Reversible covalent direct thrombin inhibitors

Abstract

Introduction: In recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.

Methods: We have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1.

Results: We demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay.

Conclusion: Compound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.

Fig 1. Structures of Compound 1, the dansylated Compound 2, and the deacylated Compound 3 described in this paper.

Fig 2. Interaction of Compound 1 with thrombin.

Compound 1 at 1 μM was incubated with excess thrombin at 5 μM at room temperature for 20 min. The reaction was then quenched and monitored by LC-MS for Compound 1 and Compound 3. Note that quantitation levels were normalized to Compound 1 without thrombin. Stochiometric conversion of Compound 1 to Compound 3 was observed.

Fig 3

Panel A: Fluorescence intensity vs time for the incubation of Compound 2 with thrombin, thrombin mutant S195A, and thrombin with PPACK. After 3 min (arrow), the dansyl-labeled thrombin inhibitor Compound 2 was added to a solution of thrombin WT (black), thrombin preincubated with an excess of the irreversible active site inhibitor PPACK (magenta), or the active site mutant thrombin S195A (green) and incubated at room temperature for 2 h. The samples were monitored by fluorescence (excitation 280 nm, emission 340 nm). The observed differences in fluorescence quenching suggest that Compound 1 targets S195. Panel B: Compound 2 incubated with WT thrombin was analyzed by SDS-PAGE. Two samples are shown at time 0 and after 30 min. In the Coomassie-stained image (top), the thrombin band is visible irrespective of compound incubation. In contrast, when viewed under UV light (bottom), the thrombin band fluoresces only after incubation, indicating the incorporation of (parts of) Compound 2 into the enzyme.

Fig 4. Structural model of the thrombin active site.

The structural model of the thrombin active site is derived from X-ray crystallography of thrombin modified by Compound 1. The S195 in the active site required for thrombin enzymatic activity is modified by the 2-methoxybenzoyl group, rendering it inactive. In the monomer shown on the right, continuous electron density is found supporting the existence of a covalent linkage. In the monomer shown on the left, electronic density for the methoxyphenyl ring indicates blockage of the active site, but covalent linkage cannot be conclusively determined due to missing electron density (red).

Fig 5. Interaction diagram for the thrombin active site modified by Compound 1.

The illustration shows that no specific contacts between the 2-methoxybenzoyl group and thrombin are made.

Fig 6. Kinetic characterization of Compound 1.

Panel A: Remaining thrombin activity following preincubation with varying concentrations of Compound 1 for one representative experiment. Panel B: Plot of the unimolecular rate constant, k_obs, determined from the slopes of the linear fits shown in panel A, as a function of initial inhibitor concentration. Shown is the fit to the Michaelis-Menten model. This method yields the kinetic parameters K_i = 3.2 ± 0.75 μM and k_inact = 0.08 ± 0.04 sec^-1. Panel C: Reaction progress curves for thrombin inhibition at varying concentrations of Compound 1 for one representative experiment. Note that the legend shows modified inhibitor concentration. Panel D: Plot of the unimolecular rate constant k_obs determined from the time constants from the one-phase exponential association fits in panel C, as a function of modified inhibitor concentration. Shown is the fit to the Michaelis-Menten model. This method yields the kinetic parameters K_i = 2.1 ± 1.8 μM and k_inact = 0.06 ± 0.02 sec^-1.

Fig 7. Spontaneous recovery of thrombin activity.

% Thrombin activity was recorded as a function of time after inhibition by Compound 1 and removal of excess Compound 1. Fitting an exponential yielded a rate constant k₃ = 1.4 x 10⁻³ min^-1, which corresponds to a half-life of about 8 h.

Fig 8. Thrombograms from the thrombin generation assay for argatroban, dabigatran, and Compound 1.

The assays were performed as described in section “Thrombin generation assay (TGA)” above.

Fig 9. Proposed mechanism of action of Compound 1.

Compound 1, an exemplary VE-DTI, binds to thrombin and orients to react with the active site S195, which results in its acylation. The modified form of thrombin is inactive until deacylation of S195.