Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.
Research article published in Biochemistry (2000)
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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
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...
Warum dies für die Hirudotherapie relevant ist
Diese Studie entwarf und testete synthetische bivalente Thrombininhibitoren und optimierte die P1'- und P3'-Reste der Linker-Region, um die Bindungsaffinität zu verbessern. Substitutionen wie D-Phenylglycin an P3' oder L-Norleucin und L-β-(2-Thienyl)alanin an P1' ergaben Ki-Werte im Bereich von ~10⁻¹³ bis 10⁻¹⁴ M. Mit einem vereinfachten 12-Aminododecansäure-Linker erreichten die L-β-(2-Thienyl)alanin- und L-Norleucin-Varianten Ki-Werte von ~1,7 × 10⁻¹⁴ M bzw. ~3,8 × 10⁻¹⁴ M – vergleichbar mit natürlichem Hirudin (Ki = 2,2 × 10⁻¹⁴ M) bei einem Drittel des Molekulargewichts (~2 kD vs. 7 kD). Kristallstrukturen von zwei Inhibitor-Thrombin-Komplexen wurden bei 2,1 Å Auflösung gelöst. Für die ASH erscheint Hirudin als Referenz für die Benchmark-Wirksamkeit; allerdings handelt es sich um eine Studie der strukturellen Biologie und medizinischen Chemie, die weder Blutegel, Blutegeltherapie noch In-vivo-Daten einbezieht.
Zitation
Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.
Slon-Usakiewicz et al. · Biochemistry, 2000
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