Cycloviolacin O16

Hookworm infection is a leading cause of maternal and child morbidity in countries of the tropics and subtropics, as well as being an important parasite in companion-animal medicine. The cyclotides are a novel family of cyclic cystine knot containing peptides from plants that have been shown to possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes of sheep. In the current study we demonstrated the in vitro effects of three representative cyclotides, kalata B1, kalata B6 and cycloviolacin O14, on the viability of larval and adult life stages of the dog hookworm Ancylostoma caninum, and larvae of the human hookworm Necator americanus. The cyclotides showed significant anthelmintic activity towards both hookworm species. The different cyclotides showed similar patterns of relative activity as that seen previously with the livestock nematode species. This study demonstrates that cyclotides have promising activity in vitro against important parasites of companion animals and humans.

The cyclotides are a novel family of backbone-cyclized cystine-knot containing peptides from plants that have been shown to possess insecticidal activity against Helicoverpa larvae, an important pest of corn and cotton. In the current study, we investigated the in vitro effects of the cyclotides on the viability of egg, larval, and adult life stages of two species of economically important gastrointestinal nematode parasites of livestock, Hemonchus contortus and Trichostrongylus colubriformis. The cyclotides showed significant activity in inhibiting development of nematode larvae and motility of adult worms. Activities were comparable to some currently used anthelmintic compounds in these in vitro assay systems. A series of alanine mutants of the prototypic cyclotide kalata B1 were assayed against larvae to determine regions of the peptide responsible for activity. It was observed that anthelmintic activity was dramatically reduced as a consequence of the mutation of a large number of residues that are found clustered on one surface. Activities toward larvae were equivalent in the naturally occurring L-isomer of kalata B1 and a synthetic all-D-isomer, indicating that there is no chiral requirement for anthelmintic activity. The clustering of important residues and the lack of chiral selectivity further support the proposed mode of action of the cyclotides, which involves a membrane-based interaction rather than an interaction at a specific receptor. The cyclotide-induced leakage of a fluorescent dye from vesicles used as a model membrane mimetic further confirms the membrane lytic ability of cyclotides. The relative potency of kalata B1 and kalata B2 in causing membrane leakage is consistent with the order of their anthelmintic activity. These results demonstrate that the cyclotides show potential for use in the control of gastrointestinal nematode parasites.

Cycloviolacin H4, a new macrocyclic miniprotein comprising 30 amino acid residues, was isolated from the underground parts of the Australian native violet Viola hederaceae. Its sequence, cyclo-(CAESCVWIPCTVTALLGCSCSNNVCYNGIP), was determined by nanospray tandem mass spectrometry and quantitative amino acid analysis. A knotted disulfide arrangement, which was designated as a cyclic cystine knot motif and characteristic to all known cyclotides, is proposed for stabilizing the molecular structure and folding. The cyclotide is classified in the bracelet subfamily of cyclotides due to the absence of a cis-Pro peptide bond in the circular peptide backbone. A model of its three-dimensional structure was derived based on the template of the homologous cyclotide vhr1 (Trabi et al. Plant Cell 2004, 16, 2204-2216). Cycloviolacin H4 exhibits the most potent hemolytic activity in cyclotides reported so far, and this activity correlates with the size of a surface-exposed hydrophobic patch. This work has thus provided insight into the factors that modulate the cytotoxic properties of cyclotides.