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Characterizing Eph receptor and ephrin antagonism by in silico modeling and cellular-based binding assays
Location: 72
Mentor: Dr. Daniel Palaez
Ephrin receptors (EphRs), the largest class of receptor tyrosine kinases, regulate axon guidance, synaptic plasticity, and cell migration. Their role in neurodegenerative diseases and cancer has made them attractive therapeutic targets, yet few approved drugs exist, and those available act on limited receptors. Given the promiscuous binding between EphRs and ephrin ligands and the overlap in signaling of the 14 known human receptors, a broad-spectrum therapeutic approach is needed. The Pelaez lab designed peptides based on ephrin binding domain homology, with prior in vivo studies demonstrating their therapeutic potential in optic neuropathy models. To characterize their targets and interactions, we used computational modeling methods, such as AlphaFold Multimer and HADDOCK, to analyze EphR ligand binding domains (LBDs). We identified conserved residues predicted to interact with our peptides, suggesting their potential for competitive antagonism of ephrin ligands in multiple EphRs. For in vitro validation, we developed stable cell lines expressing nanoLuciferase (nLuc)-EphR-mCherry fusion proteins for EphA5, EphB1, and EphB2. Using a cell-based nanoBRET assay, we assessed peptide interactions by measuring energy transfer between nLuc-tagged EphRs and fluorescently conjugated ephrin ligands. Additionally, fluorescence microscopy confirmed peptide colocalization with EphRs, further supporting their receptor affinity. These findings enhance our understanding of EphR binding dynamics and inform future studies in optic neuropathies, contributing to the development of novel pan-EphR antagonists.
