Abstract
Purpose :
Under experimental conditions, growth factor receptors, such as the Epidermal Growth Factor Receptor (EGFR), stimulate 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 constrained, in part, by ligand-mediated receptor desensitization. To overcome this limitation, we have developed a novel antagonist (compound 3-120) against c-Cbl/Cbl-b, the key E3 ubiquitin ligases that target the activated receptor for lysosomal degradation. These antagonists sustain EGFR signaling in in vitro model (immortalized corneal epithelial cells). We hypothesize compound 3-120 will sustain EGFR phosphorylation in primary corneal epithelial cells and accelerate corneal re-epithelialization and mouse models.
Methods :
Primary human corneal epithelial cells were cultured from corneas that could not be used for transplantation from consenting donors (The Eye Bank of Kentucky). Primary corneal epithelial cells were pre-treated with compound 3-120 or vehicle (0.05% DMSO) for 30 minutes, followed by treatment with 50 ng/ml EGF for 0-120 minutes. EGFR activity was measured by immunoblotting using phosphospecific EGFR antibodies. Corneal epithelial re-epithelialization was measured by monitoring the size of a 1.5 mm debridement wound in the central cornea of adult female C57/Bl6 mouse. The wound was documented by fluorescein staining and imaging by fluorescence microscopy 16 hours after wounding. Wound sizes were quantified with Image J.
Results :
The addition of compound 3-120 enhanced both the magnitude and duration of EGFR phosphorylation following EGF treatment in primary human corneal epithelial cells. Overall, there was an ~45% increase in EGFR signaling. This increase in EGFR signaling was reflected in in vivo experiments. Treatment with EGF and vehicle decrease wound size ~10%, whereas treatment with EGF and compound 3-120 decreases in wound size ~50%.
Conclusions :
Compound 3-120 represents a first in class EGFR:Cbl antagonist. Treatment with 3-120 sustains EGFR phosphorylation is cells which ultimately translates to accelerated corneal re-epithelialization. This study demonstrates that suppressing EGFR desensitization is a viable pharmacological target to accelerate corneal re-epithelialization.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.