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

FEBS Letters

DiMaio, John¹  Ni, Feng¹  Gibbs, Bernard¹  Konishi, Yasuo¹ 

[1] National Research Council of Canada, Biotechnology Research Institute, Protein Engineering Section, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada

关键词: Hirudin analog;    Thrombin inhibitor;    Pseudopeptide;    Active site;    Exosite;    NOE;    nuclear Overhauser enhancement;    Boc-AOGO;    5N-tert-butyloxycarbonyl-4-oxo-8-tosyl guanidinooctanoic acid;    TFA;    triflouroacetic acid;    AMC;    7-amino-4-methyl coumarin;    APTT;    activated partial thromboplastin time;   

DOI  :  10.1016/0014-5793(91)80441-5

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A synthetic hirudin peptide analog corresponding to N′-acetyl [D-Phe⁴⁵, ArgΨ(COCH₂)⁴⁷, Gly⁴⁸]desulfo hirudin^55–65 (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 α-thrombin (K _i=3.7±0.3 × 10⁻¹⁰ M) and bovine α-thrombin (1.8±0.7 × 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-ArgΨ(COCH₂)CH₃COOCH₃, P79L] corresponding to the amino terminal of P79 also binds competitively to the active site of α-thrombin and inhibited the proteolysis of a tripeptidyl substrate with a K ₁=17.9±2.1 μM (human) and 10.3±3.6 μM (bovine) α-thrombin. NMR experiments indicated that P79L and the corresponding amino terminal residues of P79 occcupy a mutually exclusive binding site on bovine α-thrombin while the carboxyl terminal tail of the latter adopts a similar bound conformation as the fragment hirudin??? which is known to interact with the ‘anion’ exosite. Taken together these results provide conclusive evidence that the high antithrombin activity of N???-acetyl[D-Phe⁴⁵, ArgΨ(COCH₂)⁴⁷, Gly⁴⁸]desulfo hirudin^45–65 stems from the concurrent interaction with the catalytic site and the putative ‘anion’ exosite through its respective NH₂- and COOH-terminal recognition sites.

【 授权许可】

Unknown