Modulation of human α-thrombin activity with phosphonate ester inhibitors

• Published in: Bioorganic & medicinal chemistry Vol. 5; no. 8; pp. 1531 - 1541
• Main Authors: Enyedy, Edith J., Kovach, Ildiko M.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.08.1997
• Subjects: Binding Sites; Crystallography, X-Ray; Enzyme Inhibitors - chemical synthesis; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Esters - chemistry; Esters - pharmacology; Humans; Models, Chemical; Models, Molecular; Organophosphonates - chemistry; Organophosphonates - pharmacology; Organophosphorus Compounds - chemistry; Organophosphorus Compounds - pharmacology; Protein Conformation; Stereoisomerism; Thrombin - antagonists & inhibitors; Thrombin - metabolism
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/S0968-0896(97)00099-0

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH 3; and CH 3O) inactivate human α-thrombin with rate constants 4–235 M −1 s −1 in pH 6.5, 0.025 M citrate buffer, and 0.15 M NaCl at 7.0±0.1 °C. Stereoselectivity of the inactivation of thrombin is 2–39 and favors the levorotatory enantiomers. The pH-dependence of inactivation of thrombin by (−)-PMN is sigmoidal and consistent with the participation of a catalytic residue with a p K a of 8.0±0.1 in 0.15 M NaCl and a p K a of 7.4±0.2 in 0.15 M choline chloride in the nucleophilic attack of the catalytic Ser at phosphorus. The solvent isotope effect on k i K i in the pH-independent region of the reaction is 2.26±0.17. Thrombin activity returns from the adducts on the 2–7 h time scale at 25.0±0.1 °C via a self-catalyzed intramolecular reaction. The pH dependence of reactivation is significant from the adduct formed with (−)-CH 3O-PMN and (−)-CH 3-PMN and less so from the adducts formed with the other enantiomers of the inhibitors. Kinetic p Ks ∼ 7.2, with the exception of the adducts with (−)-PMN and (−)-CH 3O-PMN, indicate that a pH-dependent conformational change affects the rate of dephosphonylation. A structural interpretation of the stereoselectivity and other mechanistic features is provided based on the energy-optimized structures of the adducts. Pharmaco-medical use of human α-thrombin covalently modified by the PMNs is suggested. Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH 3; and CH 3O) inactivate human α-thrombin selectively and efficiently. The covalent attachement to thrombin is reversible due to a self-catalyzed intramolecular attack at phosphorus by the anion of the hydrated ketone. Pharmaceutical application of the concept is suggested.