Chemical Optimization of c-Cbl Antagonists for Corneal Wound Healing

项目摘要 完整和完全分化的角膜上皮对于正常视力和保持异物至关重要 （细菌、病毒、小颗粒）。然而，对角膜上皮的损伤是导致角膜炎的原因之一。 最常见的眼部问题出现在初级保健设施中并且由多种因素引起， 包括创伤疾病和药物副作用尽管流行，不适，和潜在的 与角膜上皮扰动相关的失明，没有FDA批准的药物， 促进该组织的恢复和体内平衡。我们研究的长期目标是开发新的 促进角膜上皮伤口愈合和体内平衡的化合物。本报告的总体目标 本发明的应用是对最近鉴定的c-Cbl拮抗剂进行化学优化以增加其亲和力和效力， 同时降低细胞毒性。为了实现这一点，我们将测试中心假设，化合物， 破坏EGFR：c-Cbl相互作用将阻止EGFR泛素化，将活化的EGFR从溶酶体转移， 并延长EGFR信号传导。基于初步研究，这种抑制EGFR活性并增加 通过细胞迁移、增殖和分化恢复和维持角膜上皮。的 这些研究的基本原理是敲低c-Cbl并抑制EGFR泛素化，EGFR泛素化活性， 并增强角膜上皮伤口愈合。在对25，000，000种化合物进行计算机筛选后，我们 已经鉴定了几种结合c-Cbl并抑制配体介导的EGFR泛素化的先导化合物。我们 寻求将该顶级化合物开发成具有以下特定目的的治疗剂。在目标1中，我们 在结构上优化我们的先导化合物以增加其对c-Cbl的亲和力并破坏EGFR：c-Cbl结合。 对我们的先导化合物进行多轮化学修饰将用于鉴定新的衍生物， 以高亲和力结合重组c-Cbl并破坏EGFR：c-Cbl相互作用。在目标2中，我们将测试最高 用于破坏c-Cbl介导的EGFR泛素化和运输的亲和化合物。顶部 将测试候选物阻断配体介导的EGFR泛素化和溶酶体的能力。 降解，以及它们是否延长角膜上皮细胞中的EGFR信号传导。在目标3中，我们将确定 EGFR泛素化抑制剂促进角膜上皮创伤愈合。顶级化合物将是 使用体外、离体和体内测定来测定角膜上皮伤口愈合。我们建议的研究 在我们看来，这是创新的，因为我们将绕过EGFR占用的限制，靶向一种新的 蛋白质（c-Cbl）和分子机制（受体脱敏）。这些研究意义重大，因为 加速受损角膜的上皮再生和体内平衡将减少患者的痛苦， 最大限度地减少感染，并降低失明的发生率。角膜上皮的稳态是一个主要的公众 治疗有限的健康问题。我们的目标是开发我们的先导化合物，用于局部治疗 损害角膜上皮以恢复组织稳态。

Project Summary 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 develop new compounds that promote corneal epithelial wound healing and homeostasis. The overall objective of this application is to chemically optimize a recently identified c-Cbl antagonist to increase its affinity and potency, while decreasing cytotoxicity. To accomplish this, we will test the central hypothesis that compounds that disrupt EGFR:c-Cbl interactions will prevent EGFR ubiquitylation, divert the activated EGFR from the lysosome, and prolong EGFR signaling. Based on preliminary studies, this prolongs EGFR activity and increases the restoration and maintenance of corneal epithelium via cell migration, proliferation, and differentiation. The rationale for these studies is that knockdown of c-Cbl and inhibits EGFR ubiquitylation, prolongs EGFR activity, and enhances corneal epithelial wound healing. Following an in silico screen of 25,000,000 compounds, we have identified several lead compounds that bind c-Cbl and inhibit ligand-mediated EGFR ubiquitylation. We seek to develop the top compound into a therapeutic agent with the following specific aims. In Aim 1, we will structurally optimize our lead compound to increase its affinity for c-Cbl and disrupt EGFR:c-Cbl binding. Multiple rounds of chemical modifications to our lead compound will be used to identify new derivatives that bind recombinant c-Cbl with high affinity and disrupt EGFR:c-Cbl interactions. In Aim 2, we will test the highest affinity compounds for disruption of c-Cbl-mediated ubiquitylation and trafficking of the EGFR. The top candidates will be tested for their ability to block ligand-mediated EGFR ubiquitylation and lysosomal degradation, and if they prolong EGFR signaling in corneal epithelial cells. In Aim 3, we will determine whether inhibitors of EGFR ubiquitylation promote corneal epithelial wound healing. The top compounds will be assayed for corneal epithelial wound healing using in vitro, ex vivo, and in vivo assays. Our proposed studies are innovative, in our opinion, because we will by-pass the limitation of EGFR occupancy and target a novel protein (c-Cbl) and molecular mechanism (receptor desensitization). These studies are significant because accelerating re-epithelialization and homeostasis of compromised corneas will decrease patient distress, minimize infections, and reduce the incidence of blindness. Corneal epithelial homeostasis is a major public health issue with limited treatments. Our goal is to develop our lead compounds for the topical treatment of compromised corneal epithelium to restore tissue homeostasis.