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Brian P Ceresa, John O Trent; Biochemical Analysis of Potential c-Cbl Antagonists Identified through an in silico Screen. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2621. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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