Reversible covalent direct thrombin inhibitors.

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

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

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

Spontaneous recovery of thrombin activity.

<p>% 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 <i>k</i>₃ = 1.4 x 10⁻³ min^-1, which corresponds to a half-life of about 8 h.</p

IC₅₀s for the inhibition of endogenous substrates.

<p>IC₅₀s for the inhibition of endogenous substrates.</p

Structural model of the thrombin active site.

<p>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).</p

Proposed mechanism of action of Compound 1.

<p>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.</p

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

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

Thrombin generation assay parameters.

<p>Thrombin generation assay parameters.</p

Kinetic parameters for the inhibition of serine proteases.

<p>Kinetic parameters for the inhibition of serine proteases.</p