Identifying novel c-Cbl antagonists to promote corneal epithelial regeneration

项目摘要/摘要 完整和完全分化的角膜上皮对于正常视力和保存异物至关重要。 (细菌、病毒、小颗粒)从眼睛里流出。然而，对角膜上皮的损伤是 最常见的眼部问题出现在初级保健机构，并由各种因素引起， 包括创伤、疾病和药物的副作用。尽管患病率、不适感和潜在的 与角膜上皮扰动相关的失明，目前还没有FDA批准的药物可以 促进这一组织的修复和动态平衡。我们研究的长期目标是识别小说 调节和促进角膜上皮伤口愈合的分子机制的工具和 动态平衡。本申请的总体目标是确定能够延长EGFR的新化合物 发信号。为了实现这一点，我们将测试中心假设，即阻止c-Cbl能力的化合物 泛素化EGFR，将活化的EGFR从溶酶体转移到降解。我们认为抑制 这种相互作用将导致更大的EGFR活性，并增加角膜上皮细胞的迁移，增殖， 和分化，三种细胞生物学反应，对修复和维持 角膜上皮动态平衡。这些研究的基本原理是基于我们之前的发现 C-Cbl基因下调和泛素化抑制增强EGFR依赖的角膜上皮损伤 治愈。这项研究有以下具体目的：1)确定与 对c-Cbl的最高亲和力和2)决定最高亲和力的化合物是否最 有效的EGFR泛素化拮抗剂和最佳延长EGFR信号。我们已经完成了 对28,000,000种化合物进行电子筛查，以了解它们破坏EGFR：C-Cbl相互作用的能力。在目标1中，我们 将使用热流分析测试前50名候选人与重组、纯化的c-Cbl的结合能力 等温滴定量热法。在目标2中，我们将使用永生化的角膜上皮细胞来测试 我们的亲和力最高的化合物在阻止配体依赖的EGFR泛素化方面最有效，减缓了 受体降解率，提高角膜上皮创面愈合率。我们的建议 在我们看来，研究是创新的，因为我们将调节新的、不依赖配体的分子 增加EGFR活性的大小和持续时间的机制，作为加速恢复的一种手段 角膜上皮受损。这些研究具有重要意义，因为加速再上皮化和 受损角膜的动态平衡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。角膜上皮细胞动态平衡是一个重要的公共健康问题 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。 角膜创伤后，疾病或作为副作用的药物治疗以及提供更好的 了解调节角膜上皮内环境平衡的分子机制。

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.