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Momordicatrypsin inhibitors (TIs) such as those isolated from the seeds of the gấc 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 Momordicasubfamily. 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-to-tail cyclization or N-pyrolation, which suggests these peptides might be excellent candidates as scaffolds for epitope stabilization in drug design studies.

Cyclotides are macrocyclic cystine-knotted peptides most commonly found in the Violaceae plant family. Although Rinoreais 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 Rinoreaspecies found in Southeast Asia: R. virgataand 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.

Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.

Plants from the genus Psychotriainclude species bearing cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with cyclotides in Psychotria leiocarpaand P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpaand P. brachyceraswere monitored following herbivore- and pathogen-associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant cyclotide found in the leaves of P. leiocarpa, and also found in P. brachycerasleaves, exhibited insecticidal activity against Helicoverpa armigeralarvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity and resulted in increased rates of larval survival in treatments containing indole alkaloids. This suggests that plant alkaloids ingested by larvae might contribute to herbivore oxidative stress detoxification, corroborating, in a heterologous system with artificial oxidative stress stimulation, the antioxidant efficiency of Psychotriaalkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpaas a cyclotide-bearing species, and the absence of these peptides in P. umbellata.

Cyclotides are macrocyclic proteins produced by plants for host defense. Although they occur sparsely in other plant families, cyclotides have been detected in every Violaceae plant species so far screened. Many of the Violaceae species examined until now have been from closely related geographical regions or habitats. To test the hypothesis that cyclotides are ubiquitous in this family, two geographically isolated (and critically endangered) species of Australasian Violaceae, namely Melicytus chathamicusand M. latifolius, were examined. Surprisingly, we discovered a suite of cyclotides possessing novel sequence features, including a lysine-rich nature, distinguishing them from “conventional” cyclotides and suggesting that they might have different physiological activities in plants to those reported to date. The newly discovered cyclotides were found to bind to lipid membranes and were cytotoxic against cancer cell lines but had low toxicity against red blood cells, which is advantageous for potential therapeutic applications. This suite of novel Lys-rich cyclotides emphasizes the broad diversity of cyclotides in Violaceae species.

Five new orbitides, cyclolinopeptides 21–25, were identified in flaxseed oil (Linum usitatissimum) extracts. Their HPLC-ESIMS quasimolecular ion peaks at m/z1097.7 (21), 1115.6 (22), 1131.6 (23), 1018.6 (24), and 1034.6 (25) [M + H]+corresponded to the molecular formulae C59H89N10O10, C58H87N10O10S, C58H87N10O11S, C53H80N9O9S, and C53H80N9O10S, respectively. Their structures were elucidated by extensive HPLC-ESIMS/MS analyses, and their presence was confirmed by precursor proteins identified in flax genomic DNA sequence data. The amino acid sequences of these orbitides were confirmed as [1–10-NαC]-GILVPPFFLI, [1–10-NαC]-GMLIPPFFVI, [1–10-NαC]-GOLIPPFFVI, [1–9-NαC]-GMLVFPLFI, and [1–9-NαC]-GOLVFPLFI for cyclolinopeptides 21–25, respectively. Previously reported orbitides, [1–9-NαC]-ILVPPFFLI (1), [1–9-NαC]-MLIPPFFVI (2), [1–9-NαC]-OLIPPFFVI (3), [1–8-NαC]-MLVFPLFI (7), and [1–8-NαC]-OLVFPLFI (8), were also present in flaxseed oil. The precursors of orbitides 21, 22, and 24also produced orbitides 1, 2, and 7by alternative cyclization. Cyclolinopeptides 3, 8, 23, and 25contain MetO (O) and are not directly encoded, but are products of post-translational modification of the Met present in 2, 7, 22, and 24, respectively. Sufficient cyclolinopeptide 23was isolated for characterization via 1D (1H and 13C) and 2D (NOESY and HMBC) NMR spectroscopy. These compounds have been named as cyclolinopeptides U, V, W, X, and Y for 21, 22, 23, 24, and 25, respectively.

Cyclotides are plant proteins whose defining struc-tural features are a head-to-tail cyclized backbone and three interlocking disulfide bonds, which in combination are known as a cyclic cystine knot. This unique structural motif confers cyclotides with exceptional resistance to proteolysis. Their endogenous function is thought to be as plant defense agents, associated with their insecticidal and larval growth-inhibitory properties. However, in addition, an array of pharmaceutically relevant biological activities has been ascribed to cyclotides, including anti-HIV, anthelmintic, uterotonic, and antimicrobial effects. So far, >150 cyclotides have been elucidated from members of the Rubiaceae, Violaceae, and Cucurbitaceae plant families, but their wider distribution among other plant families remains unclear. Clitoria ternatea(Butterfly pea) is a member of plant family Fabaceae and through its usage in traditional medicine to aid childbirth bears similarity to Oldenlandia affinis, from which many cyclotides have been isolated. Using a combination of nanospray and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analyses, we examined seed extracts of C. ternateaand discovered cyclotides in the Fabaceae, the third-largest family of flowering plants. We characterized 12 novel cyclotides, thus expanding knowledge of cyclotide distribution and evolution within the plant kingdom. The discovery of cyclotides containing novel sequence motifs near the in plantacyclization site has provided new insights into cyclotide biosynthesis. In particular, MS analyses of the novel cyclotides from C. ternateasuggest that Asn to Asp variants at the cyclization site are more common than previously recognized. Moreover, this study provides impetus for the examination of other economically and agriculturally significant species within Fabaceae, now the largest plant family from which cyclotides have been described.

In recent years, the discovery of a large family of macrocyclic peptides, the cyclotides, has revealed Nature's ingenuity in molecular drug design. The incorporation of a cyclic peptide backbone and a knotted arrangement of disulfide bridges into their structures confers extraordinary chemical, thermal, and enzymatic stability on these biologically active peptides. However, these structural attributes present challenges in the identification of cyclotides. Until now, the sequencing of cyclotides has been slow and inefficient owing to inherent difficulties in the separation of these hydrophobic peptides from plants, the multiple chemical and enzymatic derivatization steps required to make them amenable to mass spectrometric sequencing, and the lack of software tools to efficiently deal with these circular permutants. The current bottleneck slowing the speed of cyclotide sequencing is the requirement for multiple HPLC purification steps before analysis. Here, we have applied proteomic strategies to fast-track the discovery of known, modified and novel sequences. Using four fractions from a previously well-characterized cyclotide-containing plant species, Viola odorata, 11 new sequences, as well as a plethora of known and modified cyclotides, were uncovered. In addition, the methodology was validated through analysis of crude leaf extracts of Oldenlandia affinisand Arabidopsis thaliana. The unambiguous identification of a suite of cyclotides in the Oldenlandia affinisextract provided the ultimate proof-of-concept for this application. Major advances in methodology include the use of optimized LC-MS/MS conditions and design of a custom-built cyclotide database, in which mature cyclotide sequences are excised, replicated and appended, marking a new “era” for cyclotide sequencing. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 592–601, 2010.