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Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Discovery and Characterization of Cyclic and Acyclic Trypsin Inhibitors from Momordica dioica Junqiao Du, Lai Yue Chan, Aaron G. Poth, and David J. Craik* Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia

J. Nat. Prod. Downloaded from pubs.acs.org by EASTERN KENTUCKY UNIV on 01/24/19. For personal use only.

S Supporting Information *

ABSTRACT: Momordica trypsin inhibitors (TIs) such as those isolated from the seeds of the gâć fruit, Momordica cochinchinensis (MCoTI-I and MCoTI-II), are widely used as scaffolds for drug design studies. To more effectively exploit these molecules in the development of therapeutics, there is a need for wider discovery of the natural sequence diversity among TIs from other species in the Momordica subfamily. Here we report the discovery of the encoding gene and six TIs from the seeds of the spiny gourd, Momordica dioica, four of which possess novel sequences (Modi 1, 3, 5, and 6) and two (Modi 2 and 4) of which are known peptides (TI-14, TI-17) previously identified in Momordica subangulata. Modi 6 is an acyclic peptide featuring a pyrrolidone carboxylic acid modification, whereas the remaining five TIs are cyclic. All Modi peptides display similar overall structures and trypsin inhibitory activities. No toxicity was observed for these peptides when tested against cancer and insect cells. All Modi peptides were exceptionally stable over 24 h in human serum, indicating a dual strategy to stabilize the peptides in nature, either head-totail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

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M. cochinchinensis (MCo) are currently divided into two categories: acyclic peptides (e.g., MCoTI-V) that contain a cystine knot and cyclic peptides (e.g., MCoTI-I and MCoTIII) that contain the cyclic cystine knot (CCK) motif.16,18 Several full gene precursors encoding MCoTI peptides (i.e., the two inhibitor peptide topologies, or TIPTOP genes) and several partial gene sequences have been reported.19,20 The full-length genes typically comprise an endoplasmic reticulum (ER) signal sequence, a prodomain, and one to seven mature peptide domains and terminate with an acyclic domain. An overview of the TIPTOP gene composition, along with its encoded MCoTI-II and MCoTI-V amino acid sequences and their corresponding structures is shown in Figure 1. Among these peptides, MCoTI-I and MCoTI-II are the most widely studied to date. These TIs are extremely stable, can penetrate cells,21,22 and are tolerant to amino acid substitutions in their loops, making them versatile scaffolds for drug design applications23−25 and the development of novel protease inhibitors.26 However, to expand our knowledge on TIs and provide more drug scaffold options, it is important to screen other species from Cucurbitaceae, including those of the Momordica genus. As extracts from Momordica dioica (a climbing plant with the same origin as M. cochinchinensis20)

rotein protease inhibitors, including trypsin inhibitors (TIs), are found in numerous organisms. Protease inhibitors isolated from plants have activities against insect larval growth, suggesting these proteins may play a role in defense.1−3 In humans, protease inhibitors are known to be essential for physiological regulation and have been associated with a range of diseases including neuropathic pain4 and skin desquamation.5 While these properties make protease inhibitors attractive candidates for developing novel therapies and insecticides, their utility is somewhat limited by their large molecular size, which makes it difficult for them to penetrate cells. Two major protein protease inhibitor families have been identified from plants: the Kunitz trypsin inhibitor (KTI) and Bowman−Birk inhibitor (BBI) families with a molecular weight of ∼20 and ∼8 kDa, respectively.6−8 A third wellestablished category of protein protease inhibitors, known as the squash (Cucurbitaceae) inhibitor family, contains peptides that are only ∼3 kDa in size (i.e., comprising 27−34 amino acids with three disulfide bonds). These smaller squash TIs may overcome some of the problems associated with the use of the larger KTIs or BBIs in drug development studies. To date, all characterized inhibitors from the squash family have been isolated from the seeds, including from bottle gourd (Lagenaria leucantha),9 bitter gourd (Momordica charantia),10 melon (Cucumis melo),11,12 ridge gourd (Luffa acutangula),13 Momordica repen,14 wax gourd (Benincasa hispida),15 and gâć fruit (Momordica cochinchinensis).16,17 The TIs isolated from © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 23, 2018

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DOI: 10.1021/acs.jnatprod.8b00716 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Schematic view of a typical TIPTOP gene and its representative encoded peptides with sequences and structures (MCoTI-II: 1HA9; MCoTI-V: 2LJS). Three-dimensional structures were created using MolMol.65 Asterisk and Z represent a pyroglutamic acid residue.

Figure 2. Gene precursor sequence and TIPTOP-derived peptides from Momordica dioica. (A) Schematic diagram of the M. dioica TI precursor and the predicted sequence of coded peptides. Sequences colored blue (Modi 2) and olive (Modi 4) are the previously reported peptides TI-14 and TI-17, respectively. The novel sequences are colored in purple (Modi 1), green (Modi 3), orange (Modi 5), and red (Modi 6). (B) Sequence alignment of known MCoTI-II, TI-24, and MCoTI-V and novel Modi peptide sequences (cyclic: Modi 1 to Modi 5; acyclic: Modi 6).20 Cysteine residues (numbered I−VI) are highlighted in orange. The N-terminal residue of the acyclic peptide (Modi 6 and TI-24, MCoTI-V) is pyrolated (Z). (C) LC-MS trace of the crude peptide extract from M. dioica seeds and their corresponding retention time.

specific band was observed from the cDNA template, possibly due to the degradation of RNA in stale seeds. There was one band produced with the gDNA template (Figure S1, Supporting Information). This precursor was a full-length transcript that encoded an ER signal peptide, followed by six cyclic peptides, and terminated with an acyclic peptide (Figure 2A). This gene composition is similar to the known precursors from other Momordica species19,20 and was named M. dioica TIPTOP (GenBank accession code: MH547558). According to the encoded order and previously described nomenclature principles,29 the peptides derived from M. dioica are named Modi 1 (3451.51 Da [M + H]+), Modi 2 (3381.47 Da [M + H]+), Modi 3 (3438.53 Da [M + H]+), Modi 4 (3437.55 Da [M + H]+), Modi 5 (3453.57 Da [M + H]+), and Modi 6 (3334.65 Da [M + H]+, pyrolated). The sequences of the six Modi peptides were determined by a combination of

have been found to possess anticancer and antidiabetic activity,27,28 this species is the focus of the current study. Here we describe a novel gene precursor that encodes both cyclic and acyclic peptides from M. dioica. This gene has a similar arrangement to previously reported genes discovered from other Momordica species.19,20 Four trypsin inhibitor peptides with novel sequences (cyclic: Modi 1, Modi 3, Modi 5; acyclic: Modi 6) and two known TI peptides (cyclic: TI-14/ Modi 2 and TI-17/Modi 4) deriving from this tandem gene were purified and sequenced. These peptides were screened in trypsin inhibition, cytotoxicity, and stability assays.

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RESULTS AND DISCUSSION Isolation and Characterization of Modi Peptides with Their Gene Precursor. Based on a previous report,20 modified primers were used to amplify the gene precursor with either genomic DNA (gDNA) or cDNA templates. No B



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Figure 3. Representative MS/MS sequencing data for Modi 5 and Modi 6. (A, B, and C) MS/MS spectra for the precursors of Modi 5, which are 487.322+, 590.752+, and 1007.773+, respectively. (D and E) Assigned MS/MS spectra of precursor ions of Modi 6, which are 584.993+ and 807.633+, respectively. Modifications of amino acids were shown as either carbamidomethylated (CAM) or pyrolated residue (Z).

enzymes, Modi 5 and Modi 6 were chosen as representative examples. Reduced and alkylated Modi 5 (3801.57 Da [M + H]+) and Modi 6 (3682.65 Da [M + H]+) were subject to digestion with trypsin, yielding three fragments for the former (487.322+, 590.752+, and 1007.773+) and two fragments for the latter (584.993+ and 807.633+). The MS/MS spectra used for characterization of these two peptides are shown in Figure 3. The resulting fragments for the other four peptides (Modi 1, Modi 2, Modi 3, and Modi 4) are listed in Table S1 (Supporting Information). Structural Analysis of Modi Peptides by NMR Spectroscopy. NMR data confirmed the peptide sequences resulting from the gene and de novo sequencing. The amide region of the 1D spectra of Modi 1 to Modi 6 all showed welldispersed peaks (Figure S2, Supporting Information), consistent with well-defined structures. The chemical shifts of these peaks were assigned using the sequential assignment procedure of Wüthrich.30 Overall, the αH chemical shifts showed the same trends as MCoTI-II (Figure 4), suggesting they have a similar fold to MCoTI-II,31 including a cystine knot (Cys I−Cys IV, Cys II−Cys V, and Cys III−Cys VI). Despite the overall similarities, there are two small differences among the peptides. Specifically, the chemical shifts of C-4

methods including gene sequencing, MS/MS sequencing, and NMR spectroscopy (Figure 2B). Modi 2 and Modi 4 share sequences with two reported peptides (TI-14 and TI-17) from Momordica subangulata, which belongs to the same Momordica subfamily as M. dioica;20 however these peptides have not been characterized at the peptide level (sufficient to be isolated and purified). The sequences of the other cyclic Modi peptides (Modi 1, Modi 3, Modi 5) have not previously been reported but are homologous to known Momordica TIs and share the same cyclization point (Gly-Asp). The N-terminus of the acyclic peptide (Modi 6) is pyrolated, as observed previously for TI-3 and TI-5 from M. cochinchinensis.16,19 All Modi peptides showed early retention times on LC-MS (Figure 2C), due to their rich composition of positively charged residues (e.g., arginine or lysine). To confirm their disulfide connectivity, all peptides were reduced with dithiothreitol and S-alkylated with iodoacetamide. Each mass increased by 348 Da, corresponding to the predicted numbers of cysteine residues, indicating the peptides contain three disulfide bonds. The alkylated samples were then digested with trypsin, chymotrypsin, or a combination of both. To illustrate digestion of the Modi peptides with the various C



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Figure 4. αH chemical shift comparison between Modi peptides and MCoTI-II. All 2D NMR spectra were recorded at 298 K. Cysteine residues are highlighted in yellow and numbered from I to VI with the connections of the disulfide bonds displayed in brackets. All spectra were assigned by CCPNMR.66 In the case of Gly residues, which have two α protons, the higher field chemical shift was used if the peaks were not overlapped.

(i.e., Cys I) and C-21 (i.e., Cys IV) of Modi 3 and Modi 5 differ slightly from those of Modi 1, Modi 2, Modi 4, and Modi 6. These two Cys residues are connected via a disulfide bond, and it is likely that this disulfide bond has a different conformation in Modi 3 and Modi 5. This is consistent with the parent structure of MCoTI-II (PDB ID: 1HA9), where the disulfide bond formed by C-4−C-21 is more flexible than the other two disulfide bonds, resulting in the flexibility of loop 6. Another difference seen in Figure 4 is for the chemical shift of S-19 of Modi 3 and Modi 5, and this is consistent with the substitution of Gly by Ser in these peptides. Trypsin Inhibitor Activity. Squash inhibitors target serine proteases with high efficiency, by specifically using an Arg/LysIle motif in loop 1 to interact with trypsin and a Leu-Ile motif to bind elastase.32 The Modi peptides all have high sequence homology at the putative trypsin inhibitory binding loop (PKILQR: Modi 1; PKILQK: Modi 2; PRILQK: Modi 3; PRILKK: Modi 4; PRILKK: Modi 5; PRILKK: Modi 6; PKILKK: MCoTI-II; and PRILKQ: MCTI-I [M. charantia trypsin inhibitor I]32). In addition, all Modi peptides had similar trypsin inhibitory activity compared to the known potent trypsin inhibitor MCoTI-II,16,33 except for Modi 2, which displayed slightly milder activity (Figure 5). This suggests that all Modi peptides share the same mechanism in terms of trypsin inhibition to MCoTI-II and other squash inhibitors, and thus, these peptides can be classified into the squash inhibitor family. Cytotoxic Activity. Cyclic TIs from plants in the Cucurbitaceae have a common feature in their structure, known as the CCK,18 and thus have been classified as cyclotides, circular miniproteins that also occur in plants from the Rubiaceae, Violaceae, Fabaceae, and Solanaceae.34,35 The natural role of cyclotides is thought to be as defense agents against insects,36 but cyclotides also display a wide range of other biological activities,37−39 including cytotoxicity to some mammalian cells, including cancer cells.40,41 Consistent with

Figure 5. Trypsin inhibitor activity of Modi peptides compared to the positive control (MCoTI-II).

them containing cyclotides, whole plant methanol extracts from M. cochinchinensis and M. dioica are cytotoxic to some cancer cell lines.27,42 Furthermore, a recent study showed a novel peptide isolated from M. charantia possessed high trypsin inhibitory activity and was cytotoxic against a colon cancer cell line.43 In addition to their cytotoxic effects on mammalian cells, squash inhibitors are presumed to have a role in plant defense mechanisms44 and inhibit several serine proteinases including trypsin, plasmin, and cathepsin G.45 On the basis of the above-mentioned studies, we investigated whether the Modi peptides were cytotoxic against either an insect cell line (Spodoptera f rugiperda) or human colon, breast, or prostate cancer cells. Interestingly, none of the six Modi peptides showed any cytotoxic effects on the mammalian or insect cell lines tested in this study (Table S2, Supporting Information). This lack of toxicity and their potent trypsin inhibitory activity suggest that the primary natural role of Modi peptides is probably to inhibit digestive proteases in animals involved in seed dispersal. This suggestion does not exclude the possibility that they could be also functioning as storage proteins in seeds.46 Serum Stability Assay. The CCK motif endows cyclotides with extremely high stability, making them ideal frameworks D



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for drug design applications.35 MCoTI-II is one of the most commonly used cyclotides in grafting studies, in which a bioactive sequence (epitope) is grafted into a stable framework to improve its stability.35,47 Based on the structural similarity between the new Modi peptides and MCoTI-II, we evaluated the stability of the Modi peptides to determine whether they might also be used as scaffolds in biopharmaceutical applications. As part of this process, the stability of the cyclic and acyclic types of inhibitors was compared. The stabilities of the peptides were tested over 24 h in 100% human serum. A short linear peptide (WQELYQLKY) was used as negative control48 and degraded within 4 h as expected. By contrast, all Modi peptides were stable in serum over 24 h (Figure 6). Interestingly, acyclic Modi 6 had similar

those targeting cancer23,54 and cardiovascular disease.48 Loop 1 of MCoTI peptides has also been used as a grafting site, particularly for targeting proteases given it naturally contains a protease (trypsin) reactive site. Indeed, loop 1 of MCoTI-II has been used for making potent inhibitors of matriptase,55 βtrypase, human leukocyte elastase,26 and foot-and-mouth disease virus (FMDV) 3C protease.56 Other grafting studies have also been based on loops 2, 3, or 5,54,57,58 further illustrating the flexibility and compatibility of MCoTI peptides as drug design scaffolds. This tolerance to residue substitutions is reflected in the new naturally occurring sequences found in the current study. Figure 7 summarizes the loop diversity of Momordica peptides reported to date along with the new Modi peptides characterized here. Loops 1, 5, and 6 show more variation than the other loops and thus might be more favorable for modification in molecular grafting experiments. Most peptides discovered in this study share the same loop sizes as previous studies (highlighted in red in Figure 7),19,20 but have a different composition. Novel loop sequences found among the six Modi peptides include loop 1 (PRILQK) from Modi 3, loop 2 (RRNSD) from Modi 3, and loop 6 (GQRV) from Modi 6. None of these variations are expected to result in major structural perturbations, but perhaps of most interest in potentially modifying flexibility is the RRNSD sequence in loop 2 of Modi 3. Analysis of MCoTI-II structure (PDB ID: 1HA9) shows that residue D-14 in loop 2 (numbered as shown in Figure 4) forms a strong hydrogen bond with the C-23 backbone nitrogen and establishes two weak hydrogen bonds with the R-13 side chain. In contrast, the corresponding residue in Modi 3, N-14, might only participate in strong hydrogen bonding with the C-23 backbone nitrogen and possibly cause loop 2 of Modi 3 to be more flexible than that of MCoTI-II. N-pyrolation of Acyclic Momordica Peptides. Literature data suggest that the pyroglutamic acid residues featured in linear cystine knotted peptides arise mainly from glutamine, with only a small number arising from glutamic acid. Examination of the Uniprot protein database (http://www. uniprot.org/) reveals that 100% of the peptides from plants annotated with pyrrolidone carboxylic acid modifications (n = 137) occur on glutamine and that only three out of a random selection of 100 (of 460) N-pyrolated animal-derived peptides are derived from an encoded N-terminal glutamic acid moiety. This is consistent with findings by Schilling et al.,59 who demonstrated that the rate of conversion of an N-glutamine substrate to a pyroglutamic acid-containing product by plant glutaminyl cyclases is over 6 orders of magnitude larger than for glutamic acid-containing substrates. While plant- and animal-derived glutaminyl cyclases can catalyze the pyrolation of synthetic substrates featuring N-terminal glutamate,60 only a small percentage of the annotated pyrolated proteins found in members of the animal kingdom occur on N-glutamate, and where they do, the result is partial (subquantitative) conversion in vivo. The lack of evidence for homologous acyclotides with unmodified N-termini alongside those with pyrolated N-termini is consistent with the pyroglutamic acid moiety in these peptides being formed exclusively from glutamine. Indeed, all genetic evidence to date shows that the pyroglutamic acid post-translational modifications found in plant-based cystine knots arise from precursor proteins encoding glutamine residues at their proto-N-termini, and we hypothesize that this may be a consistent feature of

Figure 6. Serum stability assay of Modi peptides. The short linear peptide (WQELYQLKY) was included as a control.48

stability to the cyclic Modi peptides, presumably due to the combination of its cystine knot motif and blocked (pyrolated) N-terminus. The latter modification, putatively from either a Gln or Glu to pyroglutamic acid, is a well-known posttranslational modification for stabilizing plant peptides,49 but its prevalence in cyclotide derivatives has not been widely studied; its genetic origin is discussed in more detail below. Overall, the Modi peptides reported here demonstrated remarkable stability. Colgrave et al.50 examined the relative importance of the structural features of the cyclotide kalata B1 in terms of stability and found that the cystine knot contributes to a higher degree than the cyclic backbone. Nevertheless, the termini of natural acyclic cystine knots are susceptible to cleavage by exopeptidases, as seen for TI-28 from Momordica cochinchinensis20 and for hedyotides B2−4 from Hedyotis bif lora.51 Stanger et al. 52 demonstrated equivalent stability for recombinantly expressed cyclic MCoTI-II and an acyclic MCoTI-II (modified to incorporate a sortase recognition site) when challenged with incubation at 37 or 65 °C and also in simulated gastric fluid. By contrast, an acyclic version lacking the cystine knot was found to be labile under the same conditions, and Kimura et al.53 reported that a grafted acyclic EETI-II peptide lacking N-terminal protection showed 70% degradation over 24 h in 50% mouse serum. Here we demonstrate equivalent stability in human serum for Npyrolated acyclic cystine knots versus cyclic cystine knots. “Graftability” of Momordica Peptides. MCoTI-I/II is a favorable choice as a framework for drug design applications due to its exceptional stability, tolerance to substitutions in backbone loops, and ability to penetrate cells to enable intracellular targeting.35 Loop 6 of MCoTI peptides has been most frequently chosen for grafting experiments, including E



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Figure 7. Schematic representation of loop diversities of Momordica peptides. The representative structure shown is MCoTI-II (PDB ID: 1HA9). The disulfide bonds are indicated as yellow sticks. Residues in black represent cyclic loops in cyclic peptides, while the residues of acyclic peptides are shown in light blue. Residues in red represent M. dioica peptides discovered in the current study, either sharing the same loop as the literature (arrow) or having a novel loop (new residues); the asterisk indicates the acyclic peptide. The numbers in brackets represent the total number of Momordica peptides with the same loop according to the literature.20 The dashed lines connect cyclic and acyclic peptides sharing the same loop sequence. Highlighted orange and purple circles represent the C-termini and N-termini of peptides, which are linked to the residues in loop 6, respectively. dioica peptide sequences was amplified by PCR using modified primers (forward: 5′-CGT CTT GCT AGA GAA AGG GAG-3′ and reverse: 5′-TCA GAA ACA GCA TAG CTT TCA-3′).19 The PCR product was purified using the QIA quick PCR purification kit (for the single band), cloned into pGEM-T Easy (Promega), and sequenced at the Australian Genome Research Facility (AGRF). Extraction and Purification of Peptides from M. dioica Seeds. In total, 50 g of M. dioica seeds was ground in liquid nitrogen, extracted with 50% MeCN in a 1% formic acid solution, and stirred overnight at room temperature. The crude solution was spun down for 20 min at 17000g. The supernatant was filtered through a 0.45 μm filter and then lyophilized on a freeze-dryer (CHRIST Alpha 2-4 LD freeze-dryer). The lyophilized crude material was dissolved in 1% formic acid, and the peptides were separated using C18 SPE cartridges (Phenomenex, 10 g) using a 1% formic acid/20−80% MeCN gradient elution buffer. The peptide-containing fractions were lyophilized and purified by RP-HPLC (buffer A: 0.1% trifluoroacetic acid (TFA) in H2O; buffer B: 90% MeCN with 0.1% TFA). In total, six peptides were isolated with the following total yields: Modi 1 (7.5 mg), Modi 2 (21.3 mg), Modi 3 (9.47 mg), Modi 4 (4.5 mg), Modi 5 (8.66 mg), and Modi 6 (10.71 mg). Reduction, Alkylation, and Enzymatic Digestion of Peptides. Peptides were initially dissolved in 50 μL of 0.1 M NH4HCO3, pH 8.0 (Sigma-Aldrich), prior to disulfide bond reduction with 100 mM dithiothreitol (DTT). DTT was added to the samples (1:10, v/ v), and the mixture left under nitrogen for 30 min at 60 °C. Peptides were alkylated by the addition of 250 mM iodoacetamide (IAA), and the mixture was left for 30 min at room temperature. Peptides that were reduced and alkylated were subjected to enzymatic digestion with either trypsin or chymotrypsin or a mixture of both enzymes, overnight at 37 °C (trypsin) or 30 °C (chymotrypsin). At the final stage, the reaction was quenched by adding 1% formic acid, and the

biosynthetic precursors for other pyroglutamic acid-containing plant peptides. In conclusion, this study provides new information on the structures and stabilities of new TI peptides from the Momordica plant family. The trypsin inhibitors from M. dioica described here show that nature has developed two alternative strategies (cyclization and pyroglutamyl formation from a precursor glutamine residue), each exploiting ancient, endogenous enzymatic machinery to protect peptide termini in a single precursor protein. Overall, the genetically coexpressed cyclic and acyclic cystine knot scaffolds reported here represent interesting candidates for drug design applications, either through directed modification of their intrinsic inhibitory activity or through their utilization as structural scaffolds for improving the stability of peptide epitopes. That multiple cyclic and linear peptides are encoded on the same gene opens the possibility for the efficient production via recombinant approaches of multiple synergistically acting drugs in a single expression system.

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EXPERIMENTAL SECTION

RNA Extraction, cDNA Synthesis, and DNA Amplification. M. dioica seeds (Item ID 163574113) were purchased from Advitiya Trading, 426A, Vaishali Nagar, India. Total RNA was isolated from the seeds of M. dioica according to the method described by Li et al.;61 total DNA was isolated via the cetyltrimethyl ammonium bromide (CTAB) method. Single-stranded cDNAs were synthesized using the SuperScript II reverse transcriptase kit (Invitrogen), as per the manufacturer’s instructions. The gene precursor containing the M. F



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samples were examined by 5600 or 6600 TripleTOF LC-MS/MS (AB SCIEX, Canada). NMR Analysis. Peptides (∼1 mg) were dissolved in 90% (v/v) H2O and 10% (v/v) D2O. 1D (1H) and 2D TOCSY and NOESY spectra were recorded at 298 K on a Bruker Avance 600 MHz spectrometer with similar procedures to those described previously.62 All data were processed by TOPSPIN (Bruker) and assigned by CCPNMR Analysis 2.4.1. Trypsin Inhibitor Assay. The trypsin inhibitor assay was performed as described previously.42 Briefly, bovine pancreatic trypsin was dissolved in 1 mM HCl at 4.5 mg/mL and then diluted to 0.45 mg/mL in buffer (50 mM Tris, 20 mM CaCl2, pH 8.2) and stored on ice. The substrate L-BAPNA (Na-benzoyl-L-arginine 4-nitroanilide hydrochloride, Sigma-Aldrich) was prepared at 0.435 mg/mL in 99% buffer with 1% DMSO. Peptides were prepared at a concentration of 1 mM and tested at eight different concentrations (i.e., in a 2-fold dilution series). The reaction mixture was prepared by dispensing 10 μL of buffer, 5 μL of 0.45 mg/mL trypsin, and 10 μL of the tested peptide into each well of a 96-well plate, before incubating the plate for 30 min at 37 °C. Subsequently, 125 μL of 0.435 mg/mL substrate was added and incubated for a further 10 min at room temperature. MCoTI-II was used as a positive control,63 and 100% trypsin inhibition activity (negative control) was achieved by adding trypsin alone. The reaction was stopped by adding 25 μL of 30% HOAc. Absorbance measurements were obtained at 410 nm using a Powerwave XS plate reader (Bio-Tek). The assay was done in triplicate. Cytotoxicity Assay. The cytotoxic effects of the Modi peptides were evaluated on three human cancer cell lines, including the human colorectal adenocarcinoma (HT-29), breast adenocarcinoma (MCF7), and prostate cancer (PC-3) cell lines. An insect cell line (Sf9, Spodoptera f rugiperda) was also included. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye was used in this assay. All mammalian cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS)/1% penicillin−streptomycin. Sf9 cells were cultured in Grace’s insect medium (GIBCO/Invitrogen) supplemented with 10% FBS. Cells were plated into 96-well tissue culture plates (100 μL, 2000 cells/well for mammalian cells; 50 000 cells/well for Sf9 cells) and grown for 24 h prior to treatment. The peptides were tested in triplicate, with final concentrations ranging from 0.05 to 100 μM. A solution of 1% (v/v) Triton X-100 and medium only were used as positive and negative controls, respectively. Mammalian cells and Sf9 cells were incubated for 24 and 3 h, respectively. Then, 10 μL of MTT (5 mg/mL in phosphate-buffered saline) was added to each well and further incubated for 4 h. The supernatant was removed, and 100 μL of DMSO was added into each well to dissolve the MTT formazan crystals. The absorbance was measured at 570 nm. Data were analyzed by GraphPad Prism software. Serum Stability Assay. Stability of the Modi peptides was assessed in 100% human male AB plasma, as described previously.64 All peptides, including the linear control peptide (WQELYQLKY), were tested at a final concentration of 30 μM. Peptides were incubated in the plasma for 0, 2, 4, 8, and 24 h at 37 °C. At each time point, three aliquots were taken, and 80 μL of each aliquot was injected onto a Phenomenex C18 column. Peptides were eluted using a linear gradient of 0−40% solvent B (90% MeCN with 0.1% TFA) with a 0.3 mL/min flow rate on an analytical RP-HPLC. The elution time for each peptide was determined for the 0 h time point. The remaining peptide at each time point was calculated relative to the height of the peptide peak at 215 nm for the 0 h time point and then used to determine the peptide stability.

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Additional information (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +61-7-33462019. Fax: +61-7-33462101. E-mail: d. [email protected]. ORCID

Junqiao Du: 0000-0003-3790-2975 Lai Yue Chan: 0000-0002-9346-2487 Aaron G. Poth: 0000-0002-1497-8347 David J. Craik: 0000-0003-0007-6796 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Australian Research Council (DP150100443). D.J.C. is an ARC Australian Laureate Fellow (FL150100146). L.Y.C. was supported by the Advance Queensland Women’s Academic Fund (WAF-6884942288). We thank E. K. Gilding for help in the importation process for the seeds. Editorial support was provided by J. Bates, Ph.D., of Science Write, Australia. We also acknowledge the Queensland NMR Facility for access to NMR equipment and an ARC grant (LE160100218).

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REFERENCES

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