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确的酶的有害突变引起的。在过去的十年中，我们的小组已经开发了一套创新的化学蛋白质组学和代谢组学平台，用于将功能分配给未表征的丝氨酸水解酶，并对将这些平台应用于丝氨酸水解酶特别感兴趣，这些丝氨酸水解酶与人类遗传学和神经系统疾病有因果关系。在这项拨款申请中，我们专注于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，色素性视网膜病变和白内障）中的作用。具体而言，我们已经确定，丝氨酸水解酶ABHD 12，其突变导致PHARC，是一种主要的脑溶血磷脂酰丝氨酸（溶血PS）脂肪酶。ABHD 12-/-小鼠表现出脑溶血PS含量升高以及听觉和运动缺陷，伴有神经炎症加剧，暗示溶血PS信号转导失调是PHARC的一个促成因素。因此，阻断产生溶血PS的上游酶可以提供治疗PHARC和可能的其他（神经）炎性疾病的治疗策略。我们最近确定，迄今未表征的丝氨酸水解酶ABHD 16 A是一种主要的PS脂肪酶，负责在哺乳动物系统中产生溶血PS，包括小鼠脑和PHARC受试者衍生的淋巴母细胞。该申请的目标是对TSRI的640，000+化合物库进行高通量筛选，以鉴定结构新颖的ABHD 16 A抑制剂，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测定方法，以严格评估ABHD 16 A抑制剂的效价、选择性和细胞活性。我们预计，本申请的特定目的将提供多种结构不同的ABHD 16 A抑制剂，这些抑制剂显示出适当的效力水平（体外IC 50 < 1 µM），选择性（有限数量的脱靶（小于5个）跨越丝氨酸水解酶类），和细胞活性（原位IC 50 < 10 µM），作为药物化学优化为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和将这些筛选的线索开发成选择性和体内活性抑制剂方面有着良好的记录。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ABHD 16 A及其lyso-PS产物在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.