Bifunctional Peptide Boronate Inhibitors of Thrombin:  Crystallographic Analysis of Inhibition Enhanced by Linkage to an Exosite 1 Binding Peptide

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance 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 similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human α-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final K i of 2.2 pM, k 1 of 3.51 × 107 M-1 s-1, and k - 1 of 1.81 × 10-4 s-1). The addition of hirugen peptide (20 μM) competes for exosite 1 binding and restores the k 1 and k - 1 to that of the analogous tripeptide, 0.29 × 107 M-1 s-1 and 0.13 × 10-4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with K iTry/K iThr of ∼900 compared to the analogous tripeptide, with K iTry/K iThr of ∼4. Compound 2 acts as a competitive inhibitor (K iThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human α-thrombin with compound 1 (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 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and 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 no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583−601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.