Identification of protease activated receptor-2 modulators

描述（由申请人提供）：丝氨酸蛋白酶胰蛋白酶和肥大细胞衍生的类胰蛋白酶激活外周器官中的G蛋白偶联蛋白酶激活受体2（PAR 2）可加重炎症过程和炎症性疾病，如关节炎、哮喘和过敏。 神经病理学疾病，如中风，癫痫和神经退行性疾病-涉及复杂的神经炎症过程，可加重脑损伤。 最近的研究表明，PAR 2及其活化蛋白酶的水平在损伤和损伤期间也在脑中增加，这表明PAR 2活化可能有助于脑损伤中的炎症过程。 神经炎症反应主要由反应性小胶质细胞和星形胶质细胞驱动，并且这两种细胞类型响应于PAR 2选择性活化肽而表现出显著的形态学变化和增殖。 这些观察结果共同产生了这样的假设，即PAR 2活化通过其对小胶质细胞和星形胶质细胞功能的影响在脑中起促炎作用。 尽管假设PAR 2在外周和CMS中具有有趣和重要的作用，但文献中尚未报道该受体的小分子拮抗剂或调节剂。 此外，没有小分子拮抗剂或调节剂可从商业来源获得。 为了便于评估PAR 2在CNS损伤中的作用，并探索PAR 2调节后的治疗机会，我们建议开发一种用于PAR 2变构调节剂的稳健测定法。 我们建议专门寻找以非竞争性方式调节受体的小分子，因为这些分子通常与具有丰富和选择性药理学的位点结合，并且调节剂可以提供包括增加安全性和减少临床副作用在内的优势。 该补助金的目的是：1. 确定一种可用于鉴别小分子抑制剂和增效剂的测定方法。 2. 将试验转换为384孔格式，并设计二级试验，以鉴定竞争性抑制剂以及阳性和阴性变构调节剂。

DESCRIPTION (provided by applicant): Activation of G protein-coupled protease-activated receptor-2 (PAR2) in peripheral organs by the serine proteases trypsin and mast cell-derived tryptase exacerbates inflammatory processes and inflammatory disorders such as arthritis, asthma, and allergies. Neuropathological conditions such as stroke, epilepsy, and neurodegenerative diseases- involve complex neuroinflammatory processes that can exacerbate brain damage. Recent studies suggest that levels of PAR2 and its activating proteases also increase in the brain during injury and damage, suggesting that PAR2 activation may contribute to the process of inflammation in brain injury. Neuroinflammatory responses are driven largely by reactive microglia and astrocytes, and both cell types exhibit dramatic morphological changes and proliferation in response to PAR2-selective activating peptides. These observations together give rise to the hypothesis that PAR2 activation plays a pro- inflammatory role in the brain through its effects on microglial and astrocytic functions. Despite the intriguing and important roles postulated for PAR2 in both peripheral and CMS, no small molecule antagonists or modulators have been reported for this receptor in the literature. Furthermore, no small molecule antagonists or modulators are available from commercial sources. To facilitate the evaluation of the role of PAR2 in CNS injury and explore therapeutic opportunities that follow PAR2 modulation, we propose to develop a robust assay for PAR2 allosteric regulators. We propose to specifically search for small molecules that regulate the receptor in a non-competitive fashion since such molecules often bind to sites with rich and selective pharmacology, and modulators can offer advantages including increased safety and reduced side effects in the clinic. The aims of this grant are: 1. To define an assay that will allow the identification of small molecule inhibitors and potentiators. 2. To convert assay to 384 well format and design secondary assays that will allow the identification of competitive inhibitors as well as positive and negative allosteric modulators.