Discovery, isolation and structural characterization of cyclotides from Viola sumatrana miq

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

The role of disulfide bonds in structure and activity of chlorotoxin

Background: Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxin's chemical structure-four disulfide bonds-to its tertiary structure and biological function. Results: Five disulfide bond analogs of chlorotoxin were synthesized, with l-a-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. Conclusion: The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration

From logical forms to SPARQL query with GETARUNS

We present a system for Question Answering which computes a prospective answer from Logical Forms produced by a full-fledged NLP for text understanding, and then maps the result onto schemata in SPARQL to be used for accessing the Semantic Web. As an intermediate step, and whenever there are complex concepts to be mapped, the system looks for a corresponding amalgam in YAGO classes. It is just by the internal structure of the Logical Form that we are able to produce a suitable and meaningful context for concept disambiguation. Logical Forms are the final output of a complex system for text understanding - GETARUNS - which can deal with different levels of syntactic and semantic ambiguity in the generation of a final structure, by accessing computational lexical equipped with sub-categorization frames and appropriate selectional restrictions applied to the attachment of complements and adjuncts. The system also produces pronominal binding and instantiates the implicit arguments, if needed, in order to complete the required Predicate Argument structure which is licensed by the semantic component

Discovery and characterization of cyclic and acyclic trypsin inhibitors from Momordica dioica

Momordica trypsin 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 Momordica subfamily. 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

Isolation and characterization of cyclotides from Brazilian Psychotria: significance in plant defense and co-occurrence with antioxidant alkaloids

Plants from the genus Psychotria include 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 leiocarpa and P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpa and P. brachyceras were 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. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. 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 Psychotria alkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpa as a cyclotide-bearing species, and the absence of these peptides in P. umbellata

Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold

Oral activity has been described for cyclotide-containing traditional medicines, and demonstrated for reengineered cyclotides bearing grafted therapeutic epitopes, highlighting their potential for translation to the clinic. Here we report preclinical pharmacokinetic parameters for the prototypic cyclotide kalata B1 (kB1) and two orally active grafted analogues, ckb-KAL and ckb-KIN, to provide the first in vivo dose-exposure metrics for cyclotides. Native and grafted kB1 molecules exhibited multiple compartment kinetics and measurable but limited oral bioavailability of similar magnitude to several orally administered peptide drugs in the clinic. Cyclotides are mostly associated with the central compartment, and display small (0.07-0.13 L kg for kB1 and ckb-KIN) to moderate (1 L kg for ckb-KAL) steady state volumes of distribution. This study provides new data essential to the evaluation of cyclotides as therapeutics, validating them as a viable drug design scaffold with tunable pharmacokinetic properties

Discovery and characterization of cyclotides from Rinorea species

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

The evolution of Momordica cyclic peptides

Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases

Insecticidal diversity of butterfly pea (Clitoria ternatea) accessions

Butterfly pea (Clitoria ternatea) is currently the only leguminous plant species known to produce a suite of ultrastable cyclic plant defense peptides called cyclotides. For agricultural applications, cyclotides have attracted significant interest, leading to the recent registration of a butterfly pea extract as an ecofriendly pesticide (Sero-X®). In this study, we set out to distinguish the variation in cyclotide expression and toxicity towards insect cells for butterfly pea accessions sourced worldwide. In characterizing the peptide extracts from 23 butterfly pea accessions sourced from 11 countries, we show that significant variation in cyclotide expression exists between them. For some accessions, the cyclotide Cter M, typically the most abundantly expressed cyclotide in vegetative butterfly pea tissues, is absent. Genomic and transcriptomic sequencing revealed the presence of CterM-like precursor genes in these accessions that contained missense mutations that were likely contributing to the lack of Cter M expression. Peptide profiling also showed that one accession does not produce detectable levels of other cyclotides: cliotide T1, cliotide T4, Cter A and Cter Q. A comparison of cytotoxicity against Sf9 (Spodoptera frugiperda) cells revealed that cytotoxicity is not dependent on Cter M, with accessions lacking this peptide also displaying cytotoxicity. Overall, insights from this study provides foundational knowledge about characters to be considered for selective breeding of butterfly pea with enhanced insecticidal properties