Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma Joakim E. Swedberg,‡† Guojie Wu,§† Tunjung Mahatmanto,‡# Thomas Durek,‡ Tom T. Caradoc-Davies,∥ James C. Whisstock,§* Ruby H.P. Law§* and David J. Craik‡* ‡Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia §ARC Centre of Excellence in Advanced Molecular Imaging, Department of Biochemistry and Molecular Biology, Biomedical Discovery Institute, Monash University, VIC 3800, Australia. ∥Australian Synchrotron, 800 Blackburn Road, Clayton, Melbourne, VIC 3168, Australia. †J.E.S. and G.W. contributed equally to this work. Keywords: Antifibrinolytics; Fibrinolysis; Inhibitors; Peptides; Plasmin ABSTRACT Antifibrinolytic drugs provide important pharmacological interventions to reduce morbidity and mortality from excessive bleeding during surgery and after trauma. Current drugs used for inhibiting the dissolution of fibrin, the main structural component of blood clots, are associated with adverse events due to lack of potency, high doses and non-selective inhibition mechanisms. These deficiencies warrant the development of a new generation highly potent and selective fibrinolysis inhibitors. Here we use the 14-amino acid backbone-cyclic sunflower trypsin inhibitor-1 scaffold to design a highly potent (Ki = 0.05 nM) inhibitor of the primary serine protease in fibrinolysis, plasmin. This compound displays a million-fold selectivity over other serine proteases in blood, inhibits fibrinolysis in plasma more effectively than the gold-standard therapeutic inhibitor aprotinin and is a promising candidate for development of highly specific fibrinolysis inhibitors with reduced side effects. 1 INTRODUCTION The physiological process of fibrinolysis regulates the dissolution of blood clots and thrombosis. At the core of the fibrinolytic system (also known as the ‘plasminogen-plasmin’ system), is the serine protease plasmin, that degrades fibrin, the principal structural protein of blood clots. Plasmin is produced as the zymogen plasminogen which binds to the surface of fibrin via lysine binding sites, before activation primarily by tissue plasminogen activator (tPA). Activation of the plasminogen-plasmin system during surgery or traumatic injuries results in excessive bleeding, the need for blood transfusions, and the use of antifibrinolytic drugs. The most commonly used antifibrinolytic drug is the lysine analogue tranexamic acid (TXA), which prevents binding of plasminogen to fibrin and thereby its activation by tPA, but it does not inhibit plasmin once active. In traumatic injuries, the use of TXA is reported to give a small but significant reduction in mortality, although with no reduction in the need for blood transfusions, therefore limiting its clinical applications.1 In bypass surgery TXA is given pre-operatively and shows good efficacy in reducing the need for blood transfusions, but has been associated with risks of seizures.2,3 Aprotinin is a reversible (Laskowski mechanism) plasmin active site inhibitor used for decades with good efficacy in reducing blood loss and blood transfusions,4 highlighting the advantage of using plasmin active site inhibitors as antifibrinolytics. However, the use of aprotinin is hampered by its lack of specificity, since it inhibits virtually every S1 family serine protease present in blood.5 A large clinical trial found no survival benefits of using aprotinin in surgery (despite its ability to reduce the need for blood products)4 and it has been withdrawn from general use. Consequently, there is a pressing need to develop potent and specific plasmin inhibitors to reduce bleeding after trauma and during surgery more specifically and effectively. A number of engineered plasmin inhibitors have been reported over the last two decades, but most of the more potent inhibitors suffer from poor selectivity.6,7 However, after aprotinin was withdrawn from clinical use there have been intensified efforts to design plasmin inhibitors, and a number of inhibitors with higher potency and selectivity,8,9 as well as allosteric inhibitors10 have emerged in recent years. One recent strategy that has emerged for designing potent and selective plasmin inhibitor is by producing substrate-analogues by cyclisation between the P2 and P3 residues.8 For example, a series of peptidomimetic inhibitors cyclized between the P2 and P3 residue 2 side chains has been reported, with the most promising variant having a Ki of 0.2 nM for plasmin, but with low micromolar inhibition of pKLK, FXIa and uPA.9 This lead compound was further optimized by modifying the P1 residue and the N-terminal group to produce a new series of plasmin inhibitors, some of which are more potent for plasmin. The most promising lead compound shows potent plasmin inhibition (Ki = 0.56 nM) and greatly increased selectivity over several other blood coagulation proteases.11 Although this series of inhibitors is highly promising, further selectivity optimization and evaluation is needed for therapeutic development. In an alternative approach we have been using the sunflower trypsin inhibitor-1 (SFTI-1) scaffold as a template for design of plasmin inhibitors. SFTI-1 is a 14-amino acid backbone cyclic peptide (Figure 1A) that inhibits serine proteases by the Laskowski mechanism, trapping the target protease in a futile cycle of cleavage and re-ligation of the scissile peptide bond.12,13 SFTI-1 inhibits several S1 family serine proteases such as trypsin14 (Figure 1B) and plasmin.15 Its cyclic peptide backbone and bisecting disulfide bond makes SFTI-1 highly stable and thus an attractive scaffold for design of therapeutic compounds targeting serine proteases, as well as cell surface receptors and protein-protein interactions.16 SFTI-1 has been used to engineer potent and/or selective inhibitors of a number of serine proteases, including thrombin,15 chymotrypsin,13 matriptase-1/-2,17,18 cathepsin G19 and several kallikrein-related peptidases,15,20,21 highlighting the versatility of the scaffold. In this study we use substrate-guided and structure-based design methods to engineer a series of highly potent and selective inhibitors of plasmin based on the SFTI-1 scaffold. The most promising plasmin inhibitor (Ki = 0.05 nM) has a million-fold selectivity over other serine proteases in blood and blocks fibrinolysis in human plasma with higher efficacy than aprotinin. This inhibitor is a promising lead for the development of a new generation of antifibrinolytics with higher selectivity than those currently used in the clinic. 3 RESULTS We previously reported that plasmin has a comparable, but sequence dependent (P2-P4), cleavage preference for P1 Lys or Arg in peptide substrates.22 In the current study we found that substituting the P1 residue Lys5 for Arg in SFTI-1 resulted in a compound, (1), with 13-fold reduced potency, indicating that Lys is preferred in the context of the SFTI-1 scaffold (Table 1). We have also shown that plasmin has a substrate preference for aromatic residues at the P2 position (SFTI-1 residue 4), and particularly Tyr in combination with P1 Lys.22 Substituting Thr4 with Tyr in SFTI-1 produced an inhibitor (2) with more than a 60-fold increased potency for plasmin (Ki = 0.140 nM) and over 6000-fold reduction in inhibition of trypsin. Compound 2 inhibited the neutrophil serine protease cathepsin G, coagulation factor XIa (FXIa) plasma kallikrein (pKLK), thrombin and matriptase in the micromolar range, while showing no inhibition of coagulation factors FIXa, FXa, FXIIa or the urokinase-/tissue- plasminogen activators (uPA/tPA) at 50 μM. At a concentration of ~1.6 µM23, plasminogen is one of the most abundant serine protease zymogens in blood and a highly potent and specific inhibitor is required for desirable clinical outcomes. We previously used a SFTI-based inhibitor library to show that plasmin has a P2ʹ preference for Lys (SFTI residue 7).15 Substituting Ile7 for Lys in compound 2 produced a more potent inhibitor of plasmin (compound 3; Ki = 0.051 nM) with no detectable inhibition of thrombin, FIXa, FXa, FXIa, FXIIa, tPA, uPA, pKLK or matriptase, and with improved selectivity over trypsin and cathepsin G. The structure and binding kinetics for compound 3 are shown in Figure 2 and Figure S1A, respectively. To gain a further understanding of the molecular mechanisms underpinning the high potency of compounds 2 and 3 for plasmin we produced crystal structures of these inhibitors in complex with the catalytic domain of plasmin (µ-plasmin). Our results revealed that the structures of µ-plasmin/SFTI-variant complexes adopt the typical Bowman-Birk inhibitor and type I serine protease assembly (Figures 2 and 3A and 3B), similar to the first crystal structure of µ-plasmin domain in complex with a peptide-chloromethylketone (Cα RMSD < 0.5 Å).24The µ- plasmin shows a typical trypsin-like serine protease fold consisting of two subdomains (N-terminal and C-terminal domains) which are connected by loops. Each domain is made of a 6-stranded -barrel. The catalytic domain of µ-plasmin is well ordered except for residues 14 (plasmin numbering in brackets, 560) and 15 (561), which results 4 from cleavage between residues 15 (561) and 16 (562) during the activation of µ-plasminogen to µ-plasmin by tPA. Upon cleavage, Val16 (562) moves more than 12 Å away to the activation pocket and the -amino group of Val16 (562) forms ionic bond with the side chain of Asp195 (740). This leads to a major shift of the loops at the C-terminal domain and more importantly the formation of functional catalytic triad at the interface of the two subdomains. In our structures, the catalytic triad [His57 (603), Asp102 (646) and Ser195 (741)] adopts the active conformations, similar to the structures of trypsin and matriptase complexed with SFTI-1 (PDB ID 1SFI and 3P8F, respectively) and that of the µ-plasmin structures in the PDB (PDB ID 5UGD and 5UGG; Figure S2 and S3). (Cα RMSD < 0.5 Å) The µ-plasmin/compound 2 complex diffracted to 1.43 Å (space group C121) with one binary complex in an asymmetric unit (Table S1).