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Specificity of -Cyano--Hydroxy--Methyl-N-[4-(Trifluoromethoxy)Phenyl]-Propenamide as an Inhibitor of the Epidermal Growth Factor Receptor Tyrosine Kinase

Parker Hughes Cancer Center, Departments of Structural Biology [S. G., C. M., E. A. S.], Chemistry [Y. Z.], Biochemistry [X. J., S. M.], Experimental Oncology [F. M. U.], and Drug Discovery Program [F. M. U.], Hughes Institute, St. Paul, Minnesota 55113

The epidermal growth factor receptor (EGFR) tyrosine kinase has an essential function for the survival of human breast cancer cells. In a systematic effort to design potent and specific inhibitors of this receptor family protein tyrosine kinase (PTK) as antibreast cancer agents, we recently reported the construction of a three-dimensional homology model of the EGFR kinase domain. In this model, the catalytic site is defined by two -sheets that form an interface at the cleft between the NH₂-terminal and COOH-terminal lobes of the kinase domain. Our modeling studies revealed a distinct, remarkably planar triangular binding pocket within the kinase domain with approximate dimensions of 15 Å x 12 Å x 12 Å, and the thickness of the binding pocket is 7 Å with an estimated volume of 600 ų available for inhibitor binding. Molecular docking studies had identified -cyano--hydroxy--methyl-N-[4-(trifluoromethoxy)phenyl]-propenamide (LFM-A12) as our lead inhibitor, with an estimated binding constant of 13 µM, which subsequently inhibited EGFR kinase in vitro with an IC₅₀ value of 1.7 µM. LFM-A12 was also discovered to be a highly specific inhibitor of the EGFR. Even at very high concentrations ranging from 175–350 µM, this inhibitor did not affect the enzymatic activity of other PTKs, including the Janus kinases JAK1 and JAK3, the Src family kinase HCK, the Tec family member Bruton’s tyrosine kinase, SYK kinase, and the receptor family PTK insulin receptor kinase. This observation is in contrast to the activity of a quinazoline inhibitor tested as a control, 4-(3-bromo, 4-hydroxyanilino)-6,7-dimethoxyquinazoline, which was shown to inhibit EGFR and other tyrosine kinases such as HCK, JAK3, and SYK.