Discovery and structural analysis of Eph receptor tyrosine kinase inhibitors

Abstract

The Eph family of receptor tyrosine kinases has drawn growing attention due to their role in regulating diverse biological phenomena. However, pharmacological interrogation of Eph kinase function has been hampered by a lack of potent and selective Eph kinase inhibitors. Here we report the discovery of compounds 6 and 9 using a rationally designed kinase-directed library which potently inhibit Eph receptor tyrosine kinases, particularly EphB2 with cellular EC(50)s of 40nM. Crystallographic data of EphA3 and EphA7 in complex with the inhibitors show that they bind to the 'DFG-out' inactive kinase conformation and provide valuable information for structure-based design of second generation inhibitors.

Figure 1

Chemical structures of five representative library members are shown.

Figure 2

Complex Eph structures. a) A slice of the active site of EphA3 in an orientation optimal for showing all direct ligand interactions. Protein is in green and compound 6 (ALW-II-38–3) in light orange with all nitrogen and oxygen atoms colored blue and red, respectively. b) The EphA7 and 9 (ALW-II-49–7) complex structure is similarly shown, except protein is in purple and the ligand is colored yellow. Secondary structures are shown in cartoon, protein residues in line, and compounds in stick PyMol format. All potential hydrogen-bonding interactions, with distances of less than four Angstroms, between ligand and protein are shown as black, dashed lines.

Figure 3

Compound 9 (ALW-II-49–7) inhibits EphB2 tyrosine kinase activity. The U87 glioblastoma cells were either left untreated or treated for 1 hr with indicated concentrations of ALW-II-49–7 in the presence of ephrinB1. Total lysates were subject to immunoprecipitation with an anti-EphB2 antibody, and immunoprecipitated EphB2 was analyzed by western blot with an anti-phosphotyrosine antibody (top). The blot was reprobed with an anti-EphB2 antibody to show equal loading of proteins (bottom).