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VELCADE被批准用于治疗多发性骨髓瘤，泛素途径已被证实可用于药物发现。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通过泛素化促进膜结合的上皮生长因子受体(EGFR)的降解，标志着它被溶酶体降解，而不是再循环到膜上。通过异肽酶去除泛素可以省去EGFR，有利于再循环和促进有丝分裂。这项建议的重点是AMSH，一种泛素异肽酶，可以防止EGFR的内体分选和溶酶体降解。慢性肺病，包括慢性阻塞性肺疾病(COPD)、哮喘和肺动脉高压(PAH)，以呼吸道和血管重构为特征，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有关。该项目的目的是开发通过促进EGFR的自然降解来有效对抗这些疾病的药物。在第一阶段，利用N-末端泛素融合底物报告，为AMSH活性抑制剂配置高通量筛选。对异肽酶UBPY也进行了类似的反筛选。此外，还验证了一种基于细胞的检测EGFR降解的方法，完成了第一阶段的目标。在第二阶段，首先，将使用AMSH高通量分析筛选几个化学文库，并选择选择性在二次分析中表征的HITS。接下来，将进行有效性研究；将使用人气道平滑肌(ASM)、肺血管平滑肌(PVSM)细胞和人肺成纤维细胞来评估最佳HITS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，以及这些影响是否以浓度和时间依赖的方式调节EGF诱导的细胞增殖。通过Western印迹分析，将建立EGF诱导的EGFR水平激活的浓度依赖关系。铅化合物对EGF诱导的ASM、PVSM和HLFS增殖的影响将显示出生理相关性。这项研究将提供有关选定线索的潜在疗效的关键信息。最后，药物化学将被用于建立结构-活性关系(SAR)和化学优化，从而为发展到治疗肺部疾病的临床前开发选择候选药物。 公共卫生相关性：慢性肺部疾病，包括慢性阻塞性肺疾病(COPD)、哮喘和肺动脉高压(PAH)，以呼吸道和血管重构为特征，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。这个第二阶段项目的目的是开发在细胞中导致EGFR自然降解的试剂，作为对抗肺部疾病的一种手段。这将通过使用细胞酶抑制剂AMSH间接实现，AMSH通常会使细胞内EGFR水平保持在较高水平，从而促进呼吸道炎症和肺部疾病。将对几组小化学分子进行筛选，以确定AMSH的抑制剂。筛查将使用本项目第一阶段开发的化验方法完成。这些抑制剂中最有希望的将在细胞模型中进行测试，看看它们是否如预测的那样在细胞中作用于降低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呼吸道炎症的药物。

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.