Identification of potent pan-ephrin receptor kinase inhibitors using DNA-encoded chemistry technology.

Abstract

EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that <i>EPHA2</i> and <i>EPHA4</i> expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (K_i: 4.0 nM) and EPHA4 (K_i: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC₅₀: 461 nM) and EPHA4 (IC₅₀: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (K_i: 0.13 nM) and EPHA4 (K_i: 0.38 nM) with excellent cell-based potency EPHA2 (IC₅₀: 8.0 nM) and EPHA4 (IC₅₀: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1β-mediated expression of prostaglandin synthase 2 (<i>PTGS2</i>). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.