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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：
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
项目类别：
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

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）：随着蛋白酶体抑制剂Velcade被批准用于多发性骨髓瘤治疗，泛素途径已被验证用于药物发现。另一种泛素相关降解途径是溶酶体；例如，泛素E3连接酶Cbl通过泛素化膜结合上皮生长因子受体（EGFR）来促进膜结合上皮生长因子受体（EGFR）的降解，使其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避免EGFR，有利于再循环和增强有丝分裂。该建议的重点是AMSH，一种泛素异肽酶，可阻止EGFR的内体分选和溶酶体降解。慢性肺部疾病，包括慢性阻塞性肺疾病（COPD）、哮喘和肺动脉高压（PAH），以气道和血管重构为特征，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的自然降解来开发抗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物配置了高通量筛选AMSH活性抑制剂。类似的反筛对异肽酶UBPY进行了验证。此外，一种基于细胞的检测EGFR降解的方法得到了验证，完成了第一阶段的目标。在第二阶段，首先，将使用AMSH高通量试验筛选几个化学文库，并在二级分析中选择选择性。接下来，将进行疗效研究；我们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药物促进EGFR降解和抑制EGFR活性的能力。将确定铅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的方式调节egf诱导的细胞增殖。对egf诱导的EGFR激活水平的影响的浓度依赖性将通过Western blot分析建立。先导化合物对egf诱导的ASM、PVSM和hlf增殖的影响将显示出生理相关性。本研究将提供有关所选引线潜在功效的关键信息。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临床前开发，用于治疗肺部疾病。