Abstract
Purpose :
Under experimental conditions, growth factor receptors, such as the Epidermal Growth Factor Receptor (EGFR), are critical in stimulating the re-epithelialization of the cornea. However, the addition of exogenous ligand [i.e. Epidermal Growth Factor (EGF)] has had a limited therapeutic effect. The EGFR’s response is limited, in part, by ligand-mediated receptor desensitization. In this study, we are developing new antagonists against c-Cbl/Cbl-b (referred to as CBL antagonists), the key E3 ubiquitin ligases that target the activated receptor for lysosomal degradation. We hypothesize that the clinical use of CBL antagonists will inhibit receptor desensitization and sustain EGFR signaling to increase corneal re-epithelialization and promote homeostasis
Methods :
Experiments were performed in immortalized human corneal epithelial (hTCEpi) cells. Knockout cells were engineered using CRISPR/CAS9 to knockout the E3 ubiquitin ligases, c-Cbl and Cbl-b. hTCEpi cells expressing only CAS9 were used as a control. Receptor phosphorylation was monitored via immunoblot using phospho-specific antibodies. Ligand-dependent ubiquitylation was measured by immunoprecipitating the EGFR and immunoblotting for the presence of ubiquitin. Receptor endocytosis was assessed using 125I-EGF. An in vitro wound healing assay measured monitored the rate of cell movement into an acellular area by live cell microscopy. A lead compound was identified via in silico screen to disrupt c-Cbl:EGFR interactions. Derivatives of this compound were generated by the systematic subtraction and addition of functional groups. These derivatives were screened for c-Cbl binding using microscale thermophoresis. The highest affinity compounds were tested for their effects on EGFR phosphorylation.
Results :
Knockout of c-Cbl and Cbl-b demonstrated these E3 ubiquitin ligases are the primary mediators of EGFR ubiquitylation and negatively regulate EGFR signaling. After derivation from our lead compound, we have identified the c-Cbl antagonist, compound 5-6, as having the ability to inhibit EGFR ubiquitylation, sustain receptor phosphorylation, and enhance in vitro wound healing.
Conclusions :
Here we describe 5-6 as a novel compound that antagonizes EGFR:Cbl interactions, ultimately leading to enhanced EGFR-mediated corneal epithelial biology. This study provides proof-of-principle that CBL antagonists warrant investigation using in vivo models.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.