Biochemical and Crystallographic Characterization of Homologous Non-peptidic Thrombin Inhibitors Having Alternate Binding Modes

The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, Ki = 0.42 nM, crystallographic R factor to 1.8 Å resolution, 19.6%) complexed with human α-thrombin shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3′ position of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refined crystallographic structures of the trifluoromethyl (Ki = 0.37 nM, crystallographic R factor to 2.0 Å resolution = 18.7%), fluoro (Ki = 0.60; R factor to 2.3 Å resolution = 18.4%), methoxy (Ki = 0.91 nM, R factor to 2.2 Å resolution = 19.8%) and methyl (Ki = 0.20 nM, R factor to 2.5 Å resolution = 16.9%) HPBK analogs complexed with thrombin revealed two binding modes for the closely related com-pounds. A less than 1.5-fold variation in affinity was observed for analogs (trifluoromethyl-HPBK and fluoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3′ substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity of the 3′ methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, thrombin and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.