Identifying novel c-Cbl antagonists to promote corneal epithelial regeneration

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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.