Development of c‐Cbl Antagonists to Promote Corneal Epithelial Wound Healing

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Ligand mediated desensitization is a key negative regulator of cell surface receptors. In the human cornea, epidermal growth factor receptor (EGFR) desensitization, due to the relatively high levels of endogenous EGF in the tear fluid, limits the repair and restoration of the corneal epithelium. The E3 ubiquitin ligase, c‐Cbl is a critical modulator of EGFR desensitization, as it mediates the ubiquitylation of the activated receptor and targets it to the lysosome for degradation. Ultimately, this terminates receptor signaling. We have previously demonstrated that knock down of c‐Cbl results in an enhancement of EGFR‐mediated cell migration that is necessary for healing of corneal epithelial wounds. The goal of these experiments was to identify novel compounds that antagonize c‐Cbl activity and could be developed to enhance EGFR‐mediated corneal epithelial wound healing. Using the published crystal structures of the EGFR:c‐Cbl complex, we identified the point of interaction between the two proteins. We subsequently performed an in silico screen of ~25,000,000 compounds (from the ZINC15 library) to identify compounds that are predicted to antagonize c‐Cbl binding to and ubiquitylation of the EGFR. The top 50 candidates were screened using a biochemical assay for their ability to bind c‐Cbl using Differential Scanning Fluorimetry (Thermofluor™). From that screen, six compounds were identified as c‐Cbl binders and were tested in immortalized corneal epithelial cells to determine which compounds could inhibit c‐Cbl function. Two of the six compounds blocked EGF‐mediated EGFR ubiquitylation, albeit at relatively high concentrations (IC50 were ~50 μM and ~5 μM). Further, these compounds exhibited some cytotoxicity at 100 μM. These compounds represent two lead compounds that can be chemically optimized to enhance their affinity for c‐Cbl and become more potent inhibitors of c‐Cbl activity. The long‐term goal is to use these compounds as therapeutic agents to promote the restoration of damaged corneal epithelium, to minimize patient discomfort, reduce ocular infections, and minimize the likelihood of blindness.Support or Funding InformationResearch was supported by NIH/NEI grant EY027032.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.