ABSTRACT
Factor (F) VIIa in association with tissue factor (TF) is the primary in vivo initiator of blood coagulation and activates FX and FIX to generate thrombin, which plays a key role in the pathogenesis of thrombosis. We evaluated the enzyme kinetics, antithrombotic and antihaemostatic properties of BMS-593214, an active-site, direct FVIIa inhibitor. Studies were conducted in enzymatic assays, and in anesthetised rabbit models of electrically-induced carotid arterial thrombosis (AT), thread-induced vena cava venous thrombosis (VT) and cuticle bleeding time (BT). Antithrombotic efficacy of BMS-593214 given intravenously was evaluated for both the prevention and treatment of AT and VT. BMS-593214 displayed direct, competitive inhibition of human FVIIa in the hydrolysis of a tripeptide substrate with Ki of 5 nM. However, it acted as a noncompetitive inhibitor of the activation of the physiological substrate FX by TF/VIIa with Ki of 9.3 nM. BMS-593214 showed selectivity for FVIIa and exhibited species differences in TF-FVIIa-dependent anticoagulation with similar potency in human and rabbit plasma. BMS-593214 was efficacious in the prevention and treatment models of AT and VT with ED50 values of 1.1 to 3.1 mg/kg. Furthermore, BMS-593214 exhibited a wide therapeutic window with respect to BT. These results suggest that inhibition of FVIIa with small-molecule active-site inhibitors represents a promising antithrombotic approach for the development of new therapies for the prevention and treatment of AT and VT.
Subject(s)
Benzoates/pharmacology , Carotid Artery Thrombosis/drug therapy , Enzyme Inhibitors/pharmacology , Factor VIIa/antagonists & inhibitors , Fibrinolytic Agents/pharmacology , Hemostasis/drug effects , Hemostatics/pharmacology , Heterocyclic Compounds, 4 or More Rings/pharmacology , Venous Thrombosis/drug therapy , Animals , Bleeding Time , Blood Coagulation/drug effects , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/prevention & control , Catalytic Domain , Disease Models, Animal , Dose-Response Relationship, Drug , Enzyme Inhibitors/administration & dosage , Factor VIIa/chemistry , Factor Xa/metabolism , Fibrinolytic Agents/administration & dosage , Hemostatics/administration & dosage , Humans , Injections, Intravenous , Kinetics , Male , Rabbits , Recombinant Proteins/antagonists & inhibitors , Thromboplastin/metabolism , Venous Thrombosis/blood , Venous Thrombosis/prevention & controlABSTRACT
Kex2 is the yeast prototype of a large family of serine proteases that are highly specific for cleavage of their peptide substrates C-terminal to paired basic sites. This paper reports the 2.2 A resolution crystal structure of ssKex2 in complex with an Ac-Arg-Glu-Lys-Arg peptidyl boronic acid inhibitor (R = 19.7, R(free) = 23.4). By comparison of this structure with the structure of the mammalian homologue furin [Henrich, S., et al. (2003) Nat. Struct. Biol. 10, 520-526], we suggest a structural basis for the differences in substrate recognition at the P(2) and P(4) positions between Kex2 and furin and provide a structural rationale for the lack of P(6) recognition in Kex2. In addition, several monovalent cation binding sites are identified, and a mechanism of activation of Kex2 by potassium ion is proposed.
Subject(s)
Furin/chemistry , Proprotein Convertases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Binding Sites , Boronic Acids/chemistry , Boronic Acids/metabolism , Crystallography, X-Ray , Furin/antagonists & inhibitors , Furin/metabolism , Oligopeptides/chemistry , Oligopeptides/metabolism , Proprotein Convertases/antagonists & inhibitors , Proprotein Convertases/metabolism , Protein Binding , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
This paper reports the first structure of a member of the Kex2/furin family of eukaryotic pro-protein processing proteases, which cleave sites consisting of pairs or clusters of basic residues. Reported is the 2.4 A resolution crystal structure of the two-domain protein ssKex2 in complex with an Ac-Ala-Lys-boroArg inhibitor (R = 20.9%, R(free) = 24.5%). The Kex2 proteolytic domain is similar in its global fold to the subtilisin-like superfamily of degradative proteases. Analysis of the complex provides a structural basis for the extreme selectivity of this enzyme family that has evolved from a nonspecific subtilisin-like ancestor. The P-domain of ssKex2 has a novel jelly roll like fold consisting of nine beta strands and may potentially be involved, along with the buried Ca(2+) ion, in creating the highly determined binding site for P(1) arginine.
Subject(s)
Boronic Acids/chemistry , Oligopeptides/chemistry , Proprotein Convertases , Protease Inhibitors/chemistry , Saccharomyces cerevisiae Proteins , Subtilisins/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/pharmacology , Binding Sites , Boronic Acids/metabolism , Boronic Acids/pharmacology , Calcium/chemistry , Calcium/metabolism , Crystallography, X-Ray , Models, Molecular , Oligopeptides/metabolism , Oligopeptides/pharmacology , Protease Inhibitors/metabolism , Protease Inhibitors/pharmacology , Protein Structure, Secondary , Protein Structure, Tertiary , Static Electricity , Substrate Specificity , Subtilisins/antagonists & inhibitors , Subtilisins/metabolismABSTRACT
NMR spectroscopy was used to characterize the hepatitis C virus (HCV) NS3 protease in a complex with the 24 residue peptide cofactor from NS4A and a boronic acid inhibitor, Ac-Asp-Glu-Val-Val-Pro-boroAlg-OH. Secondary-structure information, NOE constraints between protease and cofactor, and hydrogen-deuterium exchange rates revealed that the cofactor was an integral strand in the N-terminal beta-sheet of the complex as observed in X-ray crystal structures. Based upon chemical-shift perturbations, inhibitor-protein NOEs, and the protonation state of the catalytic histidine, the boronic acid inhibitor was bound in the substrate binding site as a transition state mimic. In the absence of cofactor, the inhibitor had a lower affinity for the protease. Although the inhibitor binds in the same location, differences were observed at the catalytic site of the protease.
