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Cyclotides are a class of ribosomally-synthesized plant peptides that function in plants as a defense against insects and fungal pathogens. Their unique structure comprises a cyclized peptide backbone threaded by three disulfide bonds, that imparts structural stability, a desirable quality for peptide-based therapeutics or insecticides. Producing these peptides synthetically is challenging due to the amount of chemical waste produced and inefficiency of folding certain cyclotides. Thus, it is desirable to develop a means to access cyclotide biosynthesis in their native hosts, cultured in defined conditions, at both laboratory and commercial scale. Here we developed suspension cell cultures from two species previously unexplored for cyclotide production in suspension cells, Clitoria ternatea L., Hybanthus enneaspermus F. Muell., as well as with Oldenlandia affinis (Roem. & Schult.) DC., a species reported previously to accumulate cyclotides in cell suspensions. We assessed the growth rate, cyclotide production and gene expression for the various species. We found that while many cyclotides had reduced expression in Oldenlandia affinis suspension cells when compared to plant organs, those in Clitoria ternatea and Hybanthus enneaspermus maintained or increased expression levels. The cyclotides that continued to be expressed in suspension cultures shared similar sequence and biophysical properties as a group, regardless of phylogenetic origin of the host. Of particular interest was the discovery of inducibility by NaCl of cyclotide expression in O. affinis, cycloviolacin O2 expression in O. affinis, and the scale up of cycloviolacin O2 production in H. enneaspermus. Together the results presented here highlight the utility of plant cell suspensions as modalities to produce macrocyclic peptides., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Angiogenesis is important for tumor growth, and accordingly, targeting angiogenesis has become an important pathway for antitumor therapy. A novel proapoptotic peptide, CIGB-300 (P15-Tat), has been shown to be involved in the casein kinase II phosphorylation pathway, conferring it with antiangiogenic activity. Cyclic peptides have been widely used as scaffolds in drug design studies due to their high stability and favorable biopharmaceutical properties. Here, we chose two very stable cyclic trypsin inhibitors, MCoTI-II and SFTI-1, as frameworks to incorporate the bioactive epitope P15 into various backbone loops. NMR studies revealed that all re-engineered analogs had similar secondary structures to their native cyclic frameworks. One key analog, MCoP15, displayed significant improvement for inhibiting human umbilical vein endothelial cell migration, was nontoxic, and had higher stability than the P15 epitope alone. Overall, the results show the value of P15 being engineered into cyclic trypsin inhibitor scaffolds for improving antiangiogenic activity and stability. More broadly, the study highlights the versatility of cyclic peptide frameworks in drug design for antiangiogenic therapies.

Cyclotides are plant-derived peptides found within five families of flowering plants (Violaceae, Rubiaceae, Fabaceae, Solanaceae, and Poaceae) that have a cyclic backbone and six conserved cysteine residues linked by disulfide bonds. Their presence within the Violaceae species seems ubiquitous, yet not all members of other families produce these macrocyclic peptides. The genus Palicourea Aubl. (Rubiaceae) contains hundreds of neotropical species of shrubs and small trees; however, only a few cyclotides have been discovered hitherto. Herein, five previously uncharacterized Möbius cyclotides within Palicourea sessilis and their pharmacological activities are described. Cyclotides were isolated from leaves and stems of this plant and identified as pase A - E, as well as the known peptide kalata S. Cyclotides were de novo sequenced by MALDI-TOF/TOF mass spectrometry, and their structures were solved by NMR spectroscopy. Because some cyclotides have been reported to modulate immune cells, pase A - D were assayed for cell proliferation of human primary activated T lymphocytes, and the results showed a dose-dependent antiproliferative function. The toxicity on other nonimmune cells was also assessed. This study reveals that pase cyclotides have potential for applications as immunosuppressants and in immune-related disorders.

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.

Peptide analogues derived from bioactive hormones such as somatostatin or certain growth factors have great potential as angiogenesis inhibitors for cancer applications. In an attempt to combat emerging drug resistance many FDA-approved anti-angiogenesis therapies are co-administered with cytotoxic drugs as a combination therapy to target multiple signaling pathways of cancers. However, cancer therapies often encounter limiting factors such as high toxicities and side effects. Here, we combined two anti-angiogenic epitopes that act on different pathways of angiogenesis into a single non-toxic cyclic peptide framework, namely MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II), and subsequently assessed the anti-angiogenic activity of the novel compound. We hypothesized that the combination of these two epitopes would elicit a synergistic effect by targeting different angiogenesis pathways and result in improved potency, compared to that of a single epitope. This novel approach has resulted in the development of a potent, non-toxic, stable and cyclic analogue with nanomolar potency inhibition in in vitro endothelial cell migration and in vivo chorioallantoic membrane angiogenesis assays. This is the first report to use the MCoTI-II framework to develop a 2-in-1 anti-angiogenic peptide, which has the potential to be used as a form of combination therapy for targeting a wide range of cancers.

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.

Cyclotides are a large family of plant peptides characterized by a macrocyclic backbone and knotted arrangement of three disulfide bonds. This unique structure renders cyclotides exceptionally stable to thermal, chemical and enzymatic treatments. They exhibit a variety of bioactivities, including uterotonic, anti-HIV, cytotoxic and hemolytic activity and it is these properties that make cyclotides an interesting peptide scaffold for drug design. In this study, eight new cyclotides (Viphi A-H), along with eight known cyclotides, were isolated from Viola philippica, a plant from the Violaceae family. In addition, Viba 17 and Mram 8 were isolated for the first time as peptides. The sequences of these cyclotides were elucidated primarily by using a strategy involving reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Several of the cyclotides showed cytotoxic activities against the cancer cell lines MM96L, HeLa and BGC-823. The novel cyclotides reported here: (1) enhance the known sequence variation observed for cyclotides; (2) extend the number of species known to contain cyclotides; (3) provide interesting structure-activity relationships that delineate residues important for cytotoxic activity. In addition, this study provides insights into the potential active ingredients of traditional Chinese medicines., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Cyclotides are cyclic peptides from plants in the Violaceae, Rubiaceae, Fabaceae, Cucurbitaceae, and Solanaceae families. They are sparsely distributed in most of these families, but appear to be ubiquitous in the Violaceae, having been found in every plant so far screened from this family. However, not all geographic regions have been examined and here we report the discovery of cyclotides from a Viola species from South-East Asia. Two novel cyclotides (Visu 1 and Visu 2) and two known cyclotides (kalata S and kalata B1) were identified in V. sumatrana. NMR studies revealed that kalata S and kalata B1 had similar secondary structures. Their biological activities were determined in cytotoxicity assays; both had similar cytotoxic activity and were more toxic to U87 cells compared with other cell lines. Overall, the study strongly supports the ubiquity of cyclotides in the Violaceae and adds to our understanding of their distribution and cytotoxic activity. [ABSTRACT FROM AUTHOR]