High-Throughput screening for inhibitors of neuroinflammatory lipid production

 描述(由申请人提供)：大量的遗传性神经系统疾病是由丝氨酸水解酶类特征不佳的酶的有害突变引起的。在过去的十年中，我们的团队开发了一套创新的化学蛋白质组和代谢组平台，用于分配未表征的丝氨酸水解酶的功能，并对将这些平台应用于丝氨酸水解酶特别感兴趣，这些酶因人类遗传学而与神经系统疾病存在因果联系。在这项拨款申请中，我们关注丝氨酸水解酶在神经性疾病PHARC(多发性神经病、听力损失、共济失调、视网膜色素沉着症和白内障)中所起的作用。具体地说，我们已经确定丝氨酸水解酶ABHD12是一种主要的脑溶血磷脂酰丝氨酸(lyso-PS)脂肪酶，它的突变导致PHARC。ABHD12-/-小鼠表现出脑部lyso-PS含量升高以及听觉和运动缺陷，并伴有高度的神经炎症，这表明解除调节的lyso-PS信号是PHARC的一个促成因素。因此，阻断产生溶多糖的上游酶(S)可以为治疗PHARC和可能的其他(神经)炎症性疾病提供一种治疗策略。我们最近确定，迄今为止尚未确定的丝氨酸水解酶ABHD16A是一种主要的PS脂肪酶，负责在哺乳动物系统中产生Lyso-PS，包括小鼠脑和PHARC受试者来源的淋巴母细胞。这项应用的目标是对TSRI的640,000个化合物文库进行高通量筛选，以确定结构新颖的ABHD16A抑制剂类别，这些类别可以发展为选择性和体内活性的化学探针。我们已经建立了一套多样化和创新的生化、化学蛋白质组和脂组分析来严格评估ABHD16A抑制剂的效力、选择性和细胞活性。我们期望这项应用的特定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；lt；1微米)、选择性(丝氨酸水解酶类别中有限数量的非靶标(LSS不超过五个)和细胞活性(原位IC50；10微米)，作为体内活性化学探针的药物化学优化的线索。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从这些筛选中开发出选择性和体内活性抑制剂方面有着良好的记录。因此，我们已经做好了充分的准备，使在本申请中发现的优先考虑的抑制剂朝着优化的化学探针发展，用于测试ABHD16A及其Lyso-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.