Abstract
Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.
Highlights
Cyclotides are a large family of cysteine-rich peptides, ;30 amino acids in size, derived from plants [1]
A total of 12 cyclotides were isolated from the leaves, stems, and seeds of H. enneaspermus
Hyen C was the only Möbius cyclotide isolated from H. enneaspermus
Summary
Cyclotides are a large family of cysteine-rich peptides, ;30 amino acids in size, derived from plants [1]. Cyclotides are characterized by a head-to-tail cyclic backbone and a cystine knot motif, which renders them ultrastable against degradation by harsh conditions and are amenable to residue substitutions [16,17,18] These properties make cyclotides ideal scaffolds for grafting bioactive epitopes [19], with potential applications in the agricultural and pharmaceutical industries. Cyclotides have been identified in five major plant families, namely the Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae, and Fabaceae [20,21,22] Two Möbius cyclotides (varv A and varv F), and a bracelet cyclotide (cycloviolacin O2, cyO2), have been identified as having strong
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