Abstract
Purpose :
Activation of the Epidermal Growth Factor Receptor (EGFR) is critical in corneal epithelial regeneration and homeostasis. The clinical use of EGFR ligands (e.g. epidermal growth factor – EGF) are not reliable tools to restore and maintain the epithelial layer, likely due to high endogenous EGF levels and intrinsic mechanisms of receptor down-regulation. One mediator of EGFR down-regulation is the E3 ubiquitin ligase, c-Cbl. Ubiquitylation of the EGFR targets it for lysosomal degradation; using RNAi to attenuate c-Cbl expression enhanced the kinetics of corneal epithelial wound healing. We postulate that supplementing the relatively high, endogenous levels of EGF (~2-3 ng/ml) in human tears with a c-Cbl antagonist will prevent EGFR down-regulation and enhance receptor activity and promote homeostasis of the epithelial layer. In this study, we identify and test potential c-Cbl antagonist for binding and inhibitory activity.
Methods :
Using published crystal structures of the EGFR and c-Cbl and their domains of interaction, 25,000,000 compounds were screened using an in silico assay. The top candidates were identified and tested for binding to recombinant c-Cbl using a Thermofluor® assay. EGFR ubiquitylation was measured by treating immortalized human corneal epithelial cells with EGF, immunoprecipitating the receptor, and immunoblotting for the presence of conjugated ubiquitin.
Results :
Of the ranked compounds, 50 were tested for binding to recombinant c-Cbl. Four of these bound to c-Cbl as indicated by a shift in the Thermofluor® fluorescence profile. Of these four compounds, one resulted in a decrease in ligand-mediated EGFR ubiquitylation.
Conclusions :
We have initiated our screen of potential c-Cbl antagonists and identified four compounds that directly bind c-Cbl. One compound prevents ligand-mediated EGFR ubiquitylation. Since liganded EGFRs that do not get ubiquitylated are diverted from the lysosome for degradation, EGFR signaling is sustained. This compound is a promising, first-generation c-Cbl antagonist that circumvents the limitations of exogenous ligands as a corneal epithelial wound healing therapy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.