RATIONAL DESIGN OF HIRULOG-TYPE INHIBITORS OF THROMBIN

The two crystal structures of thrombin complexed with its most potent natural inhibitor hirudin and with the active-site inhibitor D-Phe-Pro -Arg-CH2Cl [Rydel, T.J. et al., J. Mol. Biol., 221 (1991) 583; Bode, W . et al., EMBO J., 8 (1989) 3467] were used as a basis to design a new inhibitor, combining the high specificity of the polypeptide hirudin with the simpler chemistry of an organic compound. In the new inhibito r, the C-terminal amino acid residues 53-65 of hirudin are linked by a spacer peptide of four glycines to the active-site inhibitor NAPAP on yl-glycyl)-DL-p-amidinophenylalanyl-piperidine). Energy minimization t echniques served as a tool to determine the preferred configuration at the amidinophenyl-alanine and the modified piperidine moiety of the i nhibitor. The predictions are supported by the interaction energies de termined for D- and L-NAPAP in complex with thrombin, which are in goo d agreement with experimentally determined dissociation constants. The conformational flexibility of the linker peptide in the new inhibitor s was investigated with molecular dynamics techniques. A correlation b etween the P1' position and the interactions of the linker peptide wit h the protein is suggested. Modifications of the linker peptide are pr oposed based on the distribution of its main-chain torsion angles in o rder to enhance its binding to thrombin.