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Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from Carapichea ipecacuanha to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low μM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A 1-13 or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.

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.

Plant peptide protease inhibitors are important molecules in seed storage metabolism and to fight insect pests. Commonly they contain multiple disulfide bonds and are exceptionally stable molecules. In this study, a novel peptide protease inhibitor from beetroot ( Beta vulgaris ) termed bevuTI-I was isolated, and its primary structure was determined via mass spectrometry-based amino acid sequencing. By sequence homology analysis a few peptides with high similarity to bevuTI-I, also known as the Mirabilis jalapa trypsin inhibitor subfamily of knottin-type protease inhibitors, were discovered. Hence, we assessed bevuTI-I for inhibitory activity toward trypsin (IC 50 = 471 nM) and human prolyl oligopeptidase (IC 50 = 11 μM), which is an emerging drug target for neurodegenerative and inflammatory disorders. Interestingly, using a customized bioinformatics approach, bevuTI-I was found to be the missing link to annotate 243 novel sequences of M. jalapa trypsin inhibitor-like peptides. According to their phylogenetic distribution they appear to be common in several plant families. Therefore, the presented approach and our results may help to discover and classify other plant-derived cystine knot peptides, a class of plant molecules that play important functions in plant physiology and are currently being explored as lead molecules and scaffolds in drug development.

Two new bracelet cyclotides from roots of Pombalia calceolaria with potential anticancer activity have been characterized in this work. The cyclotides Poca A and B (1 and 2) and the previously known CyO4 (3) were de novo sequenced by MALDI-TOF/TOF mass spectrometry (MS). The MS 2 spectra were examined and the amino acid sequences were determined. The purified peptides were tested for their cytotoxicity and effects on cell migration of MDA-MB-231, a triple-negative breast cancer cell line. The isolated cyclotides reduced the number of cancer cells by more than 80% at 20 μM, and the concentration-related cytotoxic responses were observed with IC 50 values of 1.8, 2.7, and 9.8 μM for Poca A (1), Poca B (2), and CyO4 (3), respectively. Additionally, the inhibition of cell migration (wound-healing assay) exhibited that CyO4 (3) presents an interesting activity profile, in being able to inhibit cell migration (50%) at a subtoxic concentration (2 μM). The distribution of these cyclotides in the roots was analyzed by MALDI imaging, demonstrating that all three compounds are present in the phloem and cortical parenchyma regions.

Cyclotides are head-to-tail cyclized peptides comprising a stabilizing cystine-knot motif. To date, they are well known for their diverse bioactivities such as anti-HIV and immunosuppressive properties. Yet little is known about specific molecular mechanisms, in particular the interaction of cyclotides with cellular protein targets. Native and synthetic cyclotide-like peptides from Momordica plants are potent and selective inhibitors of different serine-type proteinases such as trypsin, chymotrypsin, matriptase, and tryptase-beta. This study describes the bioactivity-guided isolation of a cyclotide from Psychotria solitudinum as an inhibitor of another serine-type protease, namely, the human prolyl oligopeptidase (POP). Analysis of the inhibitory potency of Psychotria extracts and subsequent fractionation by liquid chromatography yielded the isolated peptide psysol 2 (1), which exhibited an IC50 of 25 μM. In addition the prototypical cyclotide kalata B1 inhibited POP activity with an IC50 of 5.6 μM. The inhibitory activity appeared to be selective for POP, since neither psysol 2 nor kalata B1 were able to inhibit the proteolytic activity of trypsin or chymotrypsin. The enzyme POP is well known for its role in memory and learning processes, and it is currently being considered as a promising therapeutic target for the cognitive deficits associated with several psychiatric and neurodegenerative diseases, such as schizophrenia and Parkinson's disease. In the context of discovery and development of POP inhibitors with beneficial ADME properties, cyclotides may be suitable starting points considering their stability in biological fluids and possible oral bioavailability.

A new orbitide named ribifolin was isolated and characterized from Jatropha ribifolia using mass spectrometry, NMR spectroscopy, quantitative amino acid analysis, molecular dynamics/simulated annealing, and Raman optical activity measurements and calculations. Ribifolin (1) and its linear form (1a) were synthesized by solid-phase peptide synthesis, followed by evaluation of its antiplasmodial and cytotoxicity activities. Compound 1 was moderately effective (IC50 = 42 μM) against the Plasmodium falciparum strain 3D7.

Cyclotides are an abundant and diverse group of ribosomally synthesized plant peptides containing a cyclic cystine-knotted structure that confers them with remarkable stability. They are explored for their distribution in plants, although little is known about the individual peptide content of a single species. Therefore, we chemically analyzed the crude extract of the coffee-family plant Oldenlandia affinis using a rapid peptidomics workflow utilizing nano-LC-MS, peptide reconstruct with database identification, and MS/MS automated sequence analysis to determine its cyclotide content. Biologically, cyclotides are mainly explored for applications in agriculture and drug design; here we report their growth-inhibiting effects on primary cells of the human immune system using biological and immunological end points in cell-based test systems. LC-MS quantification of the active O. affinis plant extract triggered the characterization of the antiproliferative activity of kalata B1, one of the most abundant cyclotides in this extract, on primary activated human lymphocytes. The effect has a defined concentration range and was not due to cytotoxicity, thus opening a new avenue to utilize native and synthetically optimized plant cyclotides for applications in immune-related disorders and as immunosuppressant peptides.