Identifying novel c-Cbl antagonists to promote corneal epithelial regeneration

鉴定新型 c-Cbl 拮抗剂以促进角膜上皮再生

基本信息

批准号：
9165379
负责人：
BRIAN P. CERESA
金额：
$ 23.1万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9165379
关键词：
Advanced Development Adverse drug effect Adverse effects Affinity Antineoplastic Agents Bacteria Binding Biological Biological Assay Biology Biomedical Engineering Blindness Calorimetry Cell Proliferation Cells Cetuximab Chemical Injury Chemicals Complement Complex Computer Simulation Cornea Corneal Injury Data Development Diabetes Mellitus Disease Distress Drug Kinetics EGF gene Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Epithelial Epithelial Cells Eye Eye Infections FDA approved Foundations Future Goals Growth Factor Receptors Health Homeostasis Human In Vitro Incidence Infection Investigation Kinetics Laboratory Study Lead Ligands Light Lysosomes Maintenance Measures Mediating Mission Modeling Molecular Natural regeneration Operative Surgical Procedures Oryctolagus cuniculus Pain Patients Pharmacodynamics Pharmacotherapy Prevalence Primary Health Care Property Public Health Reagent Receptor Signaling Recombinants Recurrence Replacement Therapy Research Retina Rodent Signal Transduction Stem cells Structure Testing Therapeutic Tissues Titrations Trauma Virus Vision Wound Healing base biophysical analysis cancer therapy cell growth cell motility corneal epithelium cytokine experience heat injury improved in vivo innovation interest knock-down limbal novel particle prevent receptor response restoration small molecule tool ubiquitin-protein ligase

项目摘要

PROJECT SUMMARY/ABSTRACT An intact and fully differentiated corneal epithelium is critical for proper vision and to keep foreign objects (bacteria, viruses, small particles) out of the eye. However, damage to the corneal epithelium is one of the most common ocular problems presented in primary care facilities and arises from a variety of factors, including trauma, disease, and a side-effect of drugs. Despite the prevalence, discomfort, and potential for blindness associated with perturbation of the corneal epithelium, there are no FDA-approved agents that promote the restoration and homeostasis of this tissue. The long-term goal of our research is to identify novel tools to modulate the molecular mechanisms that regulate and promote corneal epithelial wound healing and homeostasis. The overall objective of this application is to identify novel compounds that prolong EGFR signaling. To accomplish this, we will test the central hypothesis that compounds that block c-Cbl’s ability to ubiquitylate the EGFR, will divert the activated EGFR from the lysosome for degradation. We believe inhibition of this interaction will lead to greater EGFR activity and increase corneal epithelial cell migration, proliferation, and differentiation, three cell biological responses that are central to the restoration and maintenance of corneal epithelial homeostasis. The rationale for these studies is based on our previous findings that knockdown of c-Cbl and inhibition of ubiquitylation enhances EGFR-dependent corneal epithelial wound healing. This research has the following specific aims: 1) Identify candidate compounds that bind with the highest affinity for c-Cbl and 2) Determine whether the highest affinity compounds are the most efficacious antagonists of EGFR ubiquitylation and best prolong EGFR signaling. We have completed an in silico screen of 28,000,000 compounds for their ability to disrupt EGFR:c-Cbl interactions. In Aim 1, we will test the top 50 candidates for their ability to bind recombinant, purified, c-Cbl using a Thermofluor assay and Isothermal Titration Calorimetry. In Aim 2, we will use immortalized corneal epithelial cells to test whether our highest affinity compounds are most effective in preventing ligand-dependent EGFR ubiquitylation, slowing the rate of receptor degradation, and enhancing the rate of corneal epithelial wound healing. Our proposed studies are innovative, in our opinion, because we will modulate novel, ligand-independent molecular mechanisms to increase the magnitude and duration of EGFR activity as a means of accelerating restoration of damaged corneal epithelium. These studies are significant because accelerating re-epithelialization and homeostasis of compromised corneas will decrease the duration of patient distress, minimize the likelihood of infection, and reduce the incidence of blindness. Corneal epithelial homeostasis is a significant public health issue with limited treatment options. This research has the potential to identify new ways of treating damaged corneas following trauma, disease, or as a side-effect of drug treatment as well as provide a better understanding of the molecular mechanisms that regulate corneal epithelial homeostasis.

项目概要/摘要完整且完全分化的角膜上皮对于正常视力和保留异物至关重要（细菌、病毒、小颗粒）离开眼睛然而，对角膜上皮的损害是其中之一。初级保健机构中最常见的眼部问题是由多种因素引起的，包括创伤、疾病和药物副作用，尽管这些疾病普遍存在、不适和潜在的风险。失明与角膜上皮的扰动有关，目前尚无 FDA 批准的药物可以促进该组织的恢复和稳态我们研究的长期目标是找出新的。调节分子机制的工具，调节和促进角膜上皮伤口愈合和本申请的总体目标是鉴定能够延长 EGFR 的新型化合物。为了实现这一目标，我们将测试阻断 c-Cbl 能力的中心假设化合物。泛素化 EGFR，会将激活的 EGFR 从溶酶体中转移出来进行降解，我们相信会产生抑制作用。这种相互作用将导致更大的 EGFR 活性并增加角膜上皮细胞迁移、增殖、和分化，这三种细胞生物反应对于恢复和维持至关重要这些研究的基本原理是基于我们之前的发现：敲除 c-Cbl 和抑制泛素化可增强 EGFR 依赖性角膜上皮伤口这项研究有以下具体目的： 1) 确定与该化合物结合的候选化合物。 c-Cbl 的最高亲和力和 2) 确定最高亲和力化合物是否是最我们已经完成了 EGFR 泛素化的有效拮抗剂和最佳延长 EGFR 信号传导。在目标 1 中，我们对 28,000,000 种化合物进行了计算机筛选，以确定它们破坏 EGFR:c-Cbl 相互作用的能力。将使用 Thermoflor 测定测试前 50 名候选者结合重组、纯化、c-Cbl 的能力和等温滴定量热法在目标2中，我们将使用永生化角膜上皮细胞来测试是否。我们最高亲和力的化合物在预防配体依赖性 EGFR 泛素化、减缓受体降解速度，并提高角膜上皮伤口愈合速度。我们认为，研究是创新的，因为我们将调节新颖的、不依赖于配体的分子增加 EGFR 活性强度和持续时间的机制，作为加速恢复的手段这些研究具有重要意义，因为加速了角膜上皮的再上皮化和恢复。受损角膜的稳态将减少患者痛苦的持续时间，最大限度地减少感染，并减少失明的发生率对于公共卫生具有重要意义。这项研究有可能找到治疗受损的新方法。外伤、疾病或药物治疗副作用后的角膜，以及提供更好的保护了解调节角膜上皮稳态的分子机制。