Molecular screen for isopeptidase inhibitors to treat pulmonary disease

描述(由申请人提供)：AMSH是一种最近被描述的泛素异肽酶，与人上皮生长因子受体(EGFR)的内体转运有关。泛素途径与调节EGFR细胞活性的信号转导和转运机制密切相关。泛素E3连接酶Cbl促进EGFR的泛素化和分选到晚期内体和溶酶体，在那里它被酸性蛋白酶降解，而去泛素化酶AMSH通过从EGFR中去除泛素来消除这一作用，允许它循环到膜上，并保持其调节细胞生长和分裂的能力。已经证明，纯化的AMSH在体外可以从EGFR中去除泛素，并且siRNA介导的在HeLa细胞中去除AMSH导致了EGFR降解的增加(McCullough，Clague等人)。2004年)。因此，应该有可能发现在细胞中模拟siRNA效应的AMSH抑制剂。EGFR是癌症药物发现的一个公认的靶点，无论是作为受体还是作为一种激酶。最近有研究表明，EGFR也是发现抗粘液高分泌和慢性肺部疾病相关纤维化的化合物的一个有吸引力的靶点。在这一为期一年的第一阶段应用中，建议开发一种新的脱泛素酶AMSH的检测方法，并将其配置为高通量筛选；该检测将用于第二阶段，以发现新的抑制剂，这些抑制剂将降低EGFR的细胞水平和活性，从而通过一种新的机制发挥抗肺部疾病的活性，该新机制利用EGFR的生理调节，而不是直接攻击它。此外，在第一阶段，非特异性泛素异肽酶抑制剂将在体外测试异肽酶活性，并在基于细胞的测试中测试EGFR的衰减。开发AMSH检测的最终商业目标是发现一种对哮喘、COPD和其他慢性肺部疾病有效的药物。AMSH是一种最近被描述的泛素异肽酶，它可以从EGFR中移除泛素，防止其降解，并允许其循环到膜上，并保持其调节细胞生长和分裂的能力。最近有研究表明，EGFR已被认为是肿瘤疾病的治疗靶点，也是发现抗粘液高分泌和与慢性肺部疾病(COPD)相关的纤维化的化合物的诱人靶点。Progenra建议开发一种以高通量模式测量AMSH活性的测试方法，以便在第二阶段筛选化合物，以找到可能对COPD和其他肺部疾病具有活性的AMSH抑制剂。

DESCRIPTION (provided by applicant): AMSH is a recently described ubiquitin isopeptidase that is associated with endosomal trafficking of the human epithelial growth factor receptor (EGFR). The ubiquitin pathway is closely involved in the signal transduction and trafficking mechanisms that regulate the cellular activity of the EGFR. The ubiquitin E3 ligase Cbl promotes the ubiquitination and sorting of the EGFR to late endosomes and lysosomes, where it is degraded by acidic proteases, and the deubiquitinating enzyme AMSH abrogates this effect by removing ubiquitin from EGFR, permitting it to recycle to the membrane and retain its ability to regulate cell growth and division. It has been shown that purified AMSH removes ubiquitin from EGFR in vitro, and that siRNA mediated ablation of AMSH in HeLa cells results in increased EGFR degradation (McCullough, Clague et al. 2004). It should thus be possible to discover inhibitors of AMSH that mimic the effects of siRNA in cells. EGFR is a well-established target for cancer drug discovery, either as a receptor or a kinase. It has recently been shown that EGFR is also an attractive target for the discovery of compounds that act against mucus hypersecretion and fibrosis associated with chronic pulmonary disease. It is proposed in this one-year Phase I application to develop a novel assay for the deubiquitinase enzyme AMSH and configure it for high throughput screening; this assay will be used in Phase II to discover novel inhibitors that will reduce the cellular level and activity of EGFR and thereby have activity against pulmonary disease by a new mechanism that exploits the physiological regulation of the EGFR, as opposed to attacking it directly. In addition, in Phase I, nonspecific ubiquitin isopeptidase inhibitors will be tested in in vitro assays for isopeptidase activity and in cell based assays for attenuation of EGFR. The ultimate commercial goal of the development of an assay for AMSH is to discover a drug with efficacy against asthma, COPD, and other chronic pulmonary diseases. AMSH is a recently described ubiquitin isopeptidase, an enzyme that removes ubiquitin from EGFR, preventing its degradation and permitting it to recycle to the membrane and retain its ability to regulate cell growth and division. It has recently been shown that EGFR, already known to be a therapeutic target for neoplastic disease, is also an attractive target for the discovery of compounds that act against mucus hypersecretion and fibrosis associated with chronic pulmonary disease (COPD). Progenra proposes to develop an assay to measure AMSH activity in a high throughput mode so that, in Phase II, compounds can be screened to find inhibitors of AMSH that may have activity in COPD and other pulmonary diseases.