Design of P1 ' and P3 ' residues of trivalent thrombin inhibitors and their crystal structures

Synthetic bivalent thrombin inhibitors comprise an active site blocking seg ment, a fibrinogen recognition exosite blocking segment, and a linker conne cting these segments. Possible nonpolar interactions of the P1' and P3' res idues of the linker with thrombin S1' and S3' subsites, respectively, were identified using the "Methyl Scan" method [Slon-Usakiewicz et al. (1997) Bi ochemistry 36, 13494-13502]. A series of inhibitors (4-tert-butylbenzenesul fonyl)-Arg-(D-pipecolic acid)-Xaa-Gly-Yaa-Gly-beta Ala-Asp-Tyr-Glu-Pro-Ile- Pro-Glu-Glu-Ala-(beta-cyclohexylalanine in which nonpolar P1' residue Xaa o r 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 K-i value to (9.5 +/- 0.6) x 10(-14) or 1.3 +/- 0.5 x 10(-13) M, respectively, compared to that of a reference inhibitor with Gly residu es at Xaa and Yaa residues (K-i = (2.4 +/- 0.5) x 10(-11) M). Similarly, su bstitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanin e lowered the K-i values to (8.2 +/- 0.6) x 10-14 or(5.1 +/- 0.4) x 10(-14) M, respectively. The linker Gly-Gly-Gly-beta Ala of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvment of the K-i values to (3.8 +/- 0.6) x 10(-14) or (1.7 +/ - 0.4) x 10(-14) M, respectively. These K-i values are equivalent to that o f natural hirudin (2.2 x 10(-14) M), yet the size of the synthetic inhibito rs (2 kD) is only one-third that of hirudin (7 kD). Two inhibitors, with L- norleucine or L-beta-(2-thienyl)alanine at the P1' residue and the improved linker of 12-aminododecanoic acid, were crystallized in complex with human alpha-thrombin. The crystal structures of these complexes were solved and refined to 2.1 Angstrom resolution. The Lys(60F) Side chain of thrombin mov ed significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.