A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

Alan G Olivero,Charles Eigenbrot,Richard Goldsmith,Kirk Robarge,Dean R Artis,John Flygare,Thomas Rawson,Daniel P Sutherlin,Saloumeh Kadkhodayan,Maureen Beresini,Linda O Elliott,Geralyn G Deguzman,David W Banner,Mark Ultsch,Ulla Marzec,Stephen R Hanson,Canio Refino,Stuart Bunting,Daniel Kirchhofer

doi:10.1074/jbc.m409068200

Alan G Olivero, Charles Eigenbrot + Show 17 more

Open Access

https://doi.org/10.1074/jbc.m409068200

Copy DOI

Abstract

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.

Highlights

The most widely used anticoagulants for prevention and treatment of thrombosis include unfractionated heparin, low molecular weight heparin (LMWH)1 and orally active coumarin derivatives such as warfarin [1,2,3]
These findings led to the suggestion that specific inhibitors of TF1⁄7FVIIa may cause less bleeding because they inhibit intravascular tissue factor (TF) at concentrations that are far below those necessary to block the high amounts of the hemostatic extravascular TF [22]
Design and Synthesis of G17905—We have previously disclosed a series of sulfonamide and acylsulfonamide inhibitors of TF1⁄7FVIIa, which utilize an aminobenzamidine to bind in the S1 pocket of factor VIIa (FVIIa) [56]

Summary

EXPERIMENTAL PROCEDURES

Reagents—Except for bovine trypsin (Sigma), all enzymes used were of human origin. FX, FXa, factor XIa, thrombin, activated protein C (APC), and plasmin were from Hematologic Technologies (Essex Junction, VT). The concentration of formed FXa was calculated from standard curves with purified FXa, and the background activities, determined in experiments without the addition of FVIIa, were subtracted from each value. At various time points during the 20-min reaction period, aliquots were removed and quenched in 20 mM EDTA, 20 mM Hepes, pH 7.5, 150 mM NaCl, and the change in absorbance at 405 nm, upon the addition of S2765 (0.3 mM), was measured on a kinetic microplate reader. The concentration of newly formed FXa was calculated for each time point using a standard curve with purified FXa. For each experiment, the background activities in the absence of added FVIIa were determined and subtracted from each value.

Active Site Inhibitor of Factor VIIa

No of water molecules No of ionsd

RESULTS AND DISCUSSION

PT APTT TT RVVT

Plasma kallikrein