A new class of potent thrombin inhibitors that incorporates a scissile pseudopeptide bond

A synthetic hirudin peptide analog corresponding to N alpha-acetyl [D-Phe45, Arg psi(COCH2)47, Gly48]desulfo hirudin45-65 (P79) was synthesized. Comparative kinetic studies showed that while recombinant hirudin (HV2) is a slow-tight binding inhibitor, P79 behaves as a classical competitive inhibitor of human alpha-thrombin (Ki = 3.7 +/- 0.3 x 10(-10) M) and bovine alpha-thrombin (1.8 +/- 0.7 x 10(-9) M). P79 showed saturable inhibition of plasma APTT. The P1' subsite of P79 is isosteric with the glycine residue of the natural thrombin substrate fibrinogen, but is proteolytically stable due to the incorporation of a ketomethylene pseudopeptide bond. The model active site-directed tripeptide [D-Phe-Pro-Arg psi(COCH2)CH2COOCH3, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of alpha-thrombin and inhibited the proteolysis of a tripeptidyi substrate with a Ki = 17.9 +/- 2.1 microM (human) and 10.3 +/- 3.6 microM (bovine) alpha-thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occupy a mutually exclusive binding site on bovine alpha-thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin55-65 which is known to interact with the 'anion' exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N alpha-acetyl[D-Phe45, Arg psi(COCH2)47, Gly48]desulfo hirudin45-65 stems from the concurrent interaction with the catalytic site and the putative 'anion' exosite through its respective NH2- and COOH-terminal recognition sites.