High-Throughput screening for inhibitors of neuroinflammatory lipid production

 描述（由申请人提供）：大量遗传性神经系统疾病是由丝氨酸水解酶类中特征不明的酶的有害突变引起的。在过去的十年中，我们的小组开发了一套创新的化学蛋白质组学和代谢组学平台，用于为未表征的丝氨酸水解酶分配功能，并且特别有兴趣将这些平台应用于与人类遗传学与神经系统疾病存在因果关系的丝氨酸水解酶。在本次拨款申请中，我们重点关注丝氨酸水解酶在神经系统疾病 PHARC（多发性神经病、听力损失、共济失调、视网膜色素变性和白内障）中发挥的作用。具体来说，我们已经确定丝氨酸水解酶 ABHD12（其突变会导致 PHARC）是一种主要的脑溶血磷脂酰丝氨酸 (lyso-PS) 脂肪酶。 ABHD12-/- 小鼠表现出大脑溶血 PS 含量升高、听觉和运动缺陷以及神经炎症加剧，这表明溶血 PS 信号传导失调是 PHARC 的一个促成因素。因此，阻断产生 lyso-PS 的上游酶可以提供治疗 PHARC 和可能的其他（神经）炎症性疾病的治疗策略。我们最近确定，迄今为止尚未表征的丝氨酸水解酶 ABHD16A 是一种主要的 PS 脂肪酶，负责在哺乳动物系统（包括小鼠脑和 PHARC 受试者来源的淋巴母细胞）中产生溶血 PS。该应用的目标是对 TSRI 的 640,000 种化合物库进行高通量筛选，以确定结构新颖的 ABHD16A 抑制剂类别，这些抑制剂可以发展为选择性和体内活性化学探针。我们建立了一套多样化且创新的生化、化学蛋白质组学和脂质组学检测方法，以严格评估 ABHD16A 抑制剂的效力、选择性和细胞活性。我们预计该应用的具体目标将提供多种结构不同的 ABHD16A 抑制剂，这些抑制剂显示出适当水平的效力（体外 IC50 < 1 µM）、选择性（丝氨酸水解酶类中有限数量的脱靶（少于 5 个））和细胞活性（原位 IC50 < 10 µM），以作为药物化学优化到体内活性化学探针的先导。我们的合作研究团队在对丝氨酸水解酶进行高通量筛选以及将这些筛选的先导化合物开发成选择性和体内活性抑制剂方面拥有良好的记录。因此，我们有能力将本申请中发现的优先抑制剂发展为优化的化学探针，用于测试 ABHD16A 及其溶血 PS 产品在 PHARC 和其他（神经）炎症性疾病中的功能。

 DESCRIPTION (provided by applicant): A striking number of hereditary nervous system diseases are caused by deleterious mutations in poorly characterized enzymes from the serine hydrolase class. Over the past decade, our group has developed an innovative set of chemical proteomic and metabolomic platforms for assigning functions to uncharacterized serine hydrolases and have a special interest in applying these platforms to serine hydrolases that are causally linked by human genetics to nervous system diseases. In this grant application, we focus on the role that serine hydrolases play in the neurological disorder PHARC (polyneuropathy, hearing loss, ataxia, retinosis pigmentosa, and cataract). Specifically, we have determined that the serine hydrolase ABHD12, mutations of which cause PHARC, is a major brain lyso-phosphatidylserine (lyso-PS) lipase. ABHD12-/- mice exhibit elevated brain lyso-PS content and auditory and motor deficits coupled with heightened neuroinflammation, implicating deregulated lyso-PS signaling as a contributory factor to PHARC. Blocking the upstream enzyme(s) that produces lyso-PS could thus provide a therapeutic strategy to treat PHARC and possibly other (neuro) inflammatory diseases. We recently determined that the heretofore uncharacterized serine hydrolase ABHD16A is a major PS lipase responsible for generating lyso-PS in mammalian systems, including mouse brain and PHARC subject-derived lymphoblasts. The goal of this application is to perform a high-throughput screen of TSRI's 640,000+ compound library to identify structurally novel classes of ABHD16A inhibitors that can be progressed to selective and in vivo-active chemical probes. We have established a diverse and innovative set of biochemical, chemoproteomic, and lipidomic assays to rigorously assess the potency, selectivity, and cellular activity of ABHD16A inhibitors. We expect that the Specific Aims of this application will deliver multiple structurally distinct ABHD16A inhibitors that show suitable levels of potency (in vitro IC50 < 1 µM), selectivity (a limited number of off-targets (lss than five) across the serine hydrolase class), and cellular activity (in situ IC50 < 10 µM) for prioritization as leads for medicinal chemistry optimization into in vivo-active chemical probes. Our collaborative research team has a strong track-record of performing high-throughput screens on serine hydrolases and developing leads from these screens into selective and in vivo-active inhibitors. We are therefore well-equipped to progress the prioritized inhibitors discovered in this application toward optimized chemical probes for testing the function of ABHD16A and its lyso-PS products in PHARC and other (neuro) inflammatory disorders.