苷-柠檬酸裂解酶(ACL)，而不是ACS。这条规则最重要的例外是 癌细胞，其中ACS是乙酰辅酶A的主要来源。因此，ACS已经成为一种 抗癌靶标。尽管AR-12的发展停滞不前，但我们认为它的目标--ACS仍然值得 为进一步探索作为新型抗真菌药物的基础。为了鉴定真菌ACSS的新抑制剂， 我们开发了一种多学科的方法，基于：1)两个互补的小分子筛选 策略；2)多种病原真菌的ACS-抑制剂复合体的结构表征：3)整体 ACS功能和抑制的细胞分析，以及4)已经取得成果的药物化学策略 微摩尔血管紧张素转换酶抑制剂。新生链球菌Acs1的STD-核磁共振筛选及鉴定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歌曲的特点。作为一种并行策略，我们调整了我们的ACS活性分析以实现高通量 筛选(HTS)，目标是直接识别小分子ACS抑制剂。我们的化学计划(目标2) 在一定程度上是由一种假说指导的，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-PO3结合 AcAMP模拟物的构效关系研究 真菌ACSS的地点。从这一分析中产生的生化稳定、有效的乙酰-PO3异构体将是 与可能的ATP/AMP结合的口袋靶向片段连接以组装候选的非核苷，bi- 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 由其他强相互作用的片段组装而成，我们将优化直接在ACS中确定的抑制剂 基于活动的HTS屏幕(AIMS 2B和C)。将使用测试漏斗对新分子进行评估(目标3) 包括ACS抑制的生化特征，对一系列病原真菌的抗真菌活性， 全细胞抗急性冠脉综合征靶向活性测定，以及初步的体外毒性/ADME表征。我们的目标 是确定一种主要的ACS抑制剂支架以及用于进一步临床前开发的备份系列 广谱抗真菌药物候选药物。

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.