Highly Potent and Selective Plasmin Inhibitors Based on the

Article Cite This: J. Med. Chem. 2019, 62, 552−560

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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, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia § Australian Synchrotron, 800 Blackburn Road, Clayton, Melbourne, VIC 3168, Australia

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‡

S Supporting Information *

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 nonselective inhibition mechanisms. These drawbacks warrant the development of a new generation of 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.

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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, which 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 preoperatively and shows good efficacy in reducing the need for blood transfusions, but it 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 © 2018 American Chemical Society

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 inhibitors,10 have emerged in recent years. One recent strategy that has emerged for designing potent and selective plasmin inhibitor is by producing substrate analogues by cyclization between the P2 and P3 residues.8 For example, a series of peptidomimetic inhibitors cyclized between the P2 and P3 residue 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 Received: July 20, 2018 Published: December 6, 2018 552

DOI: 10.1021/acs.jmedchem.8b01139 J. Med. Chem. 2019, 62, 552−560

Journal of Medicinal Chemistry

Article

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.

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 are 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

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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 600-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, and FXIIa or the urokinase-/tissue- plasminogen activators (uPA/tPA) at 50 μM. At a concentration of ∼1.6 μM,23 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 an SFTI-based inhibitor library to show that plasmin has a P2′ 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 Figures 2 and 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 (μplasmin). 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 3 and 4A,B), 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 β-barrel. The catalytic domain of μ-plasmin is well ordered except for residues 14 (plasmin numbering in brackets, 560) and 15 (561), which result 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 α-amino group of Val16 (562) forms ionic bonds 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 a functional catalytic triad at the

Figure 1. Structure of SFTI-1 in complex with trypsin. (A) Stick model of SFTI-1 with atoms colored as carbon: green, nitrogen: blue, oxygen: red, and sulfur: yellow. (B) Ribbon plot of SFTI-1 (green) in complex with bovine trypsin (gray, PDB: 1SFI) with the catalytic triad of trypsin and the P1 residue of SFTI-1 shown as stick models.

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 make 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 G,19 and several kallikrein-related peptidases,15,20,21 highlighting the versatility of the scaffold. 553



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Table 1. Inhibitor Sequences and Inhibition Constants compound

peptide sequencea

SFTI-1

GRCT4K5SI7PPICFPD14

1 2

GRCT4R5SI7PPICFPD14 GRCY4K5SI7PPICFPD14

3

GRCY4K5SK7PPICFPD14

4 5 6

GRCF4K5SK7PPICFPD14 GRCW4K5SK7PPICFPD14 GRCY4K5SR7PPICFPD14

7 8 9 10 11 12 13 14 15 16 17 18

GRCY4R5SK7PPICFPD14 GRCY4R5SR7PPICFPD14 GRCY4K5SR*7PPICFPD14 GRCT4K5SR7PPICFPD14 GRCY4K5SR7PPICFPN14 GRCYKSKPPKCFPD GRCYKSKPPRCFPD GRCYKSKPPQCFPD GRCYKSKPPNCFPD GR*CYKSRPPICFPD GR*CYKSRPPICFPE DRCYKSRPPICFPD

protease

Ki (nM) ± SEM

trypsin plasmin matriptase cathepsin G thrombin FXIIa plasmin plasmin trypsin cathepsin G FXIa pKLK thrombin matriptase other proteasesb plasmin trypsin cathepsin G other proteasesc plasmin plasmin plasmin trypsin cathepsin G other proteasesc plasmin plasmin plasmin plasmin plasmin plasmin plasmin plasmin plasmin plasmin plasmin plasmin

0.017 ± 0.000327 8.9 ± 1.015 200 ± 2027 730 ± 9019 500052 130000 ± 10000 120 ± 10 0.14 ± 0.02 11 ± 1 7800 ± 2000 13000 ± 1000 32000 ± 2000 250000 ±20000 480000 ± 80000 >50 000d 0.051 ± 0.007 160 ± 10 29000 ±8000 >50 000d 0.61 ± 0.06 0.66 ±0.07 0.041 ± 0.005 28 ± 5 13000 ± 3000 >50 000d 8.3 ± 0.3 16 ± 1 0.45 ± 0.03 2.0 ± 0.3 1.2 ± 0.2 2.5 ± 0.3 4.7 ± 0.5 1.8 ± 0.2 0.66 ± 0.05 0.21 ± 0.02 1.1 ± 0.1 16 ± 1

a All sequences are backbone-cyclic via head-to-tail cyclization between residues 1 and 14 with one disulfide bond, as illustrated by compound 3 in Figure 2. Variations from SFTI-1 are shown with bold letters. Residues 4, 5, 7, and 14 are numbered in subscript. * = homo-arginine. Scanning electron microscopy (SEM) measurements are from three independent measurements performed in triplicates. bFIXa, FXa, FXIIa, thrombin, tPA, and uPA. cFIXa, FXa, FXIa, FXIIa, thrombin, tPA, uPA, pKLK, and matriptase. dNo significant inhibition at 50 μM.

position) forms hydrogen bonds with Asp189 (735) OD1 and Ser190 (736) OG deep inside the S1 pocket. Furthermore, in the μ-plasmin/compound 3 structure, Tyr4 of the SFTI scaffold plays key roles in both an intramolecular interaction within 3 and intermolecular interactions with the catalytic site of μ-plasmin (Figure 4B and Table S2). Specifically, Tyr4 OH forms a hydrogen bond with Arg2 NH2 in 3, which further constrains the compact structure of the inhibitor. Consistent with this, substituting Tyr4 in compound 3 with Phe (4) or Trp (5) results in over 10-fold reductions in potency. These findings are in agreement with our previous studies showing that intermolecular hydrogen bonds within the SFTI scaffold promotes high potency of inhibition.13,25 Furthermore, the side chain of Tyr4 forms a π-stacking interaction with Trp215 (761) and an aromatic dipole interaction with the negatively charged S2 pocket containing His57 (603), Asp102 (646), and Ser195 (760).

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; Figures 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). The μ-plasmin/compound 3 was crystallized with two protease/inhibtor complexes per asymmetric unit at 1.8 Å (space group P212121), which aligned with an RMSD of 0.121 Å over 1525 atoms. Here, we focus on the analysis of the structure of μ-plasmin/compound 3 for monomer A and its cognate inhibitor. The total buried surface area at the interface is 1239 Å2, with the main-chain of 3 forming backbone hydrogen bond interactions with μ-plasmin via residues Cys3, Lys5, Ser6, Lys7a, and Asp14, whereas the Lys5 NZ (P1 554



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S2′ pocket. These findings confirm that Lys7 stabilizes the plasmin-inhibitor complex. Accordingly, we generated an Arg7 variant (6). The inhibitory potency of compound 6 for plasmin was comparable to that of 3 and without detectable inhibition of thrombin, FIXa, FXa, FXIa, FXIIa, tPA, uPA, pKLK, or matriptase, but with reduced selectivity over trypsin and cathepsin G. In a previous plasmin screen against a P2′ library of SFTI-1 (GTCTRSXPPCNPN, X = variable residue), we found that Lys7 was preferred over Arg7, most likely because this SFTIbased library had Arg5 rather than Lys5 as the P1 residue (considerable cooperativity appears to occur between the two subsites).15 To confirm this, we synthesized Arg5 variants of compound 3 (7) and compound 6 (8) and found that the combination of Arg5/Arg7 (8) was less preferred than Arg5/ Lys7 (7) with a 2-fold reduction in potency. Further, substituting Arg7 in compound 6 with homo-Arg7 produced an inhibitor (9) with 10-fold lower potency for plasmin. Combined, these findings indicate that only the combination of Lys5/Lys7 (3) or Lys5/Arg7 (6) ideally places the basic side chains for maximum potency of inhibition. Despite the contribution of the basic P2′ residue, it appears that the intermolecular and intramolecular interactions of Tyr4 are essential for potency. To confirm this, we substituted Tyr4 with the wild-type residue Thr in one of the lead compounds (6) to produce 10, and this single substitution resulted in nearly a 50-fold reduction in potency. To understand this phenomenon, we crystalized the μ-plasmin/compound 10 complex, which diffracted to 1.32 Å, and as for the μ-plasmin/ compound 2 complex, it belonged to space group C121 with one binary complex in an asymmetric unit (Table S1). Comparison of the crystal structures of 10 (Thr4) and the lead inhibitor 3 (Tyr4) in complex with μ-plasmin (Figures 3 and 4B,C) provides an insight into this preference. Whereas the main-chain of 10 Thr4 O (P2) mediates an additional intermolecular interaction with Gln738 NE2 (Figure 4C and Table S2) and the binding surface area remains similar (1206 Å2), the aromatic π-stacking interactions between the inhibitor and plasmin were lost as was the Tyr4−Arg2 stabilizing intramolecular hydrogen bond. The μ-plasmin/compound 3 complex revealed other specific interactions where Asp14 OD2 forms a salt bridge with Arg175 (719) NE while stabilizing the inhibitor by a hydrogen bond between Asp14 OD1 and the Gly1 backbone N. We have shown previously by molecular dynamics, NMR, crystallography (PDB 4K1E and 4KEL), and binding assays that Asp14 in SFTI can be substituted with Asn to stabilize the peptide backbone (through hydrogen bonds) and improve the inhibition constants for various proteases.13,21,25,26 To evaluate the contribution of SFTI Asp14, we substituted it with Asn in the most potent inhibitor (6), and the resulting compound 11 lost 29-fold binding affinity for plasmin. To examine this, we crystalized the μ-plasmin/compound 11 complex, which diffracted to 2 Å and belongs to the P63 space group with one binary complex in an asymmetric unit (Table S1). The μplasmin/compound 11 complex revealed loss of the intramolecular Tyr4 OH Arg2 NH2 hydrogen bond as well as the salt bridge between Asp14 OD2 and Arg719 NE (Figure 4D), indicating these interactions as key for the potency of compounds 3 and 6. The crystal structure of the μ-plasmin/compound 3 complex (Figures 3 and 4B) showed that Glu60 (606) was located at the S5′ pocket of plasmin and that the P5′ Ile10 side chain was

Figure 2. Atomic structure of compound 3. Illustration of the atomic structure of compound 3 with a cyclic backbone achieved by head-totail cyclization between residues 1 and 14. The cyclic backbone and disulfide bond occur for all compounds in this study, and the residue numbering is based on wild-type SFTI-1.

Figure 3. Surface representation of crystal structures of μ-plasmin in complex with SFTI variants. The electrostatic surface representation of μ-plasmin (basic, blue; acidic, red) is shown with key binding residues of SFTI as sticks and numbered. The superimposed structures of compounds 2 (PDB 6D40), 3 (PDB 6D36), 10 (PDB 6D3Y), and 11 (PDB 6D3Z) are shown as cyan, green, yellow, and blue sticks, respectively. The structure alignment is centered at the catalytic triad, and the μ-plasmin, which is in complex with compound 2, is shown.

Another intermolecular key interaction between μ-plasmin and 3 is the positively charged side chain of Lys7 (P2′), which is positioned on top of the negatively charged S2′ pocket formed by Glu73 (623) and Glu143 (687) (without forming any hydrogen bonds). Comparing the crystal structures of 3 (Lys7) and 2 (Ile7) (Figures 3 and 4A,B), we found that 2 forms backbone hydrogen bonds as well as hydrophobic interactions with Phe41 (587) of plasmin, similar to that of 3, except without forming any electrostatic interaction with the 555



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Figure 4. 2Fo−Fc omit maps of electron density (gray) corresponding to SFTI variants were contoured at 1.0 sigma. The intramolecular interactions between the inhibitors and μ-plasmin are labeled and shown as red and black dashed lines, respectively. Compounds 2, 3, 10, and 11 are colored as in Figure 3.

affect the canonical binding mode and thus the mechanism of Laskowski inhibitors is yet to be systematically explored. These mutational studies based on μ-plasmin/inhibitor structures combined with previous substrate and inhibitor library screening studies15,22 suggested that further substitutions in compound 3 were unlikely to significantly improve the inhibition of plasmin. The dissociation rate constant (koff) for compound 3 was determined to be (9.9 ± 0.95) × 10−4 s−1, which is close to 20-fold faster than the dissociation rate constant that we previously determined for SFTI-1 and trypsin ((0.5 ± 0.1) × 10−4 s−1)13 and also faster than the koff rates determined for recently described monocyclic plasmin inhibitors by Hinkes et al.11 Conversely, the association rate constant (kon) for compound 3 was determined to be (1.9 ± 0.3) × 107 M−1 s−1, which is 10-fold faster than for SFTI-1 and trypsin ((0.2 ± 0.3) × 107 M−1 s−1)13 and more so compared to the monocyclic plasmin inhibitors reported by Hinkes et al.11 Compound 3 has a 300-fold selectivity over all serine proteases examined and over a million-fold selectivity over family S1 serine proteases from blood and was selected for further evaluation of fibrinolysis inhibition in an ex vivo fibrinolysis assay. Pooled human plasma was diluted with buffer 1:4 to allow for measurement of the fibrin content by its light scattering properties,28 and the level of inhibition was compared to the gold-standard plasmin-inhibitor aprotinin. Under these conditions, the maximum available plasminogen concentration is expected to be around 300 nM.23 Incomplete inhibition of fibrinolysis was achieved with 400 nM (70%) and 200 nM (25%) aprotinin (Figures 5 and S5), respectively, consistent with aprotinin being a promiscuous inhibitor of most blood serine proteases and thus also binding to other protease targets. Conversely, 400 nM of compound 3 completely attenuated fibrinolysis, whereas 200 nM achieved

perfectly aligned and spaced to allow for substitutions of Ile10 with residues to interact with Glu60 (606) (Figure S4). Ile10 in compound 3 was substituted with Lys (12) or Arg (13) with the aim of introducing an intermolecular salt bridge with Glu606, but these compounds lost 60-fold and 116-fold in potency, respectively. Similarly, Ile10 in compound 3 was substituted with Gln (14) or Asn (15) with the aim of introducing an intermolecular hydrogen bond with Glu60 (606). These substitutions resulted in a loss of potency of 44fold (Gln10) and 16-fold (Asn10), although less than when substituting Ile10 with basic residues. Plasmin has a preference for Arg at the P4 position (SFTI residue 2) in peptide substrates,22 and in the μ-plasmin/ compound 3 complex, Arg2 forms a perfectly aligned Tstacked cation−π interaction with the pyrrole ring of the Trp215 (761) side chain of plasmin. Substituting Arg2 for homo-Arg in one of the lead inhibitors (6) resulted in compound 16 with a 5-fold reduction in inhibition, indicating the importance of this interaction. Since the Arg2 side chain is known to interact with Asp14 to stabilize the backbone in wildtype SFTI-1,13,25 we explored the effects of combining the homo-Arg2 substitution with a Asp14 to Glu substitution; however, the resulting inhibitor (17) displayed a 27-fold loss of potency compared with compound 6, again highlighting the importance of the salt bridge between Asp14 OD2 and Arg175 (719) NE of plasmin. The μ-plasmin/compound 3 structure revealed that near Arg175 (719), another Arg residue [Arg221 (767)] is located at the S5 binding site near Gly1 in 3. Gly1 in compound 6 was substituted with Asp with the aim to promote the formation of another intramolecular salt bridge, but the resulting inhibitor 18 displayed a 390-fold loss of activity. Although we have previously shown that a Gly1 to Ala substitution in SFTI-1 may be tolerated by trypsin (3-fold loss in activity),27 whether other functional substitutions could 556



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to Arg, resulting in a plasmin inhibitor (Ki = 0.9 nM) with high selectivity over tPA, uPA, and pKLK and factors IIa, VIIa, and XIa.30 However, substituting the P2′ residue with Lys in compound 2 resulted in a modest 2.8-fold increase in potency but increased the selectivity for all off-targets. This aligns with our previous findings using an inhibitor library based on SFTI1 showing that whereas S1 family serine proteases generally prefer the wild-type P2′ residue (Ile7) in the SFTI scaffold, most proteases also have different additional preferences that may be used to modulate inhibitor specificity.15 Considering the general success of substituting the P2′ residue in the SFTI scaffold, we thought that substituting the P5′ residue might further improve potency and/or specificity. The Glu60 (606) in plasmin appears perfectly positioned to interact with a substituted P5′ residue (Ile10) in (3), and the only protease examined with an acidic residue at this position was FXIa, which is not inhibited by compound 3. However, substitutions of Ile10 with residues for potential of forming intermolecular salt bridges (Arg/Lys) or hydrogen bonds (Gln/Asn) resulted in major loss of potency. This may be due to steric hindrance since the level of loss of potency correlated with the size of the substituted residue. Another possible explanation is that introducing a residue at position 10 in SFTI that forms intermolecular interactions with plasmin has a negative effect on the reversible (Laskowski) mechanism of inhibition, trapping the protease in a futile cycle of cleavage and re-ligation of the peptide bond.12,31 We have previously shown that intramolecular hydrogen bonds in the SFTI scaffold are important for positioning the cleaved N-terminus for resynthesis of the peptide bond.13,25,26 Thus, it is possible that favorable interactions between the P5′ site of the SFTI scaffold and the S5′ site of the protease misaligns the cleaved N-terminus, reducing the efficiency of peptide bond re-ligation.

Figure 5. Inhibition of fibrinolysis in plasma. Fibrin formation and fibrinolysis induced by thrombin and tPA, respectively, were inhibited by either aprotinin (left) or compound 3 (right) and measured by fibrin light scattering. The data is shown as the mean from three independent experiments performed with 12 technical replicates. The experimental variation was omitted for clarity but is shown as mean ± standard deviation in Figure S5.

90% inhibition, consistent with a 1:1 binding to activated plasmin in the presence of other blood serine proteases.

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DISCUSSION In this study, we used the SFTI-1 scaffold to design plasmin inhibitors with high potency and exquisite selectivity over other serine proteases in the coagulation pathways. This was achieved by combining information from plasmin substrate and inhibitor library screens15,22 as well as structural studies using μ-plasmin/SFTI-variant complexes. The most selective inhibitor (3) inhibited fibrinolysis in plasma with high efficacy and provides a promising lead compound for development of an antifibrinolytic agent as well as a powerful tool to study the diverse roles of plasmin in normal physiology and disease. Plasmin’s preference for aromatic residues in the S2 pocket is clearly the most defining feature for potent and selective inhibition of this serine protease. The side chain of Tyr4 forms a perfect π-stacking interaction with Trp215 (760) and a dipole interaction with the negatively charged S2 pocket, containing His57 (603), Asp102 (646), and Ser214 (760). This finding aligns with the strong preference for aromatic P2 residues by plasmin in combinatorial29 and noncombinatorial22 peptide substrate libraries. Whereas the residues in the S2 binding pocket of plasmin are conserved among the serine proteases examined in this study (excluding Tyr215 in cathepsin G), the S2 pockets of the other serine proteases are too small to accommodate an aromatic residue because of steric hindrance from the 93−100 loop absent in plasmin (Figure S6). Indeed, the most potent and selective peptidomimetic plasmin inhibitors produced by others also have aromatic groups at the P2 position,9,11 and it is likely that capitalizing on this feature will be essential for design of highly potent and selective inhibitors in the future. Another important defining feature of plasmin is its negatively charged S2′ pocket due to the flanking Glu73 (623) and Glu143 (687) residues, and the alignment of serine proteases in this study shows that Glu73 and Glu143 are only present in plasmin (Figure S6). In another study, the P2′ residue in Kunitz domain-1 of TFPI-2 was mutated from Leu

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CONCLUSIONS Compound 3 is the most potent and selective plasmin inhibitor engineered to date and provides a promising lead compound for development of next-generation fibrinolysis inhibitors with higher efficacy and selectivity and thereby potentially reduced side effects. Although several challenges remain to be overcome in turning a promising lead into a drug, the nature of 3 as a disulfide-stabilized head-to-tail cyclic peptide places it in one of the most favorable classes of peptides for drug development.32,33 Its intrinsic advantages include high chemical stability, resistance to protease digestion, and, given its small size, ease of synthesis. The latter feature bodes well for the important commercial consideration of a low cost-of-goods. The plasminogen-plasmin system is involved in many physiological processes in addition to homeostasis, including inflammation,34 neurobiology,35 clearance of misfolded proteins,36 cell migration/tissue remodeling,37 and wound healing.38 These processes involve activation of plasminogen via interaction with numerous plasminogen receptors present on the surfaces of most cell types and have been implicated in a number of diseases related to dysregulated inflammation, autoimmunity, and cancer.39−42 Therefore, highly potent and selective plasmin inhibitors also provide invaluable tools to further define the roles of the plasminogenplasmin system in cell-based and in vivo models of physiology and disease in the future. 557



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100 mM MgCl2, and 15−21% PEG-4000 as the mother liquor, and the condition for μ-plasmin/compound 2 and μ-plasmin/compound 10 was 100 mM MES, 150 mM ammonium sulfate, and 13−17% PEG-4000. Lastly, the μ-plasmin (S741A)/compound 11 complex was crystalized in 100 mM sodium acetate pH 4.5, 1 M sodium formate. Crystals were flash cooled in liquid nitrogen in the presence of 15% glycerol. Data sets were collected at Australia Synchrotron MX beamlines and processed using XDS.46 The crystal structures of complexes were solved by molecular replacement (using 5UGG as the starting search model for μ-plasmin) and the program PHASER from CCP4.47 After refinement using the REFMAC program,48 SFTI-1 (PDB ID 1SFI) was fitted into the Fo−Fc electron density using COOT,49 and inhibitor sequences were correspondingly modified to SFTI variants. Model refinement and building were carried out using PHENIX50 and COOT. Fibrinolysis Inhibition Assays. Human pooled citrated plasma (GeneTex) was diluted with buffer (Tris−HCL, pH 7.4, 150 mM NaCl, 30 mM CaCl2) and with various concentrations of aprotinin or compound 3. The assay was initiated by addition of tPA (1.5 nM) and thrombin (2.5 nM) in buffer (final plasma concentration 1:5). Fibrin formation and fibrinolysis were monitored by light scattering at λ 405 nM using a Tecan M1000 Pro microplate reader over 1 h as previously described.51 The results are reported as the mean from three independent experiments performed using 12 technical replicates.

EXPERIMENTAL SECTION

Peptide Synthesis, Purification, and Validation. SFTI-1 and its variants were synthesized using standard 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis on 2-chlorotrityl chloride resin (0.8 mmol equiv per gram) before cyclization and disulfide bond formation as previously described.19 Briefly, coupling reactions were performed using Fmoc N-protected amino acids (4 equiv) activated with 4 equiv O-(6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate and 8 equiv N,N-diisopropylethylamine in N,N-dimethylformamide. Cyclization was performed in solution as above, with 4 equiv. 1-[Bis(dimethylamino)methylene]-1H-1,2,3triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate as the activator over 3 h. Disulfide bonds were formed by oxidation in 10% ammonium bicarbonate (pH 8.3) with 10 μM oxidized glutathione. Colorimetric peptide−pNA (para-nitroanilide) substrates were synthesized on para-phenylenediamine derivatized 2-chlorotrityl chloride resin followed by solution oxidation of the free para-amine to a para-nitro group by a modified version of the method by Abbenante et al.43 as previously described.19 Peptides were purified using reversed-phase HPLC (Shimadzu Prominence) using a 5 μm ZORBAX Extend-C18 PrepHT column (21.2 × 250 mm2) and a linear gradient of 10% acetonitrile/0.05% trifluoroacetyl (TFA) to 90% acetonitrile/0.05% TFA. SFTI variants were purified both before and after formation of the disulfide bond. Peptide purity (>95%) was confirmed by reversed-phase UPLC using a 5 μm Agilent 300 SB C18 column (2.1 × 50 mm2) at 40 °C with mobile phases as above (Figure S7). Peptide masses (Table S3) were determined by electrospray ionization mass spectroscopy (Shimadzu Prominence). Enzyme Assays. Native activated and purified proteins were obtained from Sigma-Aldrich (human plasmin, bovine cationic trypsin) and Molecular Innovations (human thrombin, pKLK, βFIXa, FXa, β-FXIIa, two chain-tPA, HMW uPA, and cathepsin G). The concentration of plasmin was determined by active-site titration using bovine aprotinin (Sigma-Aldrich). Inhibition constants for the cyclic peptide inhibitors were determined in three independent triplicate experiments with 100 μM substrate in assay buffer (100 mM Tris pH 8.0, 100 mM NaCl, and 0.005% (v/v) Triton X-100), following equilibration of proteases and inhibitors for 30 min in a 96well plate (low binding microplate wells, Corning). Protease concentrations, buffer additives, and substrates are given in Table S4. Fluorescent peptide−MCA (7-methoxycoumarin-4-yl-acetyl) substrates were obtained from Peptide Institute Inc. Substrate hydrolysis was monitored by following absorbance at 405 nm for 7 min (peptide−pNA substrates) (excluding FXIa, which was monitored for 60 min) or by fluorescence at λex = 360 nm/λem = 460 nm for 10 min (peptide−MCA substrates) using a Tecan M1000 Pro microplate reader. Inhibition constants were calculated with the Morrison equation for tight binding inhibitors using the substrate KM values given in Table S4 by nonlinear regression using Prism 7 (GraphPad). The results are reported as the mean ± SEM from three independent experiments performed in triplicates. The dissociation constant (koff) for 3 was calculated with the method derived by Baici and Gyger-Marazzi44 based on the lag phase between the steady state of hydrolysis rates preincubated protease/inhibitor versus simultaneous addition of substrate and inhibitor. A graphical representation of the method and the equation are given in Figure S1B. Protein Expression and Purification. The catalytic domain of human μ-plasminogen (residues 542−791) and its active-site mutant Ser195(741)Ala were expressed and purified from Pichia pastoris as previously described.45 Both the wild-type and active-site mutant μplasminogens were activated into μ-plasmin with tPA in the presence of the SFTI inhibitor (1:1.2 molar ratio) to generate μ-plasmin/SFTI complexes, which were further purified by size exclusion chromatography. Crystallization and Structure Determination. Purified complexes at 10 mg/mL were used for crystallization trials by the hanging drop vapor phase diffusion method at 20 °C, and crystals were obtained in the following conditions. Complex crystals of μ-plasmin with compound 3 were formed with 100 mM sodium citrate pH 4−6,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01139. Binding kinetics of compound 3 and human plasmin (Figure S1); structure alignment of inhibitor-bound forms of trypsin, matriptase, and μ-plasmin (Figure S2); structure alignment of SFTI-1 with SFTI inhibitors from this study (Figure S3); μ-plasmin/compound 3 structure (Figure S4); inhibition of fibrinolysis in plasma (with standard deviation) (Figure S5); alignment of plasmin and other S1 family serine proteases (Figure S6); analytical RP-HPLC traces confirming the purity of peptides (Figure S7); X-ray crystal structure data collection and refinement statistics (Table S1); interactions between SFTI inhibitors and μ-plasmin (Table S2); inhibitor sequences and determined masses (Table S3); protease assay conditions, substrates, and KM values (Table S4) (PDF) Accession Codes

The authors will release the PDB atomic coordinates and experimental data for μ-plasmin in complex with 2 (6D40), 3 (6D3X), 10 (6D3Y), and 11 (6D3Z) upon article publication.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.C.W.). *E-mail: [email protected] (R.H.P.L.). *E-mail: [email protected] (D.J.C.). ORCID

Joakim E. Swedberg: 0000-0003-4243-8660 Thomas Durek: 0000-0003-0686-227X David J. Craik: 0000-0003-0007-6796 558



Article

Present Address

(10) Afosah, D. K.; Al-Horani, R. A.; Sankaranarayanan, N. V.; Desai, U. R. Potent, Selective, Allosteric Inhibition of Human Plasmin by Sulfated Non-Saccharide Glycosaminoglycan Mimetics. J. Med. Chem. 2017, 60, 641−657. (11) Hinkes, S.; Wuttke, A.; Saupe, S. M.; Ivanova, T.; Wagner, S.; Knorlein, A.; Heine, A.; Klebe, G.; Steinmetzer, T. Optimization of Cyclic Plasmin Inhibitors: From Benzamidines to Benzylamines. J. Med. Chem. 2016, 59, 6370−6386. (12) Zakharova, E.; Horvath, M. P.; Goldenberg, D. P. Structure of a Serine Protease Poised to Resynthesize a Peptide Bond. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 11034−11039. (13) de Veer, S. J.; Swedberg, J. E.; Akcan, M.; Rosengren, K. J.; Brattsand, M.; Craik, D. J.; Harris, J. M. Engineered Protease Inhibitors Based on Sunflower Trypsin Inhibitor-1 (SFTI-1) Provide Insights Into the Role of Sequence and Conformation in Laskowski Mechanism Inhibition. Biochem. J. 2015, 469, 243−253. (14) Luckett, S.; Garcia, R. S.; Barker, J.; Konarev, A. V.; Shewry, P.; Clarke, A.; Brady, R. High-Resolution Structure of a Potent, Cyclic Proteinase Inhibitor From Sunflower Seeds. J. Mol. Biol. 1999, 290, 525−533. (15) de Veer, S. J.; Wang, C. K.; Harris, J. M.; Craik, D. J.; Swedberg, J. E. Improving the Selectivity of Engineered Protease Inhibitors: Optimizing the P2 Prime Residue Using a Versatile Cyclic Peptide Library. J. Med. Chem. 2015, 58, 8257−8268. (16) Wang, C. K.; Craik, D. J. Designing Macrocyclic Disulfide-Rich Peptides for Biotechnological Applications. Nat. Chem. Biol. 2018, 14, 417−427. (17) Fittler, H.; Avrutina, O.; Empting, M.; Kolmar, H. Potent Inhibitors of Human Matriptase-1 Based on the Scaffold of Sunflower Trypsin Inhibitor. J. Pept. Sci. 2014, 20, 415−420. (18) Gitlin-Domagalska, A.; Debowski, D.; Legowska, A.; Stirnberg, M.; Okonska, J.; Gutschow, M.; Rolka, K. Design and Chemical Syntheses of Potent Matriptase-2 Inhibitors Based on Trypsin Inhibitor SFTI-1 Isolated From Sunflower Seeds. Biopolymers 2017, 108, No. e23031. (19) Swedberg, J. E.; Li, C. Y.; de Veer, S. J.; Wang, C. K.; Craik, D. J. Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold. J. Med. Chem. 2017, 60, 658− 667. (20) Chen, W.; Kinsler, V. A.; Macmillan, D.; Di, W. L. Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold - a Potential Therapeutic Intervention for Skin Diseases. PLoS One 2016, 11, No. e0166268. (21) de Veer, S. J.; Furio, L.; Swedberg, J. E.; Munro, C. A.; Brattsand, M.; Clements, J. A.; Hovnanian, A.; Harris, J. M. Selective Substrates and Inhibitors for Kallikrein-Related Peptidase 7 (KLK7) Shed Light on KLK Proteolytic Activity in the Stratum Corneum. J. Invest. Dermatol. 2017, 137, 430−439. (22) Swedberg, J. E.; Harris, J. M. Plasmin Substrate Binding Site Cooperativity Guides The Design of Potent Peptide Aldehyde Inhibitors. Biochemistry 2011, 50, 8454−8462. (23) Cederholm-Williams, S. A. Concentration of Plasminogen and Antiplasmin in Plasma and Serum. J. Clin. Pathol. 1981, 34, 979−981. (24) Parry, M. A. A.; Fernandez-Catalan, C.; Bergner, A.; Huber, R.; Hopfner, K.-P.; Schlott, B.; Gührs, K.-H.; Bode, W. The Ternary Microplasmin-Staphylokinase-Microplasmin Complex is a ProteinaseCofactor-Substrate Complex in Action. Nat. Struct. Biol. 1998, 5, 917−923. (25) Swedberg, J. E.; de Veer, S. J.; Sit, K. C.; Reboul, C. F.; Buckle, A. M.; Harris, J. M. Mastering the Canonical Loop of Serine Protease Inhibitors: Enhancing Potency by Optimising The Internal Hydrogen Bond Network. PLoS One 2011, 6, No. e19302. (26) Riley, B. T.; Ilyichova, O.; Costa, M. G.; Porebski, B. T.; de Veer, S. J.; Swedberg, J. E.; Kass, I.; Harris, J. M.; Hoke, D. E.; Buckle, A. M. Direct and Indirect Mechanisms of KLK4 Inhibition Revealed By Structure and Dynamics. Sci. Rep. 2016, 6, No. 35385. (27) Quimbar, P.; Malik, U.; Sommerhoff, C. P.; Kaas, Q.; Chan, L. Y.; Huang, Y. H.; Grundhuber, M.; Dunse, K.; Craik, D. J.; Anderson,

⊥

Department of Agricultural Product Technology, Faculty of Agricultural Technology, Brawijaya University, Malang 65145, East Java, Indonesia (T.M.).

Author Contributions ∥

J.E.S. and G.W. contributed equally to this work.

Author Contributions

All authors contributed to the writing of this manuscript, and all authors have approved the final version. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Meng-Wei Kan and Simon de Veer for valuable discussions and assistance with preparing the manuscript. J.E.S. is a National Health and Medical Research Council (NHMRC) Early Career Fellow [Grant APP1069819], J.C.W. is a National Health and Medical Research Council of Australia (NHMRC) Senior Principal Research Fellow (Grant APP1059411), and D.J.C. is an Australian Research Council Laureate Fellow (Grant FL150100146).

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ABBREVIATIONS KLK, kallikrein-related peptidase; pKLK, plasma kallikrein; FXa, activated factor FIX; FIXa, activated factor FIX; FXIa, activated factor FXI; FXIIa, activated factor FXII; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; peptide−pNA, peptide−para-nitroanilide

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Highly Potent and Selective Plasmin Inhibitors Based on the

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