title: Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by benzamidine and its bis-, tris- and tetra-derivatives: thermodynamic and molecular modeling study.
abstract: The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine beta-trypsin (beta-trypsin), human alpha-thrombin (alpha-thrombin) and porcine pancreatic beta-kallikrein-B (beta-kallikrein-B) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (Ki, M-1) for beta-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for alpha-thrombin and beta-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to beta-trypsin, alpha-thrombin and beta-kallikrein-B as well as for DAPP, TAPB and TAPP association to beta-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for DAPP, TAPB and TAPP binding to alpha-thrombin and beta-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to beta-trypsin, alpha-thrombin and beta-kallikrein-B has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).
authors.affiliation: Department of Pharmaceutical Sciences, University of Ferrara, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Menegatti
authors.forename: R
authors.initials: R
authors.lastname: Ferroni
authors.forename: C
authors.initials: C
authors.lastname: Nastruzzi
authors.forename: F
authors.initials: F
authors.lastname: Bortolotti
authors.forename: S
authors.initials: S
authors.lastname: Scalia
authors.forename: G
authors.initials: G
authors.lastname: Amiconi
authors.forename: M
authors.initials: M
authors.lastname: Bolognesi
authors.forename: M
authors.initials: M
authors.lastname: Coletta
authors.forename: S
authors.initials: S
authors.lastname: Onesti
authors.forename: R
authors.initials: R
authors.lastname: Fruttero
mesh_terms: D000818:Animals; D001550:Benzamidines / Q000138:chemical synthesis* / Q000494:pharmacology*; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000037:antagonists & inhibitors; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010179:Pancreas / Q000201:enzymology; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D013552:Swine; D013816:Thermodynamics; D013917:Thrombin / Q000037:antagonists & inhibitors; D014361:Trypsin Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D014961:X-Ray Diffraction
chemical_list: D001550:Benzamidines; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; C074442:1,3-bis(4-amidinophenoxy)-2-(4-amidinophenoxymethyl)ethylpropane; C057885:1,3-di-(4-amidinophenoxy)-2,2-bis-(4-amidinophenoxymethyl)propane; D007610:Kallikreins; D013917:Thrombin; C005555:propamidine