Isolation, sequencing, and structure−activity relationships of cyclotides

Cyclotides are a topologically fascinating family of miniproteins discovered over the past decade that have expanded the diversity of plant-derived natural products. They are approximately 30 amino acids in size and occur in plants of the Violaceae, Rubiaceae, and Cucurbitaceae families. Despite their proteinaceous composition, cyclotides behave in much the same way as many nonpeptidic natural products in that they are resistant to degradation by enzymes or heat and can be extracted from plants using methanol. Their stability arises, in large part, due to their characteristic cyclic cystine knot (CCK) structural motif. Cystine knots are present in a variety of proteins of insect, plant, and animal origin, comprising a ring formed by two disulfide bonds and their connecting backbone segments that is threaded by a third disulfide bond. In cyclotides, the cystine knot is uniquely embedded within a head-to-tail cyclized peptide backbone, leading to the ultrastable CCK structural motif. Apart from the six absolutely conserved cysteine residues, the majority of amino acids in the six backbone loops of cyclotides are tolerant to variation. It has been predicted that the family might include up to 50 000 members; although, so far, sequences for only 140 have been reported. Cyclotides exhibit a variety of biological activities, including insecticidal, nematocidal, molluscicidal, antimicrobial, antibarnacle, anti-HIV, and antitumor activities. Due to their diverse activities and common structural core from which variable loops protrude, cyclotides can be thought of as combinatorial peptide templates capable of displaying a variety of amino acid sequences. They have thus attracted interest in drug design as well as in crop protection applications