Modulation of human alpha-thrombin activity with phosphonate ester inhibitors

Area of Science:

• Biochemistry
• Enzyme kinetics
• Medicinal chemistry

Background:

• Human alpha-thrombin is a key enzyme in coagulation.
• Phosphonate esters are investigated as potential enzyme inhibitors.
• Understanding enzyme-inhibitor interactions is crucial for drug development.

Purpose of the Study:

• To investigate the enantioselective inactivation of human alpha-thrombin by methylphosphonate esters.
• To elucidate the kinetic and mechanistic aspects of thrombin inhibition and reactivation.
• To explore the potential pharmaco-medical applications of modified thrombin.

Main Methods:

• Enzyme kinetics assays to determine rate constants for thrombin inactivation.
• pH-dependence studies to identify catalytic residues and pKa values.
• Solvent isotope effect measurements to probe reaction mechanisms.
• Structural analysis using energy-optimized adduct structures.

Main Results:

• Methylphosphonate esters inactivated human alpha-thrombin with varying rate constants.
• Levorotatory enantiomers showed higher inhibitory activity (stereoselectivity).
• Thrombin regained activity via a self-catalyzed intramolecular reaction, with pH-dependent reactivation rates.
• Kinetic pKs indicated pH-dependent conformational changes affecting dephosphonylation.

Conclusions:

• The study demonstrates stereoselective inhibition of human alpha-thrombin by methylphosphonate esters.
• Mechanistic insights into the inhibition and reactivation processes were obtained.
• Covalent modification of thrombin by these inhibitors suggests potential therapeutic applications.