Molecular screen for isopeptidase inhibitors to treat pulmonary disease

描述（由申请人提供）：随着蛋白酶体抑制剂万珂获批用于多发性骨髓瘤治疗，泛素途径已被验证用于药物发现。另一种泛素相关的降解途径是溶酶体;例如，泛素E3连接酶Cbl通过泛素化受体促进膜结合的上皮生长因子受体（EGFR）的降解，将其标记为溶酶体降解而不是再循环至膜。通过异肽酶去除泛素将使EGFR不受影响，有利于再循环和增强有丝分裂。该提案的重点是AMSH，一种泛素异肽酶，可防止EGFR的内体分选和溶酶体降解。包括慢性阻塞性肺疾病（COPD）、哮喘和肺动脉高压（PAH）的慢性肺疾病的特征在于气道和血管重塑，并且仍然是非常常见的疾病。EGF和EGFR与慢性肺部疾病的病理生物学相关。该项目的目的是通过促进EGFR的自然降解来开发对这些疾病具有活性的药物。在第一阶段，高通量筛选配置为AMSH活性的抑制剂，利用N-末端泛素融合的底物报告。对异肽酶UBPY进行了类似的反筛选。此外，对检测EGFR降解的基于细胞的试验进行了验证，完成了I期的目标。在第二阶段，首先，将使用AMSH高通量测定筛选几个化学文库，并在二次测定中对选择性进行表征。接下来，将进行疗效研究;将使用人气道平滑肌（ASM）、肺动脉血管平滑肌（PVSM）细胞和人肺成纤维细胞评价最佳命中物促进EGFR降解和抑制EGFR活性的能力。将确定先导化合物是否调节这些细胞中的EGFR水平，以及这些作用是否以浓度和时间依赖性方式调节EGF诱导的细胞增殖。将通过蛋白质印迹分析确定对EGF诱导的EGFR水平活化作用的浓度依赖性。先导化合物对EGF诱导的ASM、PVSM和HLF增殖的作用将指示生理相关性。本研究将提供关于所选电极导线潜在有效性的关键信息。最后，药物化学将用于建立结构-活性关系（SAR）和化学优化，从而选择候选药物进行肺部疾病治疗的临床前开发。 公共卫生相关性：包括慢性阻塞性肺疾病（COPD）、哮喘和肺动脉高压（PAH）的慢性肺疾病的特征在于气道和血管重塑，并且仍然是非常常见的疾病。一种重要的细胞生长因子（EGF）及其受体（EGFR）在慢性肺部疾病中发挥作用。该II期项目的目的是开发引起细胞中EGFR自然降解的药物，作为对抗肺部疾病的一种手段。这将通过使用细胞酶AMSH的抑制剂间接实现，AMSH通常保持EGFR的细胞水平高，从而促进气道炎症和肺部疾病。将筛选几种小化学分子的集合以鉴定AMSH的抑制剂。将使用本项目I期开发的试验完成筛选。这些抑制剂中最有前途的将在细胞模型中进行测试，以确定它们是否在细胞中起作用，以降低预期的EGFR水平和活性。将对这些抑制剂中最好的进行额外的化学修饰，以产生候选分子，用于开发治疗肺部疾病和气道炎症的药物。

DESCRIPTION (provided by applicant): With the approval of the proteasome inhibitor Velcade for multiple myeloma therapy, the ubiquitin pathway has been validated for drug discovery. An alternative ubiquitin-associated degradation pathway is lysosomal; for example, the ubiquitin E3 ligase Cbl promotes degradation of membrane bound epithelial growth factor receptor (EGFR) by ubiquitylating the receptor, marking it for lysosomal degradation rather than recycling to the membrane. Removal of ubiquitin by an isopeptidase would spare EGFR and favor recycling and enhanced mitogenesis. This proposal is focused on AMSH, a ubiquitin isopeptidase that prevents endosomal sorting and lysosomal degradation of EGFR. Chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD), asthma, and pulmonary arterial hypertension (PAH) are characterized by airway and vascular remodeling and remain extraordinarily common illnesses. EGF and EGFR are associated with the pathobiology of chronic pulmonary diseases. The aim of this project is to develop agents active against these diseases by promoting the natural degradation of EGFR. In phase I, a high throughput screen was configured for inhibitors of AMSH activity utilizing an N-terminal ubiquitin-fused substrate reporter. A similar counterscreen was validated for the isopeptidase UBPY. In addition, a cell based assay detecting EGFR degradation was validated, completing the aims of Phase I. In phase II, first, several chemical libraries will be screened using the AMSH high throughput assay, and selected hits characterized in secondary assays for selectivity. Next, efficacy studies will be performed; the ability of the best hits to promote EGFR degradation and inhibition of EGFR activity will be evaluated using human airway smooth muscle (ASM), pulmonary arterial vascular smooth muscle (PVSM) cells and human lung fibroblasts. It will be determined whether lead compounds regulate EGFR levels in these cells and whether the effects modulate EGF-induced cell proliferation in a concentration and time-dependent manner. Concentration dependence of effects on EGF-induced activation of EGFR levels will be established by Western blot analysis. Effects of lead compounds on EGF-induced ASM, PVSM and HLFs proliferation will indicate physiological relevance. This study will provide critical information about potential efficacy of the selected leads. Finally, medicinal chemistry will be employed for the establishment of structure-activity relationships (SAR) and chemical optimization, leading to the selection of candidates for progression to preclinical development for treatment of lung disease. PUBLIC HEALTH RELEVANCE: Chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD), asthma, and pulmonary arterial hypertension (PAH) are characterized by airway and vascular remodeling and remain extraordinarily common illnesses. A prominent cellular growth factor (EGF) and its receptor (EGFR) play a role in chronic pulmonary diseases. The aim of this Phase II project is to develop agents that cause the natural degradation of EGFR in cells as a means of combating pulmonary disease. This will be accomplished indirectly by using inhibitors of a cellular enzyme, AMSH, which normally keeps cellular levels of EGFR high, thereby promoting airway inflammation and pulmonary disease. Several collections of small chemical molecules will be screened to identify inhibitors of AMSH. Screening will be accomplished using an assay developed in Phase I of this project. The most promising of these inhibitors will be tested in cellular models to see whether they act in cells to reduce EGFR levels and activity as predicted. Additional chemical modification will be performed on the best of these inhibitors to generate candidate molecules for development as drugs to treat pulmonary disease and airway inflammation.