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.
Resumen
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...
Por qué esto importa para la hirudoterapia
Este estudio diseñó y evaluó inhibidores bivalentes sintéticos de la trombina, optimizando los residuos P1' y P3' de la región conectora para mejorar la afinidad de unión. Sustituciones como D-fenilglicina en P3' o L-norleucina y L-β-(2-tienil)alanina en P1' arrojaron valores de Ki del orden de ~10⁻¹³ a 10⁻¹⁴ M. Con un conector simplificado de ácido 12-aminododecanoico, las variantes con L-β-(2-tienil)alanina y L-norleucina alcanzaron valores de Ki de ~1,7 × 10⁻¹⁴ M y ~3,8 × 10⁻¹⁴ M, respectivamente, comparables a los de la hirudina natural (Ki = 2,2 × 10⁻¹⁴ M) con un tercio del peso molecular (~2 kD frente a 7 kD). Se resolvieron las estructuras cristalográficas de dos complejos inhibidor-trombina a una resolución de 2,1 Å. Para ASH, la hirudina aparece como un valor de referencia de potencia; sin embargo, se trata de un estudio de biología estructural y química medicinal que no involucra sanguijuelas, ni terapia con sanguijuelas, ni datos in vivo.
Citación
Design of P1' and P3' residues of trivalent thrombin inhibitors and their crystal structures.
Slon-Usakiewicz et al. · Biochemistry, 2000
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Añadido a la biblioteca ASH: May 28, 2026 · Última actualización del sitio: 18 de junio de 2026