Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors.

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

VE-1902-A direct thrombin inhibitor with reversible covalent mechanism of action shows efficacy with reduced bleeding in rodent models of thrombosis.

INTRODUCTION: High incidence of bleeding events remains a key risk for patients taking anticoagulants, especially those in need of long-term combination therapy with antiplatelet agents. As a consequence, patients may not receive clinically indicated combination antithrombotic therapy. Here, we report on VE-1902, a member of a novel class of precision oral anticoagulants (PROACs) that combines effective anticoagulation with reduced bleeding in preclinical testing. METHODS AND RESULTS: Acting through covalent, reversible active-site modification of thrombin similar to a previously described molecule [1], VE-1902 shows potency and selectivity for thrombin inhibition in human plasma comparable to clinically relevant direct thrombin inhibitors (DTI) such as argatroban and dabigatran (thrombin generation assay ETP EC50 = 1.3 µM compared to 0.36 µM and 0.31 µM for argatroban and dabigatran; >100-fold selectivity against related serine proteases). Unlike the current anticoagulants, VE-1902 does not significantly inhibit thrombin-mediated platelet activation in in vivo models of thrombosis. In the thrombin generation assay, the compound inhibits thrombin formation without significantly delaying the initiation phase of the clotting cascade. These features are possibly responsible for the observed reduced bleeding in tail bleeding and saphenous vein bleeding models. Consistent with this novel pharmacological profile, VE-1902 shows efficacious anticoagulation in several fibrin-driven animal models of thrombosis (arteriovenous shunt, venous stasis thrombosis, and thrombin-induced thromboembolism models), whereas it does not significantly prevent arterial occlusion in the platelet dependent FeCl3 model. CONCLUSIONS: By leaving platelet activation following vascular injury mostly unaffected, VE-1902, and the PROACs more generally, represent a new generation of precision anticoagulants with reduced bleeding risk.

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.

Discovery of ADDL--targeting small molecule drugs for Alzheimer's disease.

Amyloid beta-derived diffusible ligands (ADDLs) comprise the neurotoxic subset of soluble Abeta(1-42) oligomers, now widely considered to be the molecular cause of memory malfunction and neurodegeneration in Alzheimer's disease (AD). We have developed a screening cascade which identifies small molecule modulators of ADDL-mediated neurotoxicity. The primary screen involves a fluorescence resonance energy transfer (FRET)-based assay which selects inhibitors of Abeta1-42 oligomer assembly. The identified hits were further characterized by assessing their ability to inhibit the assembly and binding of ADDLs to cultures of primary hippocampal neurons. This approach has led to the identification of a number of small molecules which inhibit ADDL assembly and their subsequent binding to neurons. Here we describe our small molecule discovery efforts to identify ADDL assembly blocker and ADDL binding inhibitors, and to transform validated hits into pre-clinical lead compounds.