Design of P1‘ and P3‘ Residues of Trivalent Thrombin Inhibitors and Their Crystal Structures

Abstract

Synthetic bivalent thrombin inhibitors comprise an active site blocking segment, a fibrinogen recognition exosite blocking segment, and a linker connecting these segments. Possible nonpolar interactions of the P1‘ and P3‘ residues of the linker with thrombin S1‘ and S3‘ subsites, respectively, were identified using the “Methyl Scan” method [Slon-Usakiewicz et al. (1997) Biochemistry 36, 13494−13502]. A series of inhibitors (4-tert-butylbenzenesulfonyl)-Arg-(d-pipecolic acid)-Xaa-Gly-Yaa-Gly-βAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-(β-cyclohexylalanine)-(d-Glu)-OH, in which nonpolar P1‘ residue Xaa or P3‘ residue Yaa was incorporated, were designed and improved the affinity to thrombin. Substitution of the P3‘ residue with d-phenylglycine or d-Phe improved the Ki value to (9.5 ± 0.6) × 10-14 or 1.3 ± 0.5 × 10-13 M, respectively, compared to that of a reference inhibitor with Gly residues at Xaa and Yaa residues (Ki = (2.4 ± 0.5) × 10-11 M). Similarly, substitution of the P1‘ residue with l-norleucine or l-β-(2-thienyl)alanine lowered the Ki values to (8.2 ± 0.6) × 10-14 or (5.1 ± 0.4) × 10-14 M, respectively. The linker Gly-Gly-Gly-βAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvment of the Ki values to (3.8 ± 0.6) × 10-14 or (1.7 ± 0.4) × 10-14 M, respectively. These Ki values are equivalent to that of natural hirudin (2.2 × 10-14 M), yet the size of the synthetic inhibitors (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with l-norleucine or l-β-(2-thienyl)alanine at the P1‘ residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human α-thrombin. The crystal structures of these complexes were solved and refined to 2.1 Å resolution. The Lys60F side chain of thrombin moved significantly and formed a large nonpolar S1‘ subsite to accommodate the bulky P1‘ residue.