Chemical Optimization of c-Cbl Antagonists for Corneal Wound Healing

项目概要 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开发新的 促进角膜上皮伤口愈合和体内平衡的化合物。本次活动的总体目标 该应用的目的是对最近鉴定的 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。为了实现这一目标，我们将测试复合的中心假设 破坏 EGFR:c-Cbl 相互作用将阻止 EGFR 泛素化，将激活的 EGFR 从溶酶体转移， 并延长 EGFR 信号传导。根据初步研究，这会延长 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，延长 EGFR 活性， 并增强角膜上皮伤口愈合。经过 25,000,000 种化合物的计算机筛选后，我们 已经鉴定出几种结合 c-Cbl 并抑制配体介导的 EGFR 泛素化的先导化合物。我们 寻求将顶级化合物开发成具有以下特定目标的治疗剂。在目标 1 中，我们将 结构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识别新的衍生物 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，我们将测试最高的 用于破坏 c-Cbl 介导的泛素化和 EGFR 运输的亲和化合物。顶部 将测试候选者阻断配体介导的 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，我们将确定是否 EGFR泛素化抑制剂促进角膜上皮伤口愈合。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。我们提出的研究 我们认为是创新的，因为我们将绕过 EGFR 占用的限制并针对一种新颖的 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化和稳态将减少患者的痛苦， 最大限度地减少感染，并减少失明的发生率。角膜上皮稳态是一个重要的公共 治疗有限的健康问题。我们的目标是开发用于局部治疗的先导化合物 受损的角膜上皮恢复组织稳态。

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.