Cycloviolacin O37 from Viola odorata is a Cytotoxic Bracelet Cyclotide with Unique Structural Features

Abstract Cyclotides are plant-derived cyclic peptides characterized by a head-to-tail cyclized backbone and a cystine knot motif that confers exceptional stability and diverse biological activities. Here we report the discovery, structural characterization, and biological evaluation of cycloviolacin O37 (CyO37) from Viola odorata. CyO37 was isolated from leaf extracts and identified by mass spectrometry. Sequence elucidation using LC−MS/MS and transcriptomic data confirmed that CyO37 belongs to the bracelet subfamily, but exhibits unique sequence motifs in loops 2, 3, and 5, and an overall charge of +4, distinguishing it from currently reported cyclotides. Comprehensive NMR analysis demonstrated that the CyO37 sample studied is a mixture of the R- and S-methionine-sulfoxide diastereomers arising from methionine oxidation, representing the first detailed structural characterization of this modification in a cyclotide. High-resolution solution NMR structures showed that the two diastereomers adopt similar global folds but differ in local hydrogen-bonding patterns involving the sulfoxide group. Biological evaluation indicated that CyO37 displays moderate cytotoxicity against K562 leukemia cells but lacks detectable antibacterial activity. These findings correlate with reduced membrane-disruptive properties compared with other cyclotides. The discovery of CyO37 highlights underexplored structural diversity within cyclotides and suggests the existence of a broader family of higher-mass variants in V. odorata, warranting further investigation.