Bifunctional Peptide Boronate Inhibitors of Thrombin: Crystallographic Analysis of Inhibition Enhanced by Linkage to an Exosite 1 Binding Peptide
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- 作者：Emmanuel Skordalakes ; Said Elgendy ; Christopher A. Goodwin ; Donovan Green ; Michael F. Scully ; Vijay V. Kakkar ; Jean-Marie Freyssinet ; Guy Dodson ; and John J. Deadman
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：October 13, 1998
- 年：1998
- 卷：37
- 期：41
- 页码：14420 - 14427
- 全文大小：130K
- 年卷期：v.37,no.41(October 13, 1998)
- ISSN：1520-4995
文摘
The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously toenhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J., Ni,F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski,J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similarapproach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching thissegment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged,bromopropyl residue. Inhibition of human 
-thrombin by compound 1 shows slow, tight-bindingcompetitive kinetics (final Ki of 2.2 pM, k1 of 3.51 × 107 M-1 s-1, and k-1 of 1.81 × 10-4 s-1). Theaddition of hirugen peptide (20 
M) competes for exosite 1 binding and restores the k1 and k-1 to that ofthe analogous tripeptide, 0.29 × 107 M-1 s-1 and 0.13 × 10-4 s-1, respectively. Compound 1 has enhancedspecificity for thrombin over trypsin with KiTry/KiThr of ~900 compared to the analogous tripeptide, withKiTry/KiThr of ~4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selectivewith no effect on trypsin. Crystallographic analysis of complexes of human -thrombin with compound1 (1.8 Å) and compound 2 (1.85 Å) shows a covalent bond between the boron of the inhibitor and Ser195(bond lengths B-O of 1.55 and 1.61 Å, respectively). The isothiouronium group of compound 1 formsbidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov,A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming nointeractions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described forhirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of thesimilarity of binding at each of these sites to that of the analogous peptides added alone, this approachmay be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors andmay also be applicable to the design of inhibitors of other proteases.
-thrombin by compound 1 shows slow, tight-bindingcompetitive kinetics (final Ki of 2.2 pM, k1 of 3.51 × 107 M-1 s-1, and k-1 of 1.81 × 10-4 s-1). Theaddition of hirugen peptide (20 M) competes for exosite 1 binding and restores the k1 and k-1 to that ofthe analogous tripeptide, 0.29 × 107 M-1 s-1 and 0.13 × 10-4 s-1, respectively. Compound 1 has enhancedspecificity for thrombin over trypsin with KiTry/KiThr of ~900 compared to the analogous tripeptide, withKiTry/KiThr of ~4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selectivewith no effect on trypsin. Crystallographic analysis of complexes of human -thrombin with compound1 (1.8 Å) and compound 2 (1.85 Å) shows a covalent bond between the boron of the inhibitor and Ser195(bond lengths B-O of 1.55 and 1.61 Å, respectively). The isothiouronium group of compound 1 formsbidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov,A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming nointeractions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described forhirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of thesimilarity of binding at each of these sites to that of the analogous peptides added alone, this approachmay be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors andmay also be applicable to the design of inhibitors of other proteases.