Substrate-Guided Design of Selective FXIIa Inhibitors Based on the Plant-Derived Momordica cochinchinensis Trypsin Inhibitor-II (MCoTI- II) Scaffold Joakim E. Swedberg, † Tunjung Mahatmanto, †,§ Hafiza Abdul Ghani, † Simon J. de Veer, ‡,∥ Christina I. Schroeder, † Jonathan M. Harris, ‡ and David J. Craik* ,† † Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia ‡ Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane Queensland 4059, Australia * S Supporting Information ABSTRACT: Thrombosis is a leading cause of morbidity and mortality associated with cardiovascular diseases. Inhibition of factor XIIa (FXIIa) provides thrombus protection without bleeding complications. Here, we defined the extended substrate specificity of FXIIa and its close homologue factor Xa and used these data, together with inhibitor-based and structure-guided methods, to engineer selective FXIIa inhibitors based on Momordica cochinchi- nensis trypsin inhibitor-II. ■ INTRODUCTION Hemostasis is the physiological process that limits bleeding after injury to blood vessels and depends both on blood coagulation and activation of platelets. Pathological blood clotting and thrombosis rely on the same mechanisms but result in excessive and spatially indiscriminate thrombus formation that causes vascular occlusion and is associated with significant morbidity and mortality. 1 Worldwide, throm- bosis is a leading cause of morbidity and mortality associated with cardiovascular diseases such as myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. 2 There are a number of approved antithrombotic drugs on the market, but the most commonly prescribed and the only approved drug for long-term anticoagulation therapy is warfarin, 3 an orally bioavailable vitamin K antagonist. 4 Despite the efficacy of warfarin, its use carries the risk of hemorrhaging. 5 Another commonly prescribed anticoagulant drug is low molecular weight heparin (LMWH). 3 Although LMWH has a safer profile than warfarin (albeit having low risk of heparin- induced thrombocytopenia), its injection-based delivery limits its use in a clinical setting and to short-term treatment regimens. 3 These limitations of safety and ease of use have led to the development of a new generation of approved drugs that target serine proteases in the blood clotting cascade. These include the orally administered thrombin inhibitor, dabigatran, and the factor Xa (FXa) inhibitor, rivaroxaban, respectively. 3 Despite the advantages of these drugs, it is important to note that targeting FXa or thrombin still carries risk, as FXa and prothrombin have been shown to be vital for hemostasis in gene knockout experiments in mice. 6 In contrast to the vital role of FXa and thrombin, humans deficient in another serine protease in the blood clotting cascade, factor XIIa (FXIIa), appear to have normal hemostasis, 7 and, in addition, animal studies have demonstrated that FXIIa is not required for hemostasis but is important for thrombosis. This is contrary to factor XIa (FXIa), the downstream substrate of FXIIa in the clotting cascade, whose deficiency in humans causes an injury- related bleeding disorder. 8 These results open the door to development of a safer anticoagulation therapy by targeting FXIIa. 7b Recently, a peptidomimetic FXIIa inhibitor (K i = 22 nM) that blocked the intrinsic coagulation pathway in vitro was developed, 9 but further studies are needed to evaluate the potential of this inhibitor in vivo. Concurrently, a humanized FXIIa inhibitory antibody (3F7) has been extensively evaluated both in vitro and in vivo 10 and has been shown to provide thrombosis protection as efficiently as heparin in rabbits without increasing bleeding from wounds. Despite the potential of 3F7 as a promising candidate for reduction of thrombosis during cardiopulmonary bypass surgery, 10 recombinant anti- bodies are costly to produce, 11 which is a problem given that 3F7 needs to be administrated at a high dosage (7 mg/kg). In attempts to develop more cost-effective alternatives to 3F7, a range of FXIIa inhibitors have been produced, including antibodies, serpin-type nonreversible inhibitors, small molecule Received: April 12, 2016 Brief Article pubs.acs.org/jmc © XXXX American Chemical Society A DOI: 10.1021/acs.jmedchem.6b00557 J. Med. Chem. XXXX, XXX, XXX−XXX