Molecular screen for isopeptidase inhibitors to treat pulmonary disease

描述（由申请人提供）：AMSH是一种最近描述的泛素异肽酶，与人上皮生长因子受体（EGFR）的内体运输相关。泛素途径与调节EGFR细胞活性的信号转导和运输机制密切相关。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，去泛素化酶AMSH通过从EGFR去除泛素来消除这种作用，允许其再循环到膜并保留其调节细胞生长和分裂的能力。已显示纯化的AMSH在体外从EGFR去除泛素，并且HeLa细胞中siRNA介导的AMSH消融导致EGFR降解增加（McCullough，Clague等人2004）。因此，应该有可能发现AMSH的抑制剂，模拟siRNA在细胞中的作用。EGFR作为受体或激酶是癌症药物发现的公认靶标。最近已经表明，EGFR也是用于发现对抗与慢性肺病相关的粘液分泌过多和纤维化的化合物的有吸引力的靶标。在这项为期一年的I期申请中，提议开发一种用于去泛素酶AMSH的新型检测方法，并将其配置为高通量筛选;该检测方法将用于II期，以发现新型抑制剂，其将降低EGFR的细胞水平和活性，从而通过利用EGFR的生理调节的新机制对肺部疾病具有活性，而不是直接攻击EGFR。此外，在I期试验中，将在异肽酶活性的体外试验和EGFR衰减的细胞试验中检测非特异性泛素异肽酶抑制剂。开发AMSH测定法的最终商业目标是发现对哮喘、COPD和其他慢性肺部疾病有效的药物。AMSH是最近描述的泛素异肽酶，其是从EGFR去除泛素的酶，防止其降解并允许其再循环至膜并保留其调节细胞生长和分裂的能力。最近已经表明，已经已知是肿瘤疾病的治疗靶标的EGFR也是发现对抗与慢性肺病（COPD）相关的粘液分泌过多和纤维化的化合物的有吸引力的靶标。Progenra提出开发一种以高通量模式测量AMSH活性的测定方法，以便在II期中筛选化合物以发现可能在COPD和其他肺部疾病中具有活性的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.