The cyclotides are stable plant-derived mini-proteins with a topologically circular peptide backbone and a knotted arrangement of three disulfide bonds that form a cyclic cystine knot structural framework. They display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. To determine the influence of individual residues on structure and activity in the prototypic cyclotide kalata B1, all 23 non-cysteine residues were successively replaced with alanine. The structure was generally tolerant of modification, indicating that the framework is a viable candidate for the stabilization of bioactive peptide epitopes. Remarkably, insecticidal and hemolytic activities were both dependent on a common, well defined cluster of hydrophilic residues on one face of the cyclotide. Interestingly, this cluster is separate from the membrane binding face of the cyclotides. Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities.Cyclotides are a fascinating family of plant-derived mini-proteins of around 30 residues that are characterized by the topologically unique combination of a head-to-tail cyclized backbone and a cystine knot. The original discovery of the prototypic member kalata B1 (1) was guided by the indigenous medicinal use in Africa of a tea made from the plant Oldenlandia affinis (Rubiaceae) to accelerate childbirth. Kalata B1 was identified as a uterotonic component of the plant, but its unusual macrocyclic nature (2) and the fact that it was just one member of a large family of related macrocyclic peptides were elucidated only recently (3). Cyclotides have now been reported in several other species of the Rubiaceae (coffee) family (4, 5) and in all tested members of the Violaceae (violet) plant family (3, 6, 7) bringing the number of known cyclotides to over 100.

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.

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.

Featuring a circular, knotted structure and diverse bioactivities, cyclotides are a fascinating family of peptides that have inspired applications in drug design. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anti-cancer, anti-HIV, and hemolytic activities. In all of these activities, cell membranes appear to play an important role. However, the question of whether the activity of cyclotides depends on the recognition of chiral receptors or is primarily modulated by the lipid-bilayer environment has remained unknown. To determine the importance of lipid membranes on the activity of the prototypic cyclotide, kalata B1, we synthesized its all-D enantiomer and assessed its bioactivities. After the all-D enantiomer had been confirmed by (1)H NMR to be the structural mirror image of the native kalata B1, it was tested for anti-HIV activity, cytotoxicity, and hemolytic properties. The all-D peptide is active in these assays, albeit with less efficiency; this reveals that kalata B1 does not require chiral recognition to be active. The lower activity than the native peptide correlates with a lower affinity for phospholipid bilayers in model membranes. These results exclude a chiral receptor mechanism and support the idea that interaction with phospholipid membranes plays a role in the activity of kalata B1. In addition, studies with mixtures of L and D enantiomers of kalata B1 suggested that biological activity depends on peptide oligomerization at the membrane surface, which determines affinity for membranes by modulating the association-dissociation equilibrium.

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The cyclotides are a family of head-to-tail cyclized peptides that display exceptionally high stability and a range of biological activities. Acyclic permutants that contain a break in the circular backbone have been reported to be devoid of the haemolytic activity of the prototypic cyclotide kalata B1, but the potential role of the charges at the introduced termini in this loss of membraneolytic activity has not been fully determined. In this study, acyclic permutants of kalata B1 with capped N-and Ctermini were synthesized and found to adopt a native fold. These variants were observed to cause no measurable lysis of erythrocytes, strengthening the connection between backbone cyclization and haemolytic activity.

The cover picture shows mirror image forms of the cyclotide kalata B1, a 29-residue peptide with a cyclic backbone and a cystine knot. The native peptide is isolated from the African plant Oldenlandia affinis, which is used in an indigenous medicinal tea to accelerate labour, but has a range of other activities, including anti-HIV activity. The synthetic mirror image form, made from d-amino acids, was assembled by solid-phase peptide synthesis and used to demonstrate that membrane binding, rather than a chiral receptor, is implicated in cyclotide bioactivity.

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