Rapid identification of EphA2 ligand-binding domain binders through an optimized NMR fragment-screening workflow

Abstract Eph receptors are involved in the regulation of cell adhesion and migration and are implicated in cancer progression, making them important drug targets. To date, the design of drugs targeting the ligand-binding domain of Eph includes the development of peptide mimetics of the ephrin ligands and the optimization of repurposed drugs. In this work, we report the results of a fragment-based screening (FBS) campaign against the ligand-binding domain of the EphA2 receptor. We introduce the workflow for the selection, filtering and follow-up studies of FBS hits, including an NMR/X-ray hybrid approach for the structure determination of protein/ligand complexes. The proposed workflow allowed us to identify several compounds with the receptor binding affinities of 50-100 µM and IC 50 of 1 µM , better than the activities of the known repurposed drugs. Due to the low molecular weight, the newly developed hits that exhibit an initial biological effect have a high potential for further optimization.