Design of Potent and Selective Cathepsin G Inhibitors Based on the

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Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Joakim Erik Swedberg, Choi Yi Li, Simon J. de Veer, Conan K Wang, and David J Craik J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01509 • Publication Date (Web): 03 Jan 2017 Downloaded from http://pubs.acs.org on January 3, 2017

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Journal of Medicinal Chemistry

Design of Potent and Selective Cathepsin G Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Joakim E. Swedberg,* Choi Yi Li, Simon J. de Veer, Conan K. Wang, David J. Craik Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia

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ABSTRACT Neutrophils are directly responsible for destroying invading pathogens via reactive oxygen species, antimicrobial peptides and neutrophil serine proteases (NSPs). Imbalance between NSP activity and endogenous protease inhibitors is associated with chronic inflammatory disorders and engineered inhibitors of NSPs is a potential therapeutic pathway. In this study we characterized the extended substrate specificity (P4-P1) of the NSP cathepsin G using a peptide substrate library. Substituting preferred cathepsin G substrate sequences into the sunflower trypsin inhibitor-1 (SFTI-1) produced a potent cathepsin G inhibitor (Ki = 0.89 nM). Cathepsin G’s P2ʹ preference was determined by screening against a P2ʹ diverse SFTI-based library, and the most preferred residue at P2ʹ was combined in SFTI-1 with a preferred substrate sequence (P4-P2) and a non-proteinogenic P1 residue (4-guanidyl-L-phenylalanine) to produce a potent (Ki = 1.6 nM) and the most selective (≥ 360-fold) engineered cathepsin G inhibitor reported to date. This compound is a promising lead for further development of CG inhibitors targeting chronic inflammatory disorders.


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INTRODUCTION Neutrophils are a key component of the innate immune system and form the first line of defense against bacterial and fungal infections. After migrating to the site of injury, neutrophils eliminate invading organisms using cytotoxic granules that contain reactive oxygen species, antimicrobial peptides and several proteases, including neutrophil serine proteases (NSPs).1-3 At present, four NSPs have been identified, including neutrophil elastase (NE), proteinase 3 (PR3) and cathepsin G (CG),4 as well as the recently identified neutrophil serine protease 4 (NSP4).5 The NSPs, together with the granzymes (A, B, H, K and M), mast cell chymase, complement factor D, and the pseudo protease (catalytically inactive), azurocidin, belong to the S1A chymotrypsin family of serine proteases. The substrate specificity of each NSP has recently been characterized using combinatorial peptide libraries6 and protein substrates.7 NE and PR3 share an elastase-type P1 specificity with cleavage after small or branched amino acids, such as Ala, Val and Thr. By contrast, CG has an unusually broad P1 tolerance, with cleavage after aromatic, branched hydrophobic and, to some degree, basic residues.6, 7 NSP4 has been reported to have a preference for P1 Arg,6 but the protease also cleaves after aromatic residues to some degree.5 NSPs are directly responsible for destroying invading pathogens as well as degrading extracellular matrix components to allow for neutrophil infiltration during acute inflammation.8 Additionally, NSPs are involved in regulating the immune response by processing chemokines, growth factors and cell surface receptors.9 As NSPs have diverse roles that are vital for preventing infection, their activity is tightly regulated at several levels. First, NSPs are produced as inactive zymogens that require sequential removal of the N-terminal signal peptide, followed by a two amino acid pro-peptide that is cleaved by dipeptidyl peptidase I (cathepsin C).5,

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Second, NSP activity in the extracellular space is regulated by endogenous inhibitors, including 3 ACS Paragon Plus Environment


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α1-proteinase inhibitor (NE, PR3, NSP4 and CG), elafin (NE and PR3), α1-anti-chymotrypsin (CG) and secretory leukocyte protease inhibitor (CG).8 Finally, these inhibitors can also be inactivated by NSPs at high concentrations or by oxidation. For example, PR3 and NE can degrade α1-proteinase inhibitor11 whilst NE degrades α1-anti-chymotrypsin.12, 13 Additionally, the release of reactive oxygen species by activated neutrophils can inactivate secretory leukocyte protease inhibitor and α1-proteinase inhibitor by oxidizing the inhibitory loop Met73 and Met358, respectively.14, 15 Imbalance between NSP activity and endogenous protease inhibitors is associated with chronic inflammatory disorders, such as chronic obstructive pulmonary disorder (COPD), bronchiectasis, pulmonary fibrosis and cystic fibrosis.1,

8, 16-18

COPD correlates strongly with

lifestyle factors such as smoking or exposure to pollution, but genetic factors have also been identified that predispose an individual to COPD. Patients with α1-proteinase inhibitor deficiency develop COPD, and the severity of disease correlates with polymorphisms in the SERPINA1 gene,19 validating a role for proteases, including NSPs, in the development of COPD. To date, most research has focused on the development of potent and selective inhibitors of NE and a number of clinical trials using NE inhibitors have been performed or are ongoing.17 However, no NE inhibitors have been approved for clinical use, except for sivelstat, which is approved in Japan and Korea.17,

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To some extent, this may be a result of redundancy in the pro-

inflammatory signaling pathways mediated by NSPs.21 It is noteworthy that α1-proteinase inhibitor shows activity against all of the NSPs, but clinical trials for chronic inflammatory disorders have so far focused on NE alone. As NE inhibitors have only seen modest success in clinical trials, there is now growing interest in developing inhibitors for other NSPs, in particular CG.22 A dual inhibitor of CG (Ki = 4 ACS Paragon Plus Environment

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38 nM) and mast cell chymase (Ki = 2.3 nM) has been reported23 and shown to reduce inflammation in animal models of pulmonary inflammation.24 However, this inhibitor lacks selectivity and thus, it is difficult to assess whether the decrease in inflammation could be attributed to inhibition of CG, chymase or both proteases. While there are several potent endogenous inhibitors of CG, these can be inactivated by neutrophils via several mechanisms and are therefore not ideal drug candidates. Accordingly, no truly potent and selective CG inhibitors that are suitable for therapeutic development have been reported to date. Sunflower trypsin inhibitor-1 (SFTI-1), a 14 amino acid backbone cyclic peptide bisected by a disulfide bond, is the smallest known member of the Bowman-Birk family of serine protease inhibitors,25-27 and a potent inhibitor of trypsin (Ki = 0.0017 nM).28 We have previously shown that SFTI-1 is an excellent scaffold for engineering potent and selective inhibitors of specific serine proteases,25-27 or multi-target inhibitors for a family of closely related proteases.27 SFTI-1 can also be engineered to produce broad-range inhibitors towards both tryptic and chymotryptic serine proteases.27, 29 SFTI-1 has been reported to be an inhibitor of CG (Ki = 570 nM), and substituting the P1 residue in SFTI-1 from Arg5 to Phe5 produced a CG inhibitor with improved inhibition (Ki = 370 nM), albeit with limited selectivity over other serine proteases.30 This suggests that the SFTI-1 scaffold is suitable for developing potent and selective CG inhibitors, but has yet to be fully explored. In the current study we used a non-combinatorial colorimetric peptide substrate library to further define the substrate specificity of CG across the S4-S1 subsites. Preferred CG cleavage sequences were substituted into the P4-P1 subsites of SFTI-1 to produce potent CG inhibitors with sub nanomolar inhibition constants. Potency and selectivity of inhibition was further improved by screening CG against a SFTI-based inhibitor library to optimize the P2′ residue. 5 ACS Paragon Plus Environment


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The most potent and selective SFTI-variant inhibited CG with a Ki of 1.6 nM and at least 360fold selectivity over other NSPs and a panel of other serine proteases with chymotryptic and tryptic specificity. This compound is the most potent and specific CG inhibitor reported to date and is a promising lead for the development of CG inhibitors targeting chronic inflammatory disorders.

RESULTS A previous study showed that CG is inhibited by SFTI-1 (compound 1, Table 1) with a Ki of 570 nM.30 To confirm this finding, we synthesized SFTI-1 by solid-phase Fmoc synthesis and tested its activity against CG, which revealed a similar, although higher Ki (730 nM, Table 1). We previously engineered variants of SFTI-1 that were designed to display broad-range inhibition of serine proteases (i.e. compound 2 and 3).27,

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These variants were substituted at Arg2 (Thr),

Phe12 (Asn) and Asp14 (Asn) to promote intramolecular hydrogen bonding while having minimal side chain interactions with the target protease. Further, the P1 residue (Lys5) was substituted with either Arg (compound 2) or Phe (compound 3) to target proteases with trypsinlike or chymotrypsin-like specificity, respectively.29 CG is known to have mixed trypsin- and chymotrypsin-like specificity. To determine the most preferred P1 residue for designing SFTIbased inhibitors, we screened compounds 2 and 3 against CG. Both inhibitors were more potent than SFTI-1, with the P1 Phe variant (3) being the most potent (Ki = 390 nM). Considering the broad P1 specificity of CG, we also examined SFTI variants with P1 Tyr (4) and P1 Leu (5) and found that these were similarly potent to P1 Phe or Arg, respectively.


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Design and Screening of a targeted CG substrate library. We have shown that serine proteases can be screened by colorimetric para-nitroanilide (-pNA) peptide substrate libraries to determine optimal P4-P1 sequences for substituting into SFTI-1 to produce potent and selective inhibitors.25, 29, 31, 32 CG has previously been screened against a combinatorial positional scanning peptide library.6 However, since combinatorial substrate libraries only probe the preference of one protease binding subsite at once, cooperativity effects between adjacent residues in the substrate cannot be detected. To identify the most preferred substrate sequences, the findings from a positional scanning library needs to be deconvoluted using a sparse matrix library – a substrate library that contains all possible combinations of the most preferred residues for each position.25, 33 We used the information from a published positional scanning CG screen6 to design a non-combinatorial sparse matrix library of substrates to identify highly preferred CG cleavage sequences (Supporting Information, Table S1). Phe was chosen for the P1 position of the sparse matrix library since this residue produced the most potent SFTI-based inhibitor (3). Screening CG against the substrate library (Figure 2) showed that the most preferred residues overall were Asp/Thr (P4), Glu (P3) and norleucine (P2, norleucine: Nle or n). Several substrates were cleaved at significantly higher rates than the remainder of the library, and these included DTnFpNA, TEnF-pNA and IEnF-pNA. DEnF-pNA also appeared to be cleaved with high efficiency (as indicated by the yellow color after cleavage of the pNA moiety), but precipitation of the substrate prevented accurate spectrophotometric quantification. All four CG substrates were further characterized by determining their kinetic constants (Table 2). DEnF-pNA had the lowest KM (54 µM) whereas the highest kcat was seen for DTnF-pNA (1.10 s-1). DEnF-pNA was cleaved with the highest catalytic efficiency (kcat/KM), closely followed by DTnF-pNA.



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Synthesis and screening of CG peptide-aldehyde inhibitors. To explore whether the preferred substrate sequences DEnF, DTnF, TEnF and IEnF could be used to design CG inhibitors, the corresponding peptides aldehydes DEnF-H (6), DTnF-H(7), TEnF-H (8) and IEnF (9) were synthesized by solid-phase Fmoc synthesis using a solid support that produces peptide aldehydes upon acid hydrolysis. Peptide aldehydes inhibit serine proteases by forming a transition-state mimicking covalent bond with Ser195 (chymotrypsin numbering). DEnF-H, DTnF-H and IEnFH showed similar activity to the P1 substituted SFTI variants (2-5), whereas TEnF-H was 6-fold less potent most likely due to the higher KM of the corresponding substrate. Although the metabolic stability of peptide aldehydes is too low for therapeutic applications, these findings supported the use of these P4-P1 sequences for developing CG inhibitors. Substrate-guided design of potent SFTI-based CG Inhibitors. The preferred sequences DEnF, TEnF and IEnF were substituted into the P4-P1 sites of SFTI-1 as DCnF (10), TCnF (11) and ICnF (12) to preserve the vital disulfide bond (Cys3-Cys11). All variants showed improved activity compared to compounds 2-5, with compound 12 being the most potent (Ki = 3.4 nM). We have shown that Asn14 in 2 is important for maintaining the intramolecular hydrogen bond network, conformational stability and potency for kallikrein-related proteases.25, 29 Accordingly, Asp14 in compounds 11and 12 was substituted for Asn to produce 13 and 14, respectively. Compound 13 gained 12-fold potency (Ki = 0.89 nM) whereas 14 was slightly less potent. To investigate these findings further, we performed molecular dynamics simulations with 13 and 14 in complex with CG. Models of these complexes were constructed using the SFTI-1/trypsin complex and a CG crystal structure. These analyses suggested that for 13 the Asn14 side chain formed two stabilizing hydrogen bonds with the Thr2 backbone amide and side chain hydroxyl (Figure S3A), as we previously reported for 2.29 The same analysis for 14 indicated that steric


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hindrance by Ile2 reduced the hydrogen bond interaction between the Asn14 side chain and Ile2 backbone amide (Figure S3B). The most potent inhibitor (13) was screened against the other NSPs and was found to be highly selective over NE and PR3 (>10000-fold). However, screening 13 against other proteases with chymotryptic specificity revealed that chymotrypsin and KLK7 were also potently inhibited, with Ki values in the low nanomolar range (Table 3). Determining the P2ʹ preference of CG using a P2ʹ diverse SFTI-based library. We have shown that the P2ʹ residue is important for inhibitor specificity by screening diverse serine proteases against a cyclic peptide library based on 2, where P2ʹ was substituted by all proteinogenic residues (excluding Cys) or biphenylalanine (BiP; B).27 Screening CG against this P2ʹ diverse inhibitor library revealed that the P2ʹ residue in SFTI-1 (Ile7) was not the most preferred residue (Figure 3), and that CG has a broad P2ʹ specificity, which includes acidic (Asp/Glu), hydrophobic (Val/Met) and aromatic (Trp/BiP), but not basic, residues. Our previous study showed that P2ʹ Asp7 is not preferred by several serine proteases, including chymotrypsin and KLK7,27 and therefore Ile7 in 13 was substituted for Asp7, but the resulting compound 17 was unexpectedly 2-fold less potent. To rationalize this finding, we compared the Ki for the Asp7 (18) and Ile7 (2) variants of the inhibitor library, and the Asp7 variant was indeed 40-fold more potent, confirming the results from the P2ʹ diverse SFTI-based inhibitor library. The only other difference between 2 and 17 (apart from the P1 residue) is a Phe12 to Asn substitution. Consequently, Phe12 was substituted for Asn12 in 13 to produce 19, and this inhibitor showed 36-fold less activity than 13, indicating that Asn12 does not contribute to high binding affinity. These findings suggest that the P1 residue (Arg) in the P2ʹ library (Figure 3) strongly influences the P2ʹ preference, and that Asp7 is less preferred by CG when the P1 residue is Phe (17). Screening 17 against PR3 and NE revealed that this compound was slightly less selective than 13



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over these NSPs (still maintaining ~5000-fold selectivity), but with improved (~80-fold) selectivity over chymotrypsin and kallikrein-related peptidase 7 (KLK7). However, mast cell chymase was also potently inhibited by 17 (Ki = 7.7 nM). Molecular modelling of 2 and 17 showed that in the complex of CG and 2, Asp7 formed hydrogen bonds with Arg41 (chymotrypsin numbering, CG numbering in brackets (47) of CG, but not in 17 (Supporting Information, Figure S3C-F), supporting that the P1 residue modulated the P2ʹ preference of CG. This suggests that substantial cooperativity occurs between the P1 and P2ʹ residues, where the nature of the P1 residue alters the positioning of the P2ʹ residue. Design and Screening of SFTI-based CG Inhibitors with improved selectivity. With the aim of further improving potency and/or selectivity, Ile7 in 13 was substituted with Glu or BiP to produce compounds 20 and 21, respectively. The BiP7 variant (21) lost 70% potency for CG and was not further evaluated. The Glu7 variant (20) gained some 50% potency over the Asp7 variant (17), but was less selective over chymotrypsin. Molecular modelling of CG with 20 suggested that with Phe as the P1 residue Glu7 could reach into the basic S2ʹ pocket of CG to form hydrogen bonds (Supporting Information, Figure S3G-H), as seen before for 2 with P1 Arg in combination with Asp7 at the P2ʹ position. These findings further support strong cooperativity between the S1 and S2ʹ binding sites of CG. A previous study produced a dual chymotrypsin and CG (Ki = 570 nM) inhibitor by substituting the P1 residue in SFTI-1 (Lys5) for Phe.30 That study also showed that by substituting the P1 residue in SFTI-1 (Lys5) for 4-guanidyl-L-phenylalanine rather than Phe reduced the inhibitor’s activity against chymotrypsin, even though the resulting inhibitor was more potent for chymotrypsin than CG.30 With the aim of reducing the inhibition of proteases with chymotryptic S1 specificity we substituted P1 Phe in the most selective inhibitor (17) with 10 ACS Paragon Plus Environment

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4-guanidyl-L-phenylalanine, and the resulting compound 22 was similarly potent for CG as 17, but with greatly improved selectivity over chymotrypsin (>6300-fold), KLK7 (460-fold) and chymase (12000-fold) (Supporting Information, Figure S4). The least specificity of 22 was achieved for trypsin (360-fold), but this still represents >200000-fold reduction in activity compared to SFTI-1 (Ki = 0.0017 nM).28 There was no substantial inhibition of the other NSPs, thrombin or plasmin by 22, and this compound is the most potent and specific CG inhibitor reported to date. The ability of compound 22 to attenuate CG proteolysis of a physiological substrate, angiotensin I, was evaluated. CG is known to generate the pro-inflammatory octapeptide hormone angiotensin II from the biologically inactive (decapeptide) angiotensin I at the cell surface of activated neutrophils.34 CG converted angiotensin I (substrate enzyme ratio, 1000/1) to angiotensin II in 90 minutes (Figure 4A-B), whereas 100 nM of compound 22 blocked any angiotensin II generation for 5 hours (Figure 4C-D). To gain insights into the molecular determinants underpinning the potency and specificity of 22 molecular modelling of this compound in complex with CG was performed. This analysis indicated that the binding interaction relied mostly on intermolecular hydrogen bonds at the P4, P1 and P2ʹ positions. The Lys217 side chain formed hydrogen bonds with both the Thr2 side chain hydroxyl group and the Asn14 backbone carbonyl group (Figure 5A-B). At the S2ʹ pocket the P2ʹ carboxyl oxygen atoms of Asp7 formed hydrogen bonds with the guanadino group of Arg143 (148) of CG. In the S1 pocket the guanidino group of 4-guanidyl-L-phenylalanine formed hydrogen bonds with carboxylic oxygens of Glu226 (225) as well as several main chain carbonyl oxygens. This is in contrast to most trypsin-like serine proteases that recognise P1 basic residues through Asp189. NE, PR3 and NSP4 also have an acidic residue at the 226 position (Asp, chymotrypsin numbering) in combination with Gly189, but the S1 pocket is occluded by



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hydrophobic/aromatic residues. For NSP4, which is known to cleave after basic residues,5 the P1 Arg does not interact with Asp226, but rather with the hydroxyl groups of Ser192 and Ser216 (PDB 4Q7Z).35 Overlaying the CG/compound 22 model with the structure of NSP4 suggests that the side chain of 4-guanidyl-L-phenylalanine is too large to fit in the S1 pocket (Supporting Information, Figure S5A-B) and that the same binding mode as for the Phe-Phe-Argchloromethyl ketone inhibitor in the complex is unlikely. Thus, it remains to be determined to what degree compound 22 has inhibitory activity towards NSP4. DISCUSSION The design of potent inhibitors of CG that were specific over serine proteases with both trypsinand chymotrypsin-like specificity required optimization of the entire binding loop sequence (P4P2´) of SFTI-1. Substituting preferred cleavage sites into the P4-P1 positions resulted in an inhibitor (13) with improved binding affinity for CG, but without selectivity over chymotrypsin. By screening the P2ʹ preference of CG and comparing it to the corresponding preferences of other serine proteases,27 such as chymotrypsin, KLK7 trypsin, thrombin and plasmin, we identified a P2ʹ residue (Asp7) that promoted selectivity and the resulting inhibitor (20) had improved selectivity over chymotrypsin. Since the P2ʹ preference of chymase is not known selectivity over this protease was achieved by substituting a non-proteinogenic P1 residue (4guanidyl-L-phenylalanine) targeting the peculiar P1 specificity of CG. The resulting compound 22 showed at least 360-fold selectivity over serine proteases with trypsin-, chymotrypsin- and elastase-like specificity. Screening CG against SFTI-based inhibitors with diverse P1 residues (Phe, Tyr, Arg and Leu) has shown that CG has a relaxed P1 specificity for inhibitors, even more so than seen previously for substrates.6 Colorimetric tetrapeptide substrates with P1 Arg or Leu were cleaved 12 ACS Paragon Plus Environment

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with ≤33% of the rate of those with P1 Phe or Tyr.6 Conversely, in this study the activity of inhibitors with P1 Arg or Leu (2, 5) was ~80% of those with P1 Phe or Tyr (3, 4). This phenomenon is similar to what we have previously observed for KLK7 where KLK7 did not cleave with high rates after P1 Arg in colorimetric tetrapeptide substrates,36 yet SFTI-variants with P1 Arg potently inhibited KLK7 (Ki ≥ 0.8 nM).27, 29, 31 Consistent with this observation, both KLK7 and CG cleaves after basic residues in proteins to some degree after prolonged incubation.7, 37 Thus, for these two proteases it appears that when the P1 residue is presented in a loop with complementary fit to the active site and span both the prime and non-prime sides, the contribution of the P1 residue is less important. These findings have implications for engineering of inhibitors based on serpin (covalent) or standard mechanism inhibitor (reversible) protein/peptide scaffolds that rely on presenting a highly complementary binding loop. For CG and NSP4 the ability to recognize basic residues in the S1 pocket appears to have evolved independently from each other and from other serine proteases with trypsin-like specificity. For NSP4 P1 Arg binds to the hydroxyl groups of Ser192 and Ser216 rather than with Asp189 as for other trypsin-like proteases. This allows NSP4 to cleave after posttranslationally modified amino acids such as citrulline and methylarginine.35 Crystallographic studies have shown that CG can cleave after either basic or aromatic residues depending on the protonation state of Glu226. When the bound P1 residue is Lys Glu226 is unprotonated (PDB 1AU8), whereas when the bound P1 residue is Phe Glu226 is protonated (PDB 1CGH).38 This mechanism allows CG to cleave after diverse amino acids, including acidic, aliphatic, aromatic and basic residues (the MEROPS database: http://merops.sanger.ac.uk). CG cleaves with high catalytic efficiency after P1 4-guanidyl-L-phenylalanine39 and it is possible that CG can recognize and cleave after other post-translationally modified amino acids. Highly activated



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neutrophils release neutrophil extracellular traps (NETs) that contain reactive oxygen species, antimicrobial peptides, histones, DNA and NSPs that effectively contain and eliminate pathogens locally.40 Upon inflammatory stimuli enzymatic deamination of Arg to citrulline rapidly occurs for histones in NETs, resulting in unpacking of the chromatin.41 Release of reactive oxygen species by neutrophils also produces a number of chemical post-translational amino acid side chain modifications.42 Some of these post-translationally modified residues may be cleavage recognition sites for CG and/or NSP4 thus limiting proteolysis to the area of neutrophil activation. Future studies are needed to determine if this is the case, and if so, such sequences including post-translationally modified amino acids could provide leads for design of highly specific inhibitors. Screening CG against a P2ʹ diverse SFTI-based inhibitor library revealed that CG has a broad P2ʹ specificity, which includes acidic, hydrophobic and aromatic, but not basic, residues. Substituting the most preferred P2ʹ residue Asp7 into the most potent inhibitor (13) resulted in an inhibitor with 80% reduced activity (17). Conversely, substituting the similarly preferred Glu7 residue into 13 produced an inhibitor with 24% less activity (20). Molecular dynamics simulations indicated that this may be a result of cooperativity between the P1 and P2ʹ residues, where the nature of the P1 residue subtly alters the positioning of the P2ʹ residue (Supplementary Figure S3C-H). We have previously seen that the P2ʹ preference for chymotrypsin depends on the P1 residue. Screening chymotrypsin against the SFTI-based P2ʹ library with P1 Arg revealed that P2ʹ Tyr was not preferred, but P2ʹ Tyr was preferred in another SFTI-variant with P1 Phe.27 Therefore, it may be that the cooperativity between the P1 and P2ʹ positions is more pronounced than previously known, but whether this phenomenon commonly occurs for serine proteases needs to be further examined by future studies.


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CONCLUSION In this study we have defined the extended substrate specificity of CG, which allowed the design of potent macrocyclic peptide inhibitors of CG based on the SFTI-1 scaffold. One of these variants (22) showed great selectivity over other NSPs and a panel of serine proteases with chymotryptic and tryptic specificity. This variant is the most potent and specific CG inhibitor described to date and is thus a promising lead-compound for the further development of CG inhibitors targeting chronic inflammatory disorders resulting from an imbalance between NSP activity and endogenous protease inhibitors.

EXPERIMENTAL SECTION Protein Expression and Protein Sources. Proteases were sourced from Molecular Innovations (CG, NE, chymase and alpha-thrombin), BioVision (PR3) and Sigma Aldrich (bovine chymotrypsin, bovine trypsin and human plasmin). Recombinant KLK7 was expressed in zymogen form in Pichia pastoris strain X-33 and purified from the culture supernatant by cation exchange chromatography as previously described.29,

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KLK7 was activated by enterokinase

(EK Max) at 37◦C for 2 h (1 unit EK Max per 50 µg of pro-KLK) and repurified by cation exchange. The active protein was quantified by active site titration using α1-antitrypsin (SigmaAldrich) and stored in 20% glycerol (v/v) at -80°C until use. Peptide Synthesis. Peptide para-nitroanilide substrates were synthesized manually on 2chlorotrityl chloride resin (Chem-Impex, 0.8 mmol equiv/g) that had been derivatized with paraphenylenediamine (Sigma Aldrich), as previously described.44 Coupling reactions were 15 ACS Paragon Plus Environment


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performed using Fmoc N-protected amino acids (4 equiv) activated with 4 equiv O-(6chlorobenzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and 8 equiv N,N-diisopropylethylamine (DIPEA) in DMF (2 × 5 min reactions per residue). Fmoc protecting groups were removed using 30% piperidine in DMF (2 × 3 min). Assembled side chain protected peptides were cleaved from the resin using 1.25% (v/v) TFA in dichloromethane (DCM) and precipitated in diethyl ether before oxidation of the C-terminal para-aminoanilide group using 6 equiv Oxone® (Sigma Aldrich) in H2O:acetonitrile (1:1). Oxidized peptides were extracted using DCM:ethyl acetate (1:1) and the organic phase was dried using a rotary evaporator. Side-chain protecting groups were removed by cleavage using TFA/triisopropylsilane/H2O (96:2:2) (20 mL per 0.1 mmol peptide) and the peptides were collected by precipitation in diethyl ether. Peptide aldehyde inhibitors were synthesized manually on H-Phe(Boc)2-H NovaSyn TG resin (0.20 mmol/g, Novabiochem) using the same reaction conditions as for peptide para-nitroanilide substrates. On-resin removal of protecting groups and peptide cleavage was performed as previously described,33 and the final products were stored under a nitrogen atmosphere at -20°C until use. SFTI-based inhibitors were synthesized on 2-chlorotrityl chloride resin (starting at Gly1) using a Symphony automated peptide synthesizer (Protein Technologies, Inc). Peptide elongation and liberation of side-chain protected peptides from the solid support were performed as above. Head-to-tail cyclization was performed in DMF (50 mL per 0.1 mmol peptide) with 4 equiv 1[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and 8 equiv DIPEA for 3 hours. DMF and activators were removed by adding one volume of DCM and washing with 2 volumes of H2O (2-3 times). The organic phase was recovered and the DCM was removed by rotary evaporation. Side-chain protecting groups were


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removed as for peptide para-nitroanilide substrates. Formation of the disulfide bond was achieved by stirring at room temperature (3 hours) in 0.1 M ammonium bicarbonate buffer (pH 8.3) containing 10 µM oxidized glutathione (100 mL per 0.1 mmol peptide). Peptide Purification and Mass Spectrometry Analysis. Peptides were purified by reverse phase HPLC (Shimadzu Prominence) using a 5 µm ZORBAX Extend-C18 PrepHT column (21.2 × 250 mm) and a linear gradient of 10% acetonitrile/0.05% TFA to 70% acetonitrile/0.05% TFA. SFTI variants were purified twice, both before and after formation of the disulfide bond (see above). Peptide purity (> 95%) was confirmed by UPLC using a 5 µm Agilent 300 SB C18 column (2.1 × 50 mm) at 50°C with mobile phases as above (Supporting Information, Figure S1). Peptide masses were determined by electrospray ionization mass spectroscopy (Shimadzu Prominence). Substrate Library Screening. Crude tetrapeptide-pNAs were adjusted to equal molarity as measured by absorbance at 405 nm following total hydrolysis of the pNA moiety. The substrate library was screened against CG using with 150 µM substrate in 300 µL assay buffer (0.15 M NaCl, 0.1 M Tris.HCl, pH 8.0 and 0.005% (v/v) Triton X-100) with 10% acetonitrile to aid substrate solubility. Hydrolysis was monitored at λ 405 nm for 5 minutes in clear non-binding surface 96-well plates (Corning) using an Infinite® M1000 PRO microplate reader (TECAN). All assays were performed three times in triplicate Kinetic and Inhibitory Assays. Kinetic constants of substrates were determined using a serial dilution of substrate. Enzymatic activity was determined by monitoring the liberation of either pNA or 4-methylcoumaryl-7-amide (MCA) moieties. Assays with peptide-pNA substrates were performed as for the substrate library. Assays with peptide-MCA substrates were performed using 200 µl assay buffer in black non-binding surface 96-well plates (Corning) monitoring the 17 ACS Paragon Plus Environment


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fluorescence at λex 360 nm/ λem 460 nm over 10 minutes. The SFTI-based P2ʹ diverse inhibitor library (Figure 3) was screened against CG using an inhibitor concentration of 1 µM. Protease concentrations, substrates, substrate concentrations, KM values and buffer additives used are given in Table S2 (Supporting Information). Inhibition constants for peptide aldehydes or SFTI variants were determined following equilibration of inhibitor and proteases at room temperature for 30 minutes. The kinetic (Michaelis-Menten) and inhibition (Morrison Ki) constants were calculated by non-linear regression using Prism 6 (GraphPad). All assays were conducted in three independent triplicate experiments. Angiotensin II generation assay. CG (10 nM) was incubated with angiotensin I (10 µM, Sigma-Aldrich) and with or without compound 22 (100 nM) in assay buffer. Samples were taken at 1.5, 3 and 5 hours and the reaction was terminated by the addition of 2% TFA. Angiotensin I cleavage was evaluated by RP-UPLC and mass spectroscopy as described above. These assays were repeated three times with representative results shown in Figure 4. 1D NMR. Peptides were dissolved in 90% H2O/10% D2O (v/v) (compounds 1, 2 and 22) or 75% Acetonitrile-d3/25% H2O (v/v) (compounds 3-21) at approximately 1 mg/ml at pH ranging from 3–4. 1H one- and two-dimensional TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) NMR experiments were carried out on a Bruker 600 MHz spectrometer at 298K. The spectra are shown in Supplementary Figure S2 and were internally referenced using 4,4-dimethyl-4-silapentane-1-sulfonic acid. Molecular Modeling. Models of SFTI-1 variants in complex with proteases were constructed by overlaying a trypsin/SFTI-1 complex (PDB 1SFI) with CG (PDB 1T32)or NSP4 (4Q7Z) using MUSTANG45 (Cα RMSD 1.03 Å). Complexes were solvated with TIP3P water and neutralized with Na+/Cl- counter ions to a final concentration of 100 mM in VMD 1.9.2.46producing systems 18 ACS Paragon Plus Environment

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of approximately 25000 atoms including 7000 water molecules. Each complex was equilibrated using a stepwise relaxation procedure over 2.5 ns (2 fs time step) using NAMD 2.1147 and CHARMM27 force fields parameters as previously described.27 Briefly, a Langevin thermostat with a damping coefficient of 0.5 ps-1 was used to maintain the system temperature and the system pressure was maintained at 1 atm using a Langevin piston barostat. The particle mesh Ewald algorithm was used to compute long-range electrostatic interactions at every second time step and non-bonded interactions were truncated smoothly between 10.5-12 Å. Hydrogen bonds were constrained by the SHAKE algorithm (or the SETTLE algorithm for water). Production runs of 10 ns were performed under NVT conditions with otherwise identical force field and simulation parameters as above using ACEMD.48 Coordinates were saved every 500 simulation steps producing 10000 frames per trajectory. The simulation frames were clustered based on Cα RMSDs using VEGA ZZ v2.349 and the frame that most closely aligned with the largest cluster was selected representative models of the complexes (Figure 4; Supporting Information, Figure S3).



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ASSOCIATED CONTENT Supporting Information The supporting material is available free of charge on the ACS Publications website (http://pubs.acs.org). Authors will release the atomic coordinates and experimental data upon article publication. Supporting information includes peptide purification and NMR characterization, kinetic substrate and inhibitor assay methods, molecular dynamics simulations and PDB coordinates for molecular models. PDB coordinates for computational model of Figure 5A-B PDB coordinates for computational model of Figure S3A-B PDB coordinates for computational model of Figure S3C-D PDB coordinates for computational model of Figure S3E-F PDB coordinates for computational model of Figure S3G-H PDB coordinates for computational model of the table of content graphic

AUTHOR INFORMATION Corresponding Author *Dr Joakim Swedberg, Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia. Email: [email protected]. Author Contributions All authors contributed to the writing of this manuscript, and all authors have approved the final version.


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ACKNOWLEDGMENTS DJC is an Australian Research Council Laureate Fellow [FL150100146] and JES is a National Health and Medical Research Council Early Career Fellow [APP1069819].

ABBREVIATIONS USED SFTI-1, Sunflower Trypsin Inhibitor-1; CG, cathepsin G; NE, neutrophil elastase, PR3, proteinase-3, NSP4, neutrophil serine protease-4, KLK7, kallikrein related peptidase-7; -pNA, para-nitroanilide, HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; DIPEA, N,N-diisopropylethylamine.



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FIGURE LEGENDS Figure 1: Structure and sequence of SFTI-1. (A) Schematic of the SFTI-1 sequence with residues of β-strands shown in light blue, the disulfide bond shown in yellow and the scissile bond (Lys5:Ser6) is marked with a dashed line. (B) Ribbon plot of the solution structure of SFTI-1 (PDB 1JBL) with β-strands shown in light blue. The disulfide bond (yellow) and P1 residue Lys5 (carbon: grey, nitrogen: dark blue) are shown in ball and stick model. (C) Model of SFTI-1 (PDB 1SFI) shown as stick model (carbon: green, nitrogen: blue, oxygen: red, sulphur: yellow, hydrogen bonds: purple dashed lines) on the electrostatic surface of CG (PDB 1T32, positive: blue, negative: red, and neutral: white). Figure 2: Screening of CG substrate specificity against a colorimetric substrate library. Amidolytic activity of CG against a sparse matrix library of tetrapeptide-pNA substrates. The yaxis represents the relative activity of CG normalized to the cleavage rate of the substrate cleaved at the highest rate for P4 Asp (yellow), Thr (orange), Trp (green), Ile (blue) and Nle (purple) Substrate sequences for the P3-P1 subsites are given across the x-axis in the one letter amino acid code (n denotes norleucine). Data is expressed as mean ± S.E.M. from three independent experiments performed in triplicate. Figure 3: Inhibitory activity of a SFTI-based library with a variable P2ʹ residue against CG. The variable residue at the P2ʹ position is shown on the x-axis using single letter code for naturally occurring amino acids (B denotes biphenylalanine). All compounds were screened at an inhibitor concentration of 1 µM. The wild-type SFTI-1 residue Ile7 is highlighted in grey. The % inhibition (y-axis) for each variant was calculated by comparing kinetic rates to control assays without inhibitor. Data is expressed as mean ± S.E.M. from three independent experiments performed in triplicate. 28 ACS Paragon Plus Environment

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Figure 4: Compound 22 inhibits CG generation of angiotensin II. RP-UPLC trace of CG incubated with angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) at time 0 (A) and after 90 minutes (B) where all angiotensin I had been cleaved into angiotensin II (Asp-Arg-Val-TyrIle-His-Pro-Phe). The peptide ion masses (M2+) for the corresponding fractions are shown in the figure. RP-HPLC trace of CG incubated with compound 22 and angiotensin I at time 0 (C) and after 5 hours (D) where angiotensin I cleavage was completely blocked. Figure 5: Molecular dynamics simulations of models of CG/SFTI-variant complexes. (A) Stick model of 22 (carbon: green, nitrogen: blue, oxygen: red, sulphur: yellow, hydrogen bonds: purple dashed lines) on the electrostatic surface of CG (positive: blue, negative: red, and neutral: white). Residue numbering is shown for SFTI-variants (green) and CG (grey, chymotrypsin numbering) Compound 22 and CG residues are highlighted in green and grey, respectively. X denotes the side chain of 4-guanidyl-L-phenylalanine. (B) Stick model of 22 bound to CG shown as a ribbon plot (grey) with interacting residues highlighted in stick model. PDB coordinates for the computational models are included as supporting material (Figure 5.pdb).



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TABLES Table 1: Inhibitor masses and inhibition constants for CG Compound

Inhibitora

Calculated ion mass

Determined ion mass

Purity (%)

Ki (nM) ± SEM

1

GRCTKSIPPICFPD

757.9+2

757.8+2

99.9

730 ± 90

2

GTCTRSIPPICNPN

Reference 27

-

-

490 ± 60

3

GTCTFSIPPICNPN

722.8+2

722.8+2

99.9

390 ± 60

4

GTCTYSIPPICNPN

730.8+2

730.8+2

99.9

410 ± 40

5

GTCTLSIPPICNPN

705.9+2

705.8+2

98.9

480 ± 50

6

DEnF-H

507.6+1

507.4+1

97.3

630 ± 50

7

DTnF-H

479.6+1

479.4+1

99.9

530 ± 60

8

TEnF-H

493.6+1

493.4+1

97.3

3000 ± 260

9

IEnF-H

505.6+1

505.5+1

97.0

1200 ± 80

10

GDCnFSIPPICFPD

752.9+2

752.9+2

99.9

160 ± 20

11

GTCnFSIPPICFPD

745.9+2

745.9+2

99.9

7.3 ± 0.9

12

GICnFSIPPICFPD

751.9+2

751.9+2

99.9

3.4 ± 0.7

13

GTCnFSIPPICFPN

745.4+2

745.4+2

99.9

0.89 ± 0.22

14

GICnFSIPPICFPN

751.4+2

751.4+2

97.3

4.9 ± 0.7

15

GTCnFnIPPICFPD

759.0+2

758.8+2

99.9

4.8 ± 1.0

16

GTCnFnIPPICFPN

758.5+2

758.5+2

99.9

12 ± 1.5

17

GTCnFSDPPICFPN

746.4+2

746.4+2

99.9

1.7 ± 0.4

18

GTCTRSDPPICNPN

Reference 27

-

-

12 ± 1

19

GTCnFSIPPICNPN

728.9+2

728.9+2

99.9

62 ± 11

20

GTCnFSBPPICFPN

800.5+2

800.5+2

99.9

2.9 ± 0.5

21

GTCnFSEPPICFPN

753.4+2

753.4+2

99.9

1.1 ± 0.3

22

GTCnXSDPPICFPN

774.9+2

774.7+2

99.9

1.6 ± 0.2

a

Compounds1-5 and 10-22 are backbone cyclic with a Cys3-Cys11 disulfide bond. n = norleucine

b c

c

Amino acid variations from SFTI-1 (1) are shown in bold font

X = 4-guanidyl-L-phenylalanine


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Table 2: CG substrate masses and Michaelis-Menten kinetic constants Substrate

KM (µM) SEM

±

kcat(s-1) ± SEM

kcat/KM (M-1s-1)

Calculated

Determined

ion mass (M+1)

ion mass (M+1)

DEnF-pNA

643.8

643.4

54 ± 8.0

0.51 ± 0.04

9500 ± 1600

DTnF-pNA

615.8

615.4

120 ± 20

1.1 ± 0.09

9200 ± 1600

TEnF-pNA

629.8

629.4

220 ± 50

0.44 ± 0.02

2000 ± 420

IEnF-pNA

641.9

641.4

130 ± 40

0.31 ± 0.06

2400 ± 900

n = norleucine


± SEM


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Table 3: Inhibition constants for off target proteases Compound

Protease

Ki (nM) ± SEMa

Fold selectivity

NE

12000 ± 1000

13000

PR3

>10000

>11000

Chymotrypsin

1.9 ± 0.04

2.1

KLK7

69 ± 6

78

NE

8900 ± 600

5200

PR3

>10000

>5900

Chymotrypsin

140 ± 4

83

KLK7

130 ± 8

76

Chymase

7.7 ± 0.9

4.5

20

Chymotrypsin

35 ±6

31

22

NE

43000 ± 6000

27000

PR3

> 10000

> 6300

Chymotrypsin

> 10000

>6300

KLK7

740 ± 70

460

Chymase

19600 ± 2100

12000

Trypsin

580 ± 20

360

Thrombin

>10000

>6300

Plasmin

>10000

>6300

13

17

a

For inhibitors with >10000 nM there was less than 20% at

10000 nM


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FIGURES Figure 1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2


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Figure 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4


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Figure 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC GRAPHICS


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Figure 1: Structure and sequence of SFTI-1. (A) Schematic of the SFTI-1 sequence with residues of βstrands shown in light blue, the disulfide bond shown in yellow and the scissile bond (Lys5:Ser6) is marked with a dashed line. (B) Ribbon plot of the solution structure of SFTI-1 (PDB 1JBL) with β-strands shown in light blue. The disulfide bond (yellow) and P1 residue Lys5 (carbon: grey, nitrogen: dark blue) are shown in ball and stick model. (C) Model of SFTI-1 (PDB 1SFI) shown as stick model (carbon: green, nitrogen: blue, oxygen: red, sulphur: yellow, hydrogen bonds: purple dashed lines) on the electrostatic surface of CG (PDB 1T32, positive: blue, negative: red, and neutral: white). 87x190mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 2: Screening of CG substrate specificity against a colorimetric substrate library. Amidolytic activity of CG against a sparse matrix library of tetrapeptide-pNA substrates. The y-axis represents the relative activity of CG normalized to the cleavage rate of the substrate cleaved at the highest rate for P4 Asp (yellow), Thr (orange), Trp (green), Ile (blue) and Nle (purple) Substrate sequences for the P3-P1 subsites are given across the x-axis in the one letter amino acid code (n denotes norleucine). Data is expressed as mean ± S.E.M. from three independent experiments performed in triplicate. 87x154mm (300 x 300 DPI)


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Figure 3: Inhibitory activity of a SFTI-based library with a variable P2ʹ residue against CG. The variable residue at the P2ʹ position is shown on the x-axis using single letter code for naturally occurring amino acids (B denotes biphenylalanine). All compounds were screened at an inhibitor concentration of 1 µM. The wildtype SFTI-1 residue Ile7 is highlighted in grey. The % inhibition (y-axis) for each variant was calculated by comparing kinetic rates to control assays without inhibitor. Data is expressed as mean ± S.E.M. from three independent experiments performed in triplicate. 87x60mm (300 x 300 DPI)



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Figure 4: Compound 22 inhibits CG generation of angiotensin II. RP-UPLC trace of CG incubated with angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) at time 0 (A) and after 90 minutes (B) where all angiotensin I had been cleaved into angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe). The peptide ion masses (M2+) for the corresponding fractions are shown in the figure. RP-HPLC trace of CG incubated with compound 22 and angiotensin I at time 0 (C) and after 5 hours (D) where angiotensin I cleavage was completely blocked. 64x47mm (600 x 600 DPI)


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Figure 5: Molecular dynamics simulations of models of CG/SFTI-variant complexes. (A) Stick model of 22 (carbon: green, nitrogen: blue, oxygen: red, sulphur: yellow, hydrogen bonds: purple dashed lines) on the electrostatic surface of CG (positive: blue, negative: red, and neutral: white). Residue numbering is shown for SFTI-variants (green) and CG (grey, chymotrypsin numbering) Compound 22 and CG residues are highlighted in green and grey, respectively. X denotes the side chain of 4-guanidyl-L-phenylalanine. (B) Stick model of 22 bound to CG shown as a ribbon plot (grey) with interacting residues highlighted in stick model. PDB coordinates for the computational models are included as supporting material (Figure 5.pdb). 175x135mm (300 x 300 DPI)


Design of Potent and Selective Cathepsin G Inhibitors Based on the

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