Chemical Optimization of c-Cbl Antagonists for Corneal Wound Healing

项目总结

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.