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

基本信息

批准号：
8135485
负责人：
David E Sterner
金额：
$ 46.75万
依托单位：
PROGENRA, INC.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-15 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8135485
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 airway remodeling 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

项目摘要

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）：在蛋白酶体抑制剂Velcade用于多发性骨髓瘤疗法的批准下，泛素途径已被验证用于药物发现。另一种与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上皮生长因子受体（EGFR）的降解，并将其标记为溶酶体降解，而不是回收到膜。去肽酶去除泛素可以避免EGFR并有利于回收利用并增强有丝分裂发生。该建议集中在AMSH上，AMSH是一种泛素异肽酶，可防止EGFR的内体分类和溶酶体降解。慢性肺部疾病，包括慢性阻塞性肺部疾病（COPD），哮喘和肺动脉高压（PAH）的特征是气道和血管重塑，并且仍然是异常常见的疾病。 EGF和EGFR与慢性肺部疾病的病理生物学有关。该项目的目的是通过促进EGFR的自然降解来发展对这些疾病的积极影响。在第一阶段，使用N末端泛素融合的底物报道的AMSH活性抑制剂配置了高吞吐量屏幕。针对异肽酶Ubpy验证了类似的反屏幕。此外，验证了基于细胞的测定检测EGFR降解，完成了第一阶段的目标。在第二阶段，首先，将使用AMSH高吞吐量测定法对几个化学文库进行筛选，并在二级测定中以选择性为单位。接下来，将进行疗效研究；最佳命中促进EGFR降解和抑制EGFR活性的能力将使用人类气道平滑肌（ASM），肺动脉血管平滑肌（PVSM）细胞和人类肺成纤维细胞进行评估。将确定铅化合物是否调节这些细胞中的EGFR水平以及效果是否以浓度和时间依赖性方式调节EGF诱导的细胞增殖。通过Western印迹分析，将确定对EGF诱导的EGFR水平激活影响的浓度依赖性。铅化合物对EGF诱导的ASM，PVSM和HLFS增殖的影响将表明生理相关性。这项研究将提供有关所选潜在客户潜在功效的关键信息。最后，将采用药物化学来建立结构 - 活性关系（SAR）和化学优化，从而导致选择候选者以促进临床前发育以治疗肺部疾病。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）的特征是气道和血管重塑，并且仍然非常常见。突出的细胞生长因子（EGF）及其受体（EGFR）在慢性肺部疾病中起作用。该第二阶段项目的目的是开发导致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的抑制剂间接完成，该酶通常会保持EGFR高的细胞水平，从而促进气道炎症和肺部疾病。将筛选几个小型化学分子的收集，以鉴定AMSH的抑制剂。将使用该项目的第一阶段开发的测定法完成筛查。这些抑制剂的最有希望的人将在细胞模型中进行测试，以查看它们是否在细胞中起作用以降低EGFR水平和活性，如所预测的。这些抑制剂中最好的化学修饰将进行，以产生候选分子作为治疗肺部疾病和气道炎症的药物。