The Anthelmintic Activity of the Cyclotides: Natural Variants with Enhanced Activity

Abstract

The cyclotides are a family of backbone-cyclised cystine-knot-containing peptides from plants that possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematode parasites of sheep. In the current study, we investigated the in vitro effects of newly discovered natural cyclotides on the viability of larval and adult life stages of these pests. The natural variants cycloviolacin O2, cycloviolacin O3, cycloviolacin O8, cycloviolacin O13, cycloviolacin O14, cycloviolacin O15, and cycloviolacin O16 extracted from Viola odorata showed up to 18-fold greater potency than the prototypic cyclotide kalata B1 in nematode larval development assays. Cycloviolacin O2 and cycloviolacin O14 were significantly more potent than kalata B1 in adult H. contortus motility assays. The lysine and glutamic acid residues of cycloviolacin O2, the most potent anthelmintic cyclotide, were chemically modified to investigate the role of these charged residues in modulating the biological activity. The single glutamic acid residue, which is conserved across all known cyclotides, was shown to be essential for activity, with a sixfold decrease in potency of cycloviolacin O2 following methylation. The three lysine residues present in cycloviolacin O2 were acetylated to effectively mask the positive charge, resulting in a 18-fold decrease in anthelmintic activity. The relative anthelmintic activities of the natural variants assayed against nematode larvae correlated with the number of charged residues present in their sequence.

Lysine-scanning Mutagenesis Reveals an Amendable Face of the Cyclotide Kalata B1 for the Optimization of Nematocidal Activity

“…Kalata B1, B2, B6, and B7, cyclotides extracted from O. affinis, significantly inhibited the development of the larval life stages of two sheep gastrointestinal nematodes Haemonchus contortus (H. contortus) and Trichostrongylus colubriformis, as well as inhibiting motility of adult H. contortus. A subsequent study reported enhanced nematocidal activity of other natural cyclotides possessing multiple basic residues (31). Cycloviolacin O2 (containing three basic residues) was found to be the most potent cyclotide tested against sheep parasites.…”

Cyclotide Interactions with the Nematode External Surface

“…Recently, we demonstrated that cyclotides isolated from the plant Oldenlandia affinis display anthelminthic activity toward the gastrointestinal parasitic nematodes Haemonchus contortus and Trichostrongylus colubriformis (8). Subsequent studies found that cycloviolacins (a group of cyclotides isolated from Viola odorata) showed up to an 18-foldincreased potency against these nematodes relative to the prototypic cyclotide, kB1 (9). More recently, selected cyclotides have been shown to exert a toxic effect on canine and human hookworms (10).…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…[16][17][18][19] A presumed role in plant defence is also supported by recent reports of their molluscidal activity against the Golden Apple snail, a major pest of rice in South East Asia, [20] and also by their anthelmintic activities. [21,22] More broadly, their unique structural features, stability and diverse bioactivities suggest that cyclotides might be promising templates for drug design and agrochemical applications. [23,24] The term 'cyclotides' was introduced in 1999 [4] to describe the few dozen macrocyclic peptides from plants characterized at that time, but since then the number of known cyclotides has increased to more than 150.…”