Abstract:Circular peptides have attracted much interest in recent drug development efforts, particularly due to their increased stability over linear counterparts. The family of plant cyclotides represents one of the largest classes of naturally-occurring backbone-cyclized peptides displaying exceptional sequence variability and plasticity around three knotted disulfide bonds. Accordingly, a multitude of pharmaceutically as well as agrochemically relevant bioactivities has been ascribed to them. Their abundance across … Show more
“…Cyclotides have been isolated and characterized from different flowering plant species of the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families [summarized in ( Hellinger et al, 2015b ; Koehbach and Clark, 2016 )]. Inspired by their original discovery from Oldenlandia affinis , the Rubiaceae family, has been a major focus for studying the diversity and distribution of cyclotides ( Gruber et al, 2008 ; Gruber, 2010 ; Koehbach et al, 2013a ).…”
Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants’ uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules.
“…Cyclotides have been isolated and characterized from different flowering plant species of the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families [summarized in ( Hellinger et al, 2015b ; Koehbach and Clark, 2016 )]. Inspired by their original discovery from Oldenlandia affinis , the Rubiaceae family, has been a major focus for studying the diversity and distribution of cyclotides ( Gruber et al, 2008 ; Gruber, 2010 ; Koehbach et al, 2013a ).…”
Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants’ uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac (Carapichea ipecacuanha) for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRF1R). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRF1R. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V2 receptor, suggesting a CRF1R-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules.
“…Based on the peptidomics analysis the expression of cyclotides was conclusively confirmed in Allexis species (Craik and Malik, 2013;Koehbach and Clark, 2016;White and Craik, 2016). The overall similar peptide expression in four plant species might reflect conserved cyclotide precursor genes within Allexis and cyclotide genes with two or three mature domains.…”
Section: Discussionmentioning
confidence: 77%
“…Cystine-knots are reported for knottins, snakins , α-amylase or potato metalloprotease inhibitors, but these are not backbone cyclic peptides ( Tam et al, 2015 ; Correnti et al, 2018 ; Hellinger and Gruber, 2019 ). Based on the peptidomics analysis the expression of cyclotides was conclusively confirmed in Allexis species ( Craik and Malik, 2013 ; Koehbach and Clark, 2016 ; White and Craik, 2016 ). The overall similar peptide expression in four plant species might reflect conserved cyclotide precursor genes within Allexis and cyclotide genes with two or three mature domains.…”
Traditional medicine and the use of herbal remedies are well established in the African health care system. For instance, Violaceae plants are used for antimicrobial or anti-inflammatory applications in folk medicine. This study describes the phytochemical analysis and bioactivity screening of four species of the violet tribe Allexis found in Cameroon. Allexis cauliflora, Allexis obanensis, Allexis batangae and Allexis zygomorpha were evaluated for the expression of circular peptides (cyclotides) by mass spectrometry. The unique cyclic cystine-rich motif was identified in several peptides of all four species. Knowing that members of this peptide family are protease inhibitors, the plant extracts were evaluated for the inhibition of human prolyl oligopeptidase (POP). Since all four species inhibited POP activity, a bioactivity-guided fractionation approach was performed to isolate peptide inhibitors. These novel cyclotides, alca 1 and alca 2 exhibited IC50 values of 8.5 and 4.4 µM, respectively. To obtain their amino acid sequence information, combinatorial enzymatic proteolysis was performed. The proteolytic fragments were evaluated in MS/MS fragmentation experiments and the full-length amino acid sequences were obtained by de novo annotation of fragment ions. In summary, this study identified inhibitors of the human protease POP, which is a drug target for inflammatory or neurodegenerative disorders.
“…The combined -omics approach resulted in more than half of the transcript sequences being predicted at protein level. Nevertheless, to uncover complete cyclotide diversity,novel structural templates and possible PTMs in a single plant specimen, a combinatorial procedure of transcriptome analysis, LC–MS deconvolution and tandem MS sequencing and transcriptome analysis is essential 75 – 77 . Figure 6 Representative MALDI-TOF mass-spectra of HPLC-purified fraction E from crude extract of leaves of C. ternatea (Refer to Supplementary Fig.…”
Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26-37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea. Macrocyclics such as the cyclotides are a class of cyclic peptides (26-37 residues) containing three disulphide bonds. They are formed by cyclization of a gene encoded, linear precursor in specific plant species. In addition to the circular backbone, they form a cyclic cystine knot (CCK) arrangement formed by a conserved six cysteine framework (Cys I-Cys IV, Cys II-Cys V, Cys III-Cys VI) 1-3. This knotted arrangement along with the circular backbone, renders these cyclotides exceptionally stable against enzymatic, chemical and thermal degradation, with their principal function thought to be in plant defense 4. Thus, cyclotides are potential candidates for peptide-based drugs; either as scaffolds to stabilize s...
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