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

• 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
• 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

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，角膜上皮损伤是造成角膜上皮损伤的原因之一。 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、疾病和药物的副作用。尽管普遍存在、不适和潜在的 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颖的 调节分子机制的工具，调节和促进角膜上皮伤口愈合和 体内平衡。本申请的总体目标是鉴定能够延长 EGFR 的新型化合物 发信号。为了实现这一目标，我们将测试中心假设，即阻碍 c-Cbl 能力的化合物 泛素化 EGFR，会将激活的 EGFR 从溶酶体转移以进行降解。我们相信抑制 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 角膜上皮稳态。这些研究的基本原理是基于我们之前的发现： 敲低 c-Cbl 和抑制泛素化可增强 EGFR 依赖性角膜上皮伤口 康复。本研究有以下具体目标： 1) 鉴定与 c-Cbl 的最高亲和力和 2) 确定最高亲和力化合物是否是最 EGFR 泛素化的有效拮抗剂，可最好地延长 EGFR 信号传导。我们已经完成了 对 28,000,000 种化合物进行计算机筛选，以确定它们破坏 EGFR:c-Cbl 相互作用的能力。在目标 1 中，我们 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。我们提出的 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 角膜上皮受损。这些研究意义重大，因为加速了上皮化和 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，减少失明的发生率。角膜上皮稳态是一个重要的公共卫生问题 治疗选择有限的问题。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。