Molecular screen for isopeptidase inhibitors to treat pulmonary disease

治疗肺部疾病的异肽酶抑制剂的分子筛选

• 批准号: 7908583
• 负责人: David E Sterner
• 金额: $ 51.71万
• 依托单位: PROGENRA, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7908583
• 关键词: Antineoplastic Agents; Applications Grants; Asthma; Binding; Biological Assay; Blood Vessels; Cell Nucleus; Cell Proliferation; Cell model; Cells; Chemicals; Chronic; Chronic Obstructive Airway Disease; Collection; Cytosol; Degradation Pathway; Dependence; Development; Disease; Disease model; EGF gene; Endosomes; Enzymes; Epidermal Growth Factor Receptor; Epithelial; Excision; Fibroblasts; Gefitinib; Gleevec; Goals; Growth Factor; Growth Factor Receptors; Homeostasis; Human; Hydrolase; In Vitro; Individual; Lead; Libraries; Lung; Lung diseases; Lysosomes; Maintenance; Membrane; Modification; Molecular; Multiple Myeloma; N-terminal; Organelles; Pathway interactions; Peptide Hydrolases; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Physiological; Play; Proteasome Inhibitor; Proteins; Receptor Activation; Recycling; Reporter; Research; Role; Screening procedure; Smooth Muscle Myocytes; Sorting - Cell Movement; Specificity; Stimulation of Cell Proliferation; Structure-Activity Relationship; System; Testing; Therapeutic; Time; USP8 gene; Ubiquitin; Vascular Smooth Muscle; Vascular remodeling; Velcade; Western Blotting; airway inflammation; base; candidate selection; cell growth; cell growth regulation; combat; counterscreen; design; drug discovery; enzyme pathway; high throughput screening; in vitro Model; inhibitor/antagonist; interest; isopeptidase; meetings; multicatalytic endopeptidase complex; new therapeutic target; novel; novel strategies; pre-clinical; preclinical evaluation; prevent; protein degradation; public health relevance; pulmonary arterial hypertension; receptor; respiratory smooth muscle; small molecule libraries; therapy development; ubiquitin isopeptidase; ubiquitin-protein ligase

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.

描述（由申请人提供）：随着蛋白酶体抑制剂 Velcade 用于多发性骨髓瘤治疗的批准，泛素通路已被验证用于药物发现。另一种泛素相关降解途径是溶酶体；例如，泛素 E3 连接酶 Cbl 通过泛素化受体来促进膜结合上皮生长因子受体 (EGFR) 的降解，将其标记为溶酶体降解而不是循环到膜上。通过异肽酶去除泛素将保护 EGFR，并有利于回收和增强有丝分裂发生。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。慢性肺部疾病，包括慢性阻塞性肺病（COPD）、哮喘和肺动脉高压（PAH），其特征是气道和血管重塑，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学相关。该项目的目的是通过促进 EGFR 的自然降解来开发针对这些疾病的活性药物。在第一阶段，利用 N 端泛素融合底物报告基因对 AMSH 活性抑制剂进行高通量筛选。针对异肽酶 UBPY 验证了类似的反筛选。此外，还验证了检测 EGFR 降解的基于细胞的测定，完成了第一阶段的目标。在第二阶段，首先，将使用 AMSH 高通量测定筛选几个化学文库，并在二次测定中筛选选定的命中以进行选择性。接下来，将进行功效研究；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来评估最佳命中促进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水平以及其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水平激活的影响的浓度依赖性。先导化合物对 EGF 诱导的 ASM、PVSM 和 HLF 增殖的影响将表明生理相关性。这项研究将提供有关所选先导潜在功效的关键信息。最后，药物化学将用于建立构效关系（SAR）和化学优化，从而选择候选药物以进行肺部疾病治疗的临床前开发。 公共卫生相关性：慢性肺部疾病，包括慢性阻塞性肺病 (COPD)、哮喘和肺动脉高压 (PAH)，其特点是气道和血管重塑，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着重要作用。该二期项目的目标是开发能够导致细胞中 EGFR 自然降解的药物，作为对抗肺部疾病的手段。这将通过使用细胞酶 AMSH 的抑制剂来间接实现，AMSH 通常使 EGFR 的细胞水平保持在较高水平，从而促进气道炎症和肺部疾病。将筛选几个小化学分子集合来鉴定 AMSH 抑制剂。筛选将使用该项目第一阶段开发的检测方法来完成。这些抑制剂中最有前途的将在细胞模型中进行测试，看看它们是否能像预测的那样在细胞中发挥作用，降低 EGFR 水平和活性。将对这些最好的抑制剂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。