CHARACTERIZATION OF THE TWO-STEP PATHWAY FOR INHIBITION OF THROMBIN BY ALPHA -KETOAMIDE TRANSITION STATE ANALOGS

Abstract The interaction of thrombin with several potent and selective α-ketoamide transition state analogs was characterized. L-370,518 (H-N-Me-d-Phe-Pro-t-4-aminocyclohexylglycylN-methylcarboxamide) a potent (K i = 90 pm) and selective (>104-foldversus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed thatk −1, the rate constant for dissociation ofEI1, was comparable tok 2, the rate constant for conversion ofEI1 to EI2 (0.049 and 0.035 s−1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (K i = 4 nm) largely due to an increase in k −1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1′N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K −1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (K i = 40 pm) than L-370,518 due to its 16-fold higherk 2 and 10-fold lowerk −2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The K i value for L-371,912 and the K −1 value for its α-ketoamide analog, L-370,518, were similar (5 and 14 nm, respectively). Likewise, the K i value for L-372,011 and the K −1 value for its α-ketoamide analog, L-372,051, were similar (330 and 285 nm, respectively). These observations are consistent with the view that the α-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the α-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.