title: 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-betaAla-Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala- (be ta-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 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 residues at Xaa and Yaa residues (K(i) = (2.4 +/- 0.5) x 10(-11) M). Similarly, substitution of the P1' residue with L-norleucine or L-beta-(2-thienyl)alanine 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-betaAla of the inhibitors in the previous sentence was simplified with 12-aminododecanoic acid, resulting in further improvement 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 of natural hirudin (2.2 x 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-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 A resolution. The Lys(60F) side chain of thrombin moved significantly and formed a large nonpolar S1' subsite to accommodate the bulky P1' residue.
authors.affiliation: Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada.
authors.forename: J J
authors.initials: JJ
authors.lastname: Slon-Usakiewicz
authors.forename: J
authors.initials: J
authors.lastname: Sivaraman
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: M
authors.initials: M
authors.lastname: Cygler
authors.forename: Y
authors.initials: Y
authors.lastname: Konishi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000138:chemical synthesis* / Q000737:chemistry; D000991:Antithrombins / Q000138:chemical synthesis* / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D003460:Crystallization; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry
chemical_list: D000596:Amino Acids; D000991:Antithrombins; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D013917:Thrombin