Characterization of the Two-step Pathway for Inhibition of Thrombin by α-Ketoamide Transition State Analogs

Abstract

The interaction of thrombin with several potent and selective ␣-ketoamide transition state analogs was characterized. L-370,518 (H-N-Me-D-Phe-Pro-t-4-aminocyclohexylglycyl N-methylcarboxamide) a potent (K i ‫؍‬ 90 pM) and selective (>10 4 -fold versus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI 1 ) rearranges to a more stable, final complex (EI 2 ). A novel sequential stopped-flow analysis showed that k ؊1 , the rate constant for dissociation of EI 1 , was comparable to k 2 , the rate constant for conversion of EI 1 to EI 2 (0.049 and 0.035 s ؊1, respectively) indicating that formation of the initial complex EI 1 is partially rate controlling. Replacement of the Nterminal 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 EI 1 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 EI 1 . 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 higher k 2 and 10-fold lower k ؊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 ␣-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 ␣-ketoamide analog, L-372,051, were similar (330 and 285 nM, respectively). These observations are consistent with the view that the ␣-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 ␣-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct.Our pursuit of novel antithrombotic agents has focused on direct inhibitors of thrombin, a trypsin-like serine proteinase that plays a key role in thrombosis. One strategy for the design of potent thrombin inhibitors is to replace the substrate P1 1 carboxamido group with an electrophilic keto or aldehyde group (1). Cyclotheonamide A is a naturally occurring proteinase inhibitor from a marine sponge (Theonella) that contains such a potency-enhancing keto group (2-4). Cyclotheonamide A is a potent (K i ϭ 1 nM) reversible inhibitor of thrombin (5, 6) and several other trypsin-like proteinases (5, 7). Various ␣-ketoamide derivatives of tripeptide substrates of thrombin have been prepared in an attempt to identify selective, potent, reversible inhibitors of thrombin (8, 9). Placement of a t-4-AChxGly 2 residue at the P1 position in ␣-ketoamide thrombin inhibitors provided excellent selectivity for thrombin, relative to tryspin...

SCH 503034, a Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease, Suppresses Polyprotein Maturation and Enhances the Antiviral Activity of Alpha Interferon in Replicon Cells

“…1) showed timedependent inhibition of the single-chain NS3 protease characteristic of ketoamide inhibitors of serine proteases (Fig. 2) (19,33,37,44). As crystallographic analysis had indicated the formation of a covalent adduct with the active-site serine (Fig.…”

Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

“…The apparent K I thus represents the true K I and requires no correction for substrate binding. 29 The measured K I for binding of the nonbiotinylated EP42675 to thrombin was 61 Ϯ 20pM ( Table 2). The biotin label of EP217609 increased the affinity to 43 Ϯ 16pM, although the significance of these differences was marginal because of the large errors in K I at the protease concentration used (ϳ 20 ϫ K I ).…”

“…No evidence for any biphasic slow inhibition kinetics reflecting a possible 2-step inhibition mechanism was observed for any of the EP compoundprotease interactions. 29 The compounds thus appear to inhibit all proteases through a simple one-step reversible equilibrium. Data for EP217609 only are presented for the 4 proteases whose activity was insignificantly inhibited over the same EP compound concentration range.…”

“…29 The fitted parameters were v o , n, and K I *. It should be noted that, under conditions where inhibitor dissociation from the protease is slow relative to the time over which substrate hydrolysis is measured, the substrate will only interact with the free protease and not compete with the inhibitor-bound protease and therefore K I * will be equal to K I .…”

“…29 K I * is related to K I by a factor that accounts for the competitive effect of substrate binding:…”