Biochemical and crystallographic characterization of homologous non-peptidic thrombin inhibitors having alternate binding

The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl )]-Gly-boroLys-OH (HPBK, K-i=0.42 nM, crystallographic R factor to 1.8 Angs trom resolution, 19.6%) complexed with human a-thrombin shows that the boro n adopts a tetrahedral geometry and is covalently bonded to the active seri ne, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interact ion with the indole side chain of Trp215. Four HPBK analogs containing eith er electron-withdrawing or electron-donating substitutents at the 3' positi on of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refine d crystallographic structures of the trifluoromethyl (K-i = 0.37 nM, crysta llographic R factor to 2.0 Angstrom resolution = 18.7%), fluoro (K-i = 0.60 ; R factor to 2.3 Angstrom resolution = 18.4%), methoxy (K-i = 0.91 nM, R f actor to 2.2 Angstrom resolution = 19.8%) and methyl (K-i = 0.20 nM, R fact or to 2.5 Angstrom resolution = 16.9%) HPBK analogs complexed with thrombin revealed two binding modes for the closely related compounds. A less than 1.5-fold variation in affinity was observed for analogs (triflouromethyl-HP BK and flouro-HPBK) binding with the edge-to-face interaction. The slight i nductive modulation is consistent with the overall weak nature of the edge- to-face interaction. Owing to an unexpected rotation of the phenethyl aroma tic ring, the 3' substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity o f 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 t he subtle interplay of weak forces that determine the equilibrium binding o rientation of inhibitor, solvent and protein.