title: Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.
abstract: Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.
authors.affiliation: Department of Chemistry, University of Napoli, Italy.
authors.forename: G
authors.initials: G
authors.lastname: De Simone
authors.forename: G
authors.initials: G
authors.lastname: Balliano
authors.forename: P
authors.initials: P
authors.lastname: Milla
authors.forename: C
authors.initials: C
authors.lastname: Gallina
authors.forename: C
authors.initials: C
authors.lastname: Giordano
authors.forename: C
authors.initials: C
authors.lastname: Tarricone
authors.forename: M
authors.initials: M
authors.lastname: Rizzi
authors.forename: M
authors.initials: M
authors.lastname: Bolognesi
authors.forename: P
authors.initials: P
authors.lastname: Ascenzi
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology*; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology*; D013816:Thermodynamics; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry*
chemical_list: D006834:Hydrazines; C107136:N-carbobenzoxy-prolyl-azalysine-4-nitrophenyl ester; C101147:N-ethoxycarbonyl-phenylalanyl-prolyl-alpha-azalysine 4-nitrophenyl ester; D009842:Oligopeptides; D013917:Thrombin