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
This study designed and tested synthetic bivalent thrombin inhibitors, optimizing the P1' and P3' residues of the linker region to improve binding affinity. Substitutions such as D-phenylglycine at P3' or L-norleucine and L-β-(2-thienyl)alanine at P1' yielded Ki values in the range of ~10⁻¹³ to 10⁻¹⁴ M. With a simplified 12-aminododecanoic acid linker, L-β-(2-thienyl)alanine and L-norleucine variants achieved Ki values of ~1.7 × 10⁻¹⁴ M and ~3.8 × 10⁻¹⁴ M, respectively—comparable to natural hirudin (Ki = 2.2 × 10⁻¹⁴ M) at one-third the molecular weight (~2 kD vs. 7 kD). Crystal structures of two inhibitor-thrombin complexes were solved to 2.1 Å resolution. For ASH, hirudin appears as a benchmark potency reference; however, this is a structural biology and medicinal chemistry study involving no leeches, no leech therapy, and no in vivo data.
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: June 18, 2026