Discovery and Characterization of Cyclotides from Rinorea Species

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

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Discovery and Characterization of Cyclotides from Rinorea Species Ploypat Niyomploy,†,‡ Lai Yue Chan,† Peta J. Harvey,† Aaron G. Poth,† Michelle L. Colgrave,§,⊥ and David J. Craik*,† †

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand § CSIRO Agriculture and Food, 306 Carmody Road, St. Lucia, Queensland 4067, Australia ⊥ School of Science, Edith Cowan University, 270 Joondalup Drive, Joondalup, WA 6027, Australia ‡

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S Supporting Information *

ABSTRACT: Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinorea is the second-largest genera within the Violaceae family, few studies have examined whether or not they contain cyclotides. To further our understanding of cyclotide diversity and evolution, we examined the cyclotide content of two Rinorea species found in Southeast Asia: R. virgata and R. bengalensis. Seven cyclotides were isolated from R. virgata (named Rivi1−7), and a known cyclotide (cT10) was found in R. bengalensis. Loops 2, 5, and 6 of Rivi1−4 contained sequences not previously seen in corresponding loops of known cyclotides, thereby expanding our understanding of the diversity of cyclotides. In addition, the sequence of loop 2 of Rivi3 and Rivi4 were identical to some related noncyclic “acyclotides” from the Poaceae plant family. As only acyclotides, but not cyclotides, have been reported in monocotyledons thus far, our findings support an evolutionary link between monocotyledon-derived ancestral cyclotide precursors and dicotyledon-derived cyclotides. Furthermore, Rivi2 and Rivi3 had comparable cytotoxic activities to the most cytotoxic cyclotide known to date: cycloviolacin O2 from Viola odorata; yet, unlike cycloviolacin O2, they did not show hemolytic activity. Therefore, these cyclotides represent novel scaffolds for use in future anticancer drug design.

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yclotides1 are a large family of plant-derived circular mini-proteins that have a range of biological activities, including uterotonic,2 anti-HIV,3−6 antimicrobial,7 antifouling,8 hemolytic,9 immunosuppressive,10,11 and cytotoxic.12−15 Although the natural role of cyclotides in plants is not yet fully defined, they are thought to be host defense agents based on their insecticidal, nematocidal, and molluscicidal activities.16−18 Different cyclotides are expressed in different plant tissues,19 and it has been postulated that this reflects tuning of the cyclotide suite to different defense tasks.20 The prototypical cyclotide, kalata B1, was discovered in a traditional Congolese medicine “kalata−kalata” by Lorents Gran, a Norwegian doctor, in the 1970s. Derived from a boiled leaf extract of the herb Oldenlandia aff inis DC. (Rubiaceae), this preparation was used to accelerate childbirth.21,22 One of the active uterotonic agents, kalata B1, was later characterized as a 29 amino acid cyclic peptide and its three-dimensional structure elucidated using NMR spectroscopy.23 This discovery led to efforts to find similar examples of cyclic peptides in other plants, and the term “cyclotide” was defined in 1999, when it became apparent that related macrocyclic peptides were present in a large number of plants.24 A further 20 cyclotides have since been reported in O. aff inis,25 and ∼400 cyclotides have been sequenced from a variety of other plants (www.cybase.org.au).26 More recent pharmacology analysis © XXXX American Chemical Society and American Society of Pharmacognosy

have confirmed the uterotonic activity of O. aff inis extract, including kalata B7, for inducing strong contractility on human uterine smooth muscle cells.27 Cyclotides are typically comprised of 28−37 amino acids with a head-to-tail cyclized backbone and incorporate six cysteines that form three disulfide bonds. Two of the disulfide bonds (CysI−CysIV and CysII−CysV) and their connecting backbone segments form a small embedded ring that is penetrated by the third disulfide bond (CysIII−CysVI). This unique structure is known as a cyclic cystine knot (CCK) motif and engenders cyclotides with high resistance to chemical, thermal, or enzymatic degradation, making them suitable candidates for pharmaceutical applications.28−30 Such applications typically involve the chemical synthesis of modified cyclotides in which one or more of the six backbone loops (the sequences between conserved Cys residues) are substituted with bioactive peptide sequences.31 Cyclotides are classified into three subfamilies, termed the Möbius, bracelet, or trypsin inhibitor cyclotides.32 The names of the Möbius and bracelet subfamilies are derived from their structures: the presence of a cis-proline amide bond in loop 5 causes a “Möbius” twist in the peptide backbone; otherwise, a Received: July 13, 2018

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DOI: 10.1021/acs.jnatprod.8b00572 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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conventional “bracelet” structure is formed. These two subfamilies also differ subtly in the type and number of amino acid residues in the backbone loops between the cysteine residues; however, they have high sequence homology to one another with a conserved glutamic acid (Glu) and aspartic acid/asparagine (Asp/Asn) in loops 1 and 6, respectively.33,34 The most well studied members of the trypsin inhibitor subfamily are MCoTI-I and MCoTI-II, which are extracted from seeds of the tropical vine Momordica cochinchinensis. Like the Möbius and bracelet cyclotides, members of this subfamily contain a CCK motif, but they share little sequence homology with the other two subfamilies apart from the six conserved Cys residues.35,36 The Violaceae plant family is the most abundant source of cyclotides with 317 of the more than 400 reported cyclotides found in this family. Cyclotides have also been identified in the Rubiaceae (85 cyclotides), Fabaceae (18 cyclotides), Cucurbitaceae (five cyclotides), and Solanaceae (12 cyclotides) plant families, 37−39 and Gruber et al. postulated that the Apocynaceae may also be a source.40 The Violaceae family comprises 25 genera,41 but to date, only six (i.e., Gloeospermum spp., Hybanthus spp., Leonia spp., Melicytus spp., Viola spp., and Rinorea spp.) have been reported to contain cyclotides.42 Rinorea consists of 225−275 species43 and is the second-largest genus of the Violaceae family. Despite its size, only two studies have reported cyclotides from Rinorea spp.; namely, the characterization (at the peptide level by MS/MS) of riden A from R. dentata44 and the cDNA prediction of rigra A from R. gracilipes and rili A and B from R. lindeniana.42 In the current study, we examined two species from the Rinorea genus not previously examined for cyclotides: R. virgata and R. bengalensis. R. virgata is commonly found in Southeast Asian countries, including Myanmar, Thailand, and Vietnam, and R. bengalensis is also endemic in Australia and nearby Pacific islands. Previous work on these plants focused on the morphological and biological activities of R. virgata (www.efloras.org) and the accumulation of nickel in R. bengalensis.45 The latter species is of particular interest as some cyclotides have been reported to have metal-binding capabilities.46,47 In addition, R. virgata was considered an interesting candidate for cyclotide discovery as it is classified in a different clade well-separated from most other Rinorea species.43 The discovery of cyclotides from R. virgata and R. bengalensis has the potential to further our understanding of cyclotide evolution and diversity, and novel cyclotides might have biological activities with potential pharmaceutical applications or be useful in defining the diversity of cyclotide sequences. Herein, we report the discovery of seven cyclotides from R. virgata (Rivi1−7) and one known cyclotide from R. bengalensis (which matched the sequence of cT10,48 also known as CT1249). Four of the novel cyclotides (Rivi1−4) were structurally characterized and screened for biological activity in a range of in vitro assays.

Figure 1. Total ion chromatogram (TIC) of crude protein extracted from R. virgata leaves analyzed by LC-MS/MS. A linear gradient of 1−45% solvent B over 40 min was employed before a column cleaning step from 45 to 80% solvent B over 5 min at a flow rate of 0.18 mL min−1.

respectively. Four additional peaks were definitively identified as the cyclotides Rivi4−7, eluting at 28.63, 38.65, 38.90, and 41.80 min. Many other peaks in the chromatogram were not recognized as cyclotides because they did not contain three disulfide bonds, as revealed by reduction and alkylation. A crude protein extract from R. bengalensis was more viscous and difficult to handle than the R. virgata extracts and, as such, required additional purification steps before analysis by mass spectrometry (MS). Consequently, there was less material to work with; nevertheless, one cyclotide was identified from the R. bengalensis family (described below). The crude peptides from R. virgata and R. bengalensis were partially purified by solid-phase extraction (SPE). Most cyclotides from R. virgata eluted in the 20 and 30% acetonitrile fractions (Figure S1), whereas only one cyclotide from R. bengalensis was found in the 30% acetonitrile SPE fraction (Figure S2). Five peptides from R. virgata were selected for further sequence characterization, but only one peptide sequence from R. bengalensis was subjected to MS/MS analysis. Sequence analysis of the peptide from R. bengalensis, using uHPLC-ESI-MS/MS, yielded an MS/MS spectrum matching the previously identified cyclotide cT1048 (also known as CT1249) from the Fabaceae family plant Clitoria ternatea (Figure S3) after automated database searching using the ProteinPilot search engine. De novo peptide sequencing by MALDI-TOF/TOF analysis subsequently confirmed the amino acid sequence of this cyclotide (Figure S3). The cyclotide-containing fractions from the SPE of R. virgata extract were purified by several rounds of HPLC, yielding four pure peptides, Rivi1−4 (Figure S4), and a mixed fraction of three peptides, Rivi5−7. The samples, which were within the molecular weight range of cyclotides (2800−3500 Da), were reduced and alkylated using dithiothreitol and iodoacetamide successively before being digested with endo-Glu-C, trypsin, or chymotrypsin and analyzed using tandem MS sequencing. The pure fractions containing Rivi1−4 were each readily sequenced. From the mixed fraction, only the sequence of Rivi5 could be deduced in part because it was the most abundant component in the fraction but primarily because it contained a single trypsin cleavage point and yielded a +18 Da product on digestion of the reduced cyclic peptide. Rivi6 and Rivi7 appeared to contain multiple trypsin cleavage points, and

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RESULTS AND DISCUSSION Cyclotide Isolation and Sequencing. Extracts of both Rinorea species were prepared from fresh leaves (see Experimental Section). The LC-MS chromatogram of a crude extract of R. virgata leaves revealed at least seven cyclotides (Rivi1−7) among the multitude of peaks present (Figure 1). The most abundant peaks corresponded to Rivi1− 3 with retention times at 33.42, 33.63, and 33.72 min, B



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residues, except for Rivi4, which has 30 residues. Rivi4 belongs to the bracelet subfamily, and Rivi1, 2, 3, and 5 to the Möbius subfamily, based on the presence of a loop 5 proline (confirmed to be in the cis configuration by NMR analysis of Rivi1−3). Although unable to be sequenced, Rivi6 and 7 were classified as cyclotides based on their mass shift after EndoGlu-C enzymatic digestion (Figure S5) as well as the fact that Rivi6 shares its molecular weight with kalata B2, and Rivi7 has the same molecular weight as the known cyclotides Mra23 and chassatide C6.26 Because many possible cyclotide sequences match those masses, the sequences of Rivi6 and Rivi7 remain undetermined and it is unclear whether they are known or novel cyclotides. For the sequencing procedure to be illustrated in detail, the sequence of Rivi3, the most abundant cyclotide in R. virgata, was characterized using LC-ESI-MS/MS analysis of three different enzymatic digests. The assigned b- and y- ion series are shown in Figure 2. Similarly, the MS/MS spectra that enabled the elucidation of the peptide sequences from Rivi1, 2, 4, and 5 are provided in Figures S6−S9, respectively. During the purification of Rivi7, evidence for the presence of Asn deamidation, a phenomenon that can sometimes occur during proteomic sample preparation50,51 and has previously been reported for cyclotides,52,53 was identified (Figure S5). For discriminating the isobaric Leu/Ile residues in the proposed Rivi 1−4 sequences, a combination of NMR (see

this feature combined with their low abundance in the mixture rendered them not amenable to sequencing. The sequences of Rivi1−5 are compared to the prototypic cyclotide kalata B1 in Table 1. All contain 29 amino acid Table 1. Amino Acid Sequences of Novel Cyclotides from R. virgata

a

All experimental masses are within 10 ppm of theoretical values. Kalata B1 was not determined in the current study and is shown here for sequence comparison. cSequences aligned using Clustal Omega. d Cycloviolacin O2 was not determined in the current study and is shown here for sequence comparison. b

Figure 2. MS/MS spectra of Rivi3 proteolytic fragments. Rivi3 was reduced, alkylated, and digested to yield (A) the endo-Glu-C peptide of m/z 696.3 representing the 5+ charge state of the intact but linearized cyclotide, (B) the chymotryptic peptide KNGLPICGETCL at m/z 681.32+, (C) the tryptic peptide CYTPGCSCR at m/z 580.72+, and (D) the tryptic peptide RPVCYK at m/z 411.72+. C



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Classification of Novel Rivi Cyclotides Based on Secondary Structure. The novel peptides Rivi1−4 were examined by NMR spectroscopy to assess their secondary structure in solution. Following acquisition of TOCSY and NOESY spectra of each of the isolated peptides at 298 K and pH 4, backbone chemical shifts were fully assigned and used to calculate secondary αH shifts (Figure 3). The amino acid spin systems and positions of Ile and Leu in the novel sequences were also identified using NMR and found to be consistent with the results from MS and amino acid analysis (Table S1− S4), respectively. On the basis of the inter-residue NOE correlations, all prolines were assigned as having trans peptide bond conformations apart from those in loop 5 of the sequence that had a cis conformation. Hence, Rivi1−3 peptides are classified as Möbius subfamily members by analogy with kalata B1. In contrast, the proline of loop 5 is absent in Rivi4, and consequently, this peptide is defined as a bracelet cyclotide. A proline is also absent in Rivi2 albeit in loop 6, and these modifications in proline content affect the local chemical environment of these regions, as reflected in the differences in the secondary αH shifts between the novel peptides and kalata B1 (Figure 3). For example, Rivi2 appears to be more disordered across part of loop 6, and the secondary shifts of Rivi4 are highly variable across the entirety of loop 5. Rivi1 and Rivi3 also lack the strongly downfield shifted Pro 24 because this proline is preceded by an arginine residue, unlike kalata B1 and Rivi2, whose Pro 24 chemical shifts are affected by the aromatic side chain of the adjacent tryptophan. Therefore, R. virgata contains two unique cyclotides from the Möbius subfamily, namely, Rivi1 and Rivi3, each with an “RRP” sequence in loop 5. Rivi3 Has a Unique Loop 5 Conformation. Rivi3, as the most abundant cyclotide in R. virgata, was synthesized chemically to provide sufficient peptide for complete structural analysis by NMR spectroscopy. The three-dimensional solution structure of Rivi3 was calculated from 295 distance restraints, 39 dihedral angle restraints, and four hydrogen bond pairs. The final family of structures has good structural and energy statistics, as shown in Table S5, and overlay well over the entire molecule with an RMSD for the backbone atoms of 0.74 ± 17 Å (Figure 3B). Comparison of the Rivi3 structure (PDB ID: 6DHR; BMRB ID: 30470) with that of kalata B1 (PDB ID: 1NB1) (Figure 3C) revealed that the backbone structures of these two cyclotides overlay well with an RMSD of 0.69 Å. This data presents the first 3D solution structure of a loop 5 arginine-proline bond in the cis configuration. On the basis of similarities in the secondary αH chemical shifts (Figure 3A), it is likely that Rivi1 will have a similar structure. Cytotoxicity and Hemolytic Activities. For their potential bioactivities to be investigated, cyclotide-containing fractions from R. virgata (eluted from SPE cartridges with 20 and 30% acetonitrile) were screened in an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell cytotoxicity assay against a range of mammalian cells, including two normal cell lines (i.e., human foreskin fibroblast [HFF-1] and human umbilical vein endothelial cell [HUVEC]) and three cancer cells lines (i.e., breast cancer [MDA-MB-231], melanoma [MM96L], and colon cancer [HT-29]). The two fractions had similar cytotoxic activity against all cells except the breast cancer (MDA-MB-231) cells, which were only effectively killed by the 30% acetonitrile SPE fraction (Figure 4). However, this fraction contained a range of other molecules

below) and amino acid analysis was used (Tables S1−S4). Specifically, amino acid analysis defined the relative content of Ile versus Leu residues and NMR localized the Ile or Leu residues in the peptide sequence. This analysis was not possible for Rivi 5, and the positions of Ile and Leu residues were assigned by homology with known cyclotides. Sequence Diversity of Novel Cyclotides. The five novel sequences were compared to all entries in the Uniprot database using BLASTp similarity searches with default settings.54 The best quality matches with 100% query coverage belong to known cyclotides from the Viola genus. The percentage sequence identities of top scoring full length matches were Rivi1 showing 82.7% identity to mden-F (from Melicytus dentatus), Rivi2 showing 82.0% identity to mela-1 (from Melicytus latifolius), Rivi3 and 4 showing 82.0 and 73.0% identity, respectively, to vodo peptide N (from V. odorata) and mobo-B (from Melicytus obovatus), and Rivi5 showing 89.0% identity to mech-7 (from Melicytus chathamicus). Rivi4 had the lowest sequence identity, which is likely due to the lack of a proline and the presence of the extra amino acid residue in loop 5. The individual loop sequences from Rivi1−4 were compared to the corresponding loops of known cyclotides in Cybase.26 Loop 6 of all four peptides was found to contain new sequences as did loop 2 of Rivi1 and Rivi2 and loop 5 of Rivi1, 3, and 4 (Table 2). The remaining loops of all four peptides Table 2. Comparison of Rivi1−4 Loop Sequences to Those of Known Cyclotidesa cyclotide

loop 1

loop 2

loop 3

loop 4

loop 5

loop 6

Rivi1 Rivi2 Rivi3 Rivi4

V,R,F,P V,R,F,P V,R,F,P V,R,F,P

*b * P P

V,R V,R,F,P V,R,F,P V,R,F,P

V,R,F,P V,R V,R,F,P V,R

* V,R,F * *

* * * *

a

V, R, F, P denotes similarity with Violaceae, Rubiaceae, Fabaceae and Poaceae families, respectively. bAsterisk indicates that the loop contains a new sequence not seen in this loop of other cyclotides.

contained known sequences from cyclotides of the Violaceae, Rubiaceae, and Fabaceae families. Interestingly, the known loop sequence “LLGK”, which was previously identified in an acyclotide (i.e., a backbone linear cyclotide derivative) from the Poaceae family,55 is also found in loop 2 of Rivi3 and Rivi4. The presence of this sequence in two different plant lineages may provide further insight into the evolutionary relationships between cyclotides in monocotyledon and dicotyledon plants. So far, only acyclotides, but not cyclotides, have been reported in monocots, and it is not known why dicots but not monocots have evolved a cyclization ability. It is also not understood what all of the evolutionary advantages of cyclic vs acyclic cyclotide derivatives might be, although previous studies have established that synthetic acyclic permutants of cyclotides typically lack the activity of the parent cyclic molecule, as reported for anti-HIV56 and hemolytic activity.57 It seems likely that cyclization engenders the peptides with both increased stability and enhanced bioactivity. Therefore, the similarity between the loop 2 sequences of Rivi3−4 to the known loop sequence of an acyclotide supports an evolutionary link between monocot-derived ancestral cyclotide precursors and dicot-derived cyclotides. Further studies are required to confirm such a link. D



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Figure 3. Structural analysis of Rivi peptides. (A) Secondary αH shifts of Rivi1 (white bars), Rivi2 (gray bars), Rivi3 (blue bars), and Rivi4 (diagonal blue bars) compared with kalata B1 (chemical shifts obtained from Cybase,26 black bars). Cysteines are highlighted in yellow. All secondary shifts were calculated using the random coil 1H NMR chemical shifts of Wishart et al.72 (B) Backbone superposition of the 20 lowest energy structures of Rivi3. (C) Ribbon diagram of Rivi3 (blue) overlaid with kalata B1 (red, PDB ID: 1NB1). Disulfide bonds are highlighted in yellow and labeled by cysteine residue numbers.

Compared to melittin, a positive control for red blood cell lysis, Rivi1−3 had no effect on red blood cells at concentrations up to 10 μM, suggesting minimal toxicity toward these cells as compared to the tested cancer cell lines. Conclusions. Overall, one known cyclotide was found in R. bengalensis, and seven cyclotides were found in R. virgata with five of the seven sequences found to be novel. This work reports the largest number of cyclotides discovered in any single Rinorea species to date and is likely to be the lower limit of the number of cyclotides in this species, as many unidentified peaks remained. Of the four isolated and structurally characterized sequences, only Rivi4 was found to belong to the bracelet subfamily due to the absence of a proline in its loop 5. Although the majority of Möbius cyclotides have a “WP” sequence in this loop (i.e., 74% of this subfamily according to Cybase26), Rivi1 and Rivi3 are unique among identified cyclotides as having an “RRP” sequence. Indeed, this work presents the first three-dimensional structure of a loop 5 arginine-proline bond in the cis configuration. Rivi1−4 have similar toxicity profiles when tested against noncancerous and cancerous cell lines. Although toxicity was shown toward normal cells, this could be overcome by mutation studies in the future to potentially identify residues responsible for this activity. In summary, the novel cyclotides from the Rinorea species presented herein have significantly expanded our understanding of the diversity of known cyclotide sequences and support a possible evolutionary link between monocot-derived ancestral cyclotide precursors and dicot-derived cyclotides. Moreover, on the basis of their strong cytotoxicity profile and low hemolytic activity, Rivi2 and Rivi3 should be explored as

as well as Rivi1−4 (Figure S1) that may have exerted activity toward the breast cancer cell line. Despite the minimal effect of the 20% acetonitrile SPE fraction against the breast and colon cells, further purification was warranted because of the concentration-dependent manner in which this fraction exerted its activity against normal human skin cells (HFF-1 and MM96L). Hence, the 20% fraction was further purified using HPLC and the purity of isolated peptides confirmed by analytical HPLC (see Figure S4). Isolated peptides (Rivi1−4) were tested on normal cells (HUVEC and HFF-1) to determine if they have less toxicity against these cell lines compared to that of cancer cells. Paclitaxel, a commercial drug commonly used for anticancer applications,58,59 was tested as a positive control. In addition, previously published results for cycloviolacin O2 (cO2)3,60 are also included in Table 3 for comparison as it is the most potently cytotoxic cyclotide demonstrated to date. Rivi1 was the least toxic of the Rivi peptides with a considerably higher IC50 than reported for cO2. In contrast, Rivi2−4 had activity comparable to cO2 against each of the cell lines with IC50 values in the low micromolar range, although Rivi4 was ∼2-fold less potent against MDA-MB-231 cells and Rivi2 (the only isolated peptide fully tested against HT-29 cells) was more than 5-fold less potent against human colon adenocarcinoma cells. In general, Rivi 2−4 had only slightly higher IC50 values than cO2 and showed minimal selectivity between the cell lines. Meanwhile, paclitaxel had high potency and specificity against breast and colon cancer cells compared to those of all other peptides and cO2. All peptides (except Rivi4 due to the limited amount of isolated peptide) were tested in a hemolytic assay to examine the toxicity of the peptides toward red blood cells (Table 3). E



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Chanthaburi Province, Thailand, and the Brisbane Botanical Gardens, Australia, in January 2014 and April 2015, respectively. Fresh leaves from R. virgata (14.06 g) and R. bengalensis (124.33 g) were cooled and ground in liquid nitrogen to each yield a fine powder. The powders were then dissolved in 100 mL of 50% (v/v) acetonitrile and 1% (v/v) formic acid and stirred overnight at 4 °C. The supernatant from each crude solution was collected after centrifugation at 8,000 rpm for 45 min before being lyophilized using a freeze dryer (Christ Alpha 2−4 LD freeze-dryer). The crude extract from R. virgata was separated chromatographically on a uHPLC (Shimadzu Prominence uHPLC, Japan) directly coupled to a Triple TOF 5600 mass spectrometer (AB SCIEX, Canada) equipped with duoelectrospray ion source. The crude peptide solution (5 μL) was injected onto a Zorbac 300SB-C18 column (2.1 mm × 100 mm, 1.8 μm, 0.18 mL min−1 flow rate, Agilent, USA) and eluted with a linear gradient from 1 to 45% solvent B over 40 min before a column cleaning step from 45 to 80% solvent B over 5 min (where solvent A was 0.1% formic acid and solvent B comprised 90% acetonitrile, 0.1% formic acid) at a flow rate of 0.18 mL min−1. The eluate from the uHPLC was analyzed by a full-scan TOF-MS over the mass range 350−2000 Da and processed by Analyst TF 1.6 software (AB SCIEX). Solid-Phase Extraction, RP-HPLC/Analytical HPLC Purification. The crude extracts from both plants were initially purified by SPE followed by reversed-phase high-performance liquid chromatography (RP-HPLC). First, C18 SPE cartridges (Waters, 500 mg) were activated and equilibrated with 10 mL of methanol and 1% (v/v) formic acid, respectively. Crude extract (2 g) was dissolved in 20 mL of 1% (v/v) formic acid and loaded into the cartridge. The crude solution was eluted sequentially with 10 mL of 20−80% (v/v) acetonitrile and 1% (v/v) formic acid for cyclotide separation. Cyclotide-containing fractions of R. virgata were freeze-dried and redissolved in 0.05% (v/v) trifluoroacetic acid/water (solvent A) before loading on a Phenomenex C18 semipreparative column (250 mm × 10 mm, 10 μm, 300 Å, 3 mL min−1 flow rate) on an RP-HPLC (LC10, Shimadzu) and run at 0.5 and 1% min−1 gradients in 20−80% solvent B (0.045% trifluoroacetic acid/90% acetonitrile) to yield pure cyclotides, which were detected at 215 and 280 nm. This purification step was repeated several times to obtain high-purity cyclotides, and finally, all cyclotides were subjected to HPLC (Shimadzu) with a flow rate of 0.3 mL/min on a Vydac 218TP C18 5 μm column using a 2% gradient of 0−80% solvent B for assessing peptide purity before use in a range of biological assays and further peptide characterization. Peptide Synthesis. For a sufficient amount of Rivi3 to be obtained for NMR spectroscopy analysis, this peptide was synthesized with Fmoc-based chemistry according to the method described by Cheneval et al.64 Reduction, Alkylation, and Enzymatic Digestion. The purified cyclotide from R. virgata was dissolved in 100 μL of MilliQ water and adjusted to pH 8 with 10 mM NH4HCO3. The peptide

Figure 4. Cytotoxic activity of the fraction eluted with 20% (closed symbol) and 30% (open symbol) acetonitrile with 1% formic acid from R. virgata against the cell line (A) human foreskin fibroblasts (HFF-1), a noncancerous cell line, (B) human melanoma cancer cell line (MM96L), (C) breast cancer cell line (MDA-MB-231), and (D) human colon adenocarcinoma cell line (HT-29). Concentration− response curves were run in triplicate (n = 3). Standard deviation were determined by fitting concentration−response curves with the nonlinear regression equation.

novel scaffolds for future drug design and grafting application studies.

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EXPERIMENTAL SECTION

Isolation, Extraction, and LC-MS Analysis of Cyclotides. Fresh leaves of R. virgata and R. bengalensis were collected from

Table 3. IC50 Values of Cyclotides from R. virgata in Cell Cytotoxicity and Hemolytic Assays IC50 (μM) ± SDa cyclotide Rivi1 Rivi2 Rivi3 Rivi4 cO2i paclitaxel (24 h) paclitaxel (48 h) melittin

HUVEC ≫3 1.6 2.6 2.8 0.35 ≫10 ≫10 ND

± ± ± ±

b

0.02 0.06 0.02 0.0661

HFF-1 ≫3 2.1 1.9 1.7 1.3 ≫10 ≫10 ND

± ± ± ±

c

0.02 0.02 0.01 0.0912

MM96Ld ≫3 1.3 1.1 0.93 0.65 ≫10 ≫10 ND

± ± ± ±

0.01 0.05 0.02 0.0660

MDA-MB-231e ≫10 0.95 2.3 4.4 1.560 0.20 0.001 ND

± 0.10 ± 0.01 ± 0.04 ± 0.11 ± 0.10

HT-29f ≫10 5.9 NDh ND 0.87 0.07 0.01 ND

RBCg

± 0.04

± 0.0862 ± 0.13 ± 0.10

≫10 ≫10 ≫10 ND 3663 ND ND 0.96 ± 0.03

a Data represent the mean ± standard deviation (S.D.) from three replicates. These are biological replicates. bHUVEC: human umbilical vein endothelial cells. cHFF-1: human foreskin fibroblast cells. dMM96L: melanoma cells. eMDA-MB-231: breast cancer cells. fHT-29: colon cancer cells. gRBC: red blood cells. hND: not determined, as the amount of peptide was insufficient for the experiments. iCycloviolacin O2 (cO2): we emphasize that caution should be exercised when comparing the literature data cO2 with the Rivi peptides as no direct side-by-side comparison was made.

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solution was mixed with 10 μL of 100 mM dithiothreitol and incubated at 60 °C under nitrogen for 30 min to reduce the disulfide bonds. The reduced peptide was then mixed with 10 μL of 250 mM iodoacetamide and incubated for 30 min at room temperature. Reduced and alkylated peptides were digested with endo-Glu-C, trypsin, chymotrypsin, and a mixture of endo-Glu-C and trypsin before being incubated at 37 °C for 6 h. For single-enzyme digestion, 2 μL of 1 μg/μL endo-Glu-C, trypsin, or chymotrypsin were added to a 20 μL sample. For double-enzyme digestions, 20 μL of sample were mixed with 2 μL of both enzymes (endo-Glu-C and trypsin). All peptides, after reduction, alkylation, and enzymatic digestion, were desalted with C18 Ziptips (Millipore) and eluted in 10 μL of 80% (v/ v) acetonitrile with 1% formic acid. The desalted cyclotide was mixed with 7 mg/mL of CHCA matrix (α-cyano-4-hydroxycinnamic acid [CHCA] in 50% [v/v] acetonitrile/1% [v/v] formic acid) in a 1:1 ratio before spotting on a MALDI target and applied to MALDI-TOF MS (Bruker UltrafleXtreme TOF/TOF MS) to determine the molecular weight of each cyclotide. De Novo Peptide Sequencing Using Nanoelectrospray MS/ MS. The amino acid sequences of all digested peptides from R. virgata were determined using nanoelectrospray MS/MS. Digested peptides (10 μL) were loaded into Thermo Fisher Scientific ES380 nanoES metal-coated spray capillaries before undergoing nanospray using a QSTAR Pulsar mass spectrometer (AB SCIEX) equipped with a Proxeon II nano ion source for peptide sequencing. The nanospray voltage was set to 900 V with a declustering potential of 70 V. TOFMS data were acquired in the mass range of 400−2000 Da, and MS/ MS data were acquired by increasing the collision energy (from 10− 55 V) to obtain fragment ion coverage with enhanced signal. Analyst QS (AB SCIEX) software packages were used to acquire and process MS/MS data. The cyclotide-containing 30% acetonitrile SPE fraction from R. bengalensis was reduced, alkylated, and digested with endoGlu-C (as described above) before analysis by uHPLC-MS/MS on a Triple TOF 5600 mass spectrometer (AB SCIEX) and MALDI-TOF MS (Bruker UltrafleXtreme TOF/TOF MS) to obtain MS/MS spectra, which were analyzed using the Analyst TF 1.6 (AB SCIEX) and Flex Analysis (Bruker, Germany) software packages, respectively. The MS/MS spectra generated on the Triple TOF 5600 mass spectrometer were searched using the ProteinPilot database search engine. Secondary Structure Analysis Using NMR and ThreeDimensional Structure Calculation. Isolated peptides Rivi1−4 were prepared for NMR analysis by dissolving in 90% H2O/10% D2O. 1 H NMR spectra and two-dimensional spectra (TOCSY and NOESY) were recorded at mixing times of 80 and 200 ms, respectively, on a Bruker Avance 600 MHz spectrometer at 298 K. NMR data were processed using TOPSPIN 2.1 (Bruker), and assignments were made using CCPNMR (version 2.4.4) according to methods described by Wüthrich et al.65 Initial three-dimensional structures of Rivi3 were calculated using the program CYANA with distance restraints derived from NOESY spectra and backbone φ and ψ dihedral angles generated using the program TALOS-N.66 CNS was then used to generate a final set of structures using torsion angle dynamics and refinement and energy minimization in explicit solvent67 followed by assessment of final structures for stereochemical quality using MolProbity.68 Amino Acid Analysis. Dried peptide samples (Rivi1−4) were analyzed by gas-phase hydrolysis with 6 M HCl at 110 °C for 24 h using Waters AccQTag Ultra chemistry on a Waters Acquity UPLC for amino acid analysis of each peptide. The reported amount of Asn/ Asp and Gln/Glu is the sum of both amino acids in each pair because Asn and Gln are hydrolyzed to Asp and Glu, respectively. Cys and Trp were not analyzed using this method. Cytotoxicity Assays. Cyclotide-containing fractions from SPE (eluted at 20 and 30% acetonitrile in 1% formic acid) and purified cyclotides (Rivi1−4) from R. virgata were used to determine cytotoxicity against MDA-MB-231 (breast cancer), MM96L (melanoma), HT-29 (colon cancer), and HFF-1 (human foreskin fibroblast) cells using an MTT assay (Sigma). Purified cyclotides Rivi1−4 were also tested against human umbilical vein endothelial

cells (HUVECs). All cells were plated in 96-well plates at 3 × 103 cells/well (100 μL) in different media, including 10% FBS/EBM-2 media supplemented with SingleQuots (which includes growth factors, cytokines, and antibiotics; Lonza) for HUVECs, 15% FBS/ DMEM (Dulbecco’s modified Eagle medium; Gibco) for HFF-1 cells, 10% FBS/DMEM for HT-29 and MDA-MB-231 cells, and 10% FBS/ RPMI with 2 mM L-glutamine and 1 mM sodium pyruvate (Gibco) for MM96L cells. Cells were incubated at 37 °C in 5% CO2 for 24 h prior to the assay. Medium was first removed and replaced with fresh serum-free EBM-2 and DMEM media (100 μL well−1) followed by the addition of 10 μL of fraction from SPE at concentrations ranging from 0.75 to 100 μg/mL with the exception of HT-29, which was used from 0.35 to 50 μg/mL, and Rivi 1−4, which was used from 0.075 to 10 μM. The plate was incubated for 2 h before a 10 μL aliquot of MTT (5 mg mL−1 in phosphate buffered saline [PBS]) was added to each well and incubated for a further 3 h. The supernatant was removed, and the formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO). The plate was measured at 600 nm using a BioTek PowerWave XS spectrophotometer. Paclitaxel (Sigma) was used as a positive control at concentrations ranging from 0.00075 to 10 μM. Paclitaxel was incubated with cells for either 24 or 48 h.69,70 Peptides and paclitaxel were tested in triplicate, and the data were analyzed using the statistical software package GraphPad Prism to obtain IC50 values from the sigmoidal concentration−response curves. Hemolytic Assay. Rivi1−3 were prepared at concentrations ranging from 0.003 to 10 μM. Melittin (0.15−20 μM; Sigma) was included as a positive control to determine toxicity to human red blood cells according to the method described by Chan et al.71

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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.jnatprod.8b00572. Amino acid analysis of Rivi1−4 (Tables S1−S4); statistical analysis of Rivi3 structures (Table S5); MS profile data of 20 and 30% acetonitrile SPE fraction from R. virgata (Figure S1) and a cyclotide (cT10 or CT12) isolated from R. bengalensis (Figure S2); MS/MS data profile of a cyclotide (cT10 or CT12) isolated from R. bengalensis (Figure S3); analytical HPLC chromatogram of Rivi1−4 (Figure S4); MS profile data of Rivi6 and 7 (Figure S5); and MS/MS profile data of Rivi1, 2, 4, and 5 (Figures S6−S9) (PDF)

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AUTHOR INFORMATION

Corresponding Author

* Tel: +61 7 3346 2019. Fax: +61 7 3346 2101. E-mail: d. [email protected]. ORCID

Aaron G. Poth: 0000-0002-1497-8347 Michelle L. Colgrave: 0000-0001-8463-805X 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 two grants from the Australian Research Council (ARC) (DP150100443 and LE160100218). 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). The work was facilitated using infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure G



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Discovery and Characterization of Cyclotides from Rinorea Species

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