Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

Abstract

Cyclotides are plant-derived peptides characterized by a ~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 one 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, 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.

Quantitative Estimation of Cyclotide-Induced Bilayer Membrane Disruption by Lipid Extraction with Mesoscopic Simulation

“…The animation at [87] and figure 9 demonstrate the activity decrease due to "hydrophobic patch" mutations: The increasingly polar/hydrophilic transformation of "hydrophobic patch" amino acids still allows for cyclotide oligomerization but leads to a decreased tendency to aggregate into networks: This in turn diminishes the "hydrophobic vicinities" so that the collective lipid extraction abilities are more and more reduced. The importance of the "hydrophobic patch" surface region for a cyclotide's bioactivity has also been emphasized by experimental studies [8,14,19,34] where the activity decrease due to polar/hydrophilic amino acid replacements/modifications is in agreement with experimental findings.…”

“…However, a collective interaction of multiple cyclotides is able to distort up to disrupt the bilayer structure. Different modes of action depending on the cyclotide/membrane system are to be expected: These comprise cyclotide membrane binding [5, 6, 8-10, 12, 14, 15, 17-19, 21, 23-25, 36] and intrusion [27,31,32,34] as well as cell penetration [6,21,27,32,30] up to the final ability to disrupt a membrane by a postulated formation of pores [3, 5-7, 9, 11, 13, 14, 16, 17, 19-24, 26, 29, 31, 35-38] or by distinct lipid extraction [17,35,37,38].…”

Cyclotides Isolated From Violet Plants of Cameroon Are Inhibitors of Human Prolyl Oligopeptidase

“…in Humbert (Niyomploy et al, 2018), only to mention a few examples. In addition, cyclotides from Violaceae have been investigated in various biological assays and several bioactivities have been reported to date, such as antimicrobial (Tam et al, 2015), anti-tumor (Lindholm et al, 2002;Du et al, 2020), immunosuppressive (Grundemann et al, 2013) as well as protease inhibition (Hellinger et al, 2015a). Since proteases are interesting drug targets and many cystineknot peptides have anti-protease activity, we were intrigued by studying Allexis cyclotides as protease inhibitors (Hellinger and Gruber, 2019).…”

“…Indeed, this plant family is one of the prototypic cyclotide expressing plants with many positively confirmed species, e.g. V. tricolor ( Hellinger et al, 2015b ), V. odorata ( Colgrave et al, 2010 ), H. enneaspermus ( Du et al, 2020 ) or Rinorea bengalensis (Wall.) Gagnep.…”

“…in Humbert ( Niyomploy et al, 2018 ), only to mention a few examples. In addition, cyclotides from Violaceae have been investigated in various biological assays and several bioactivities have been reported to date, such as antimicrobial ( Tam et al, 2015 ), anti-tumor ( Lindholm et al, 2002 ; Du et al, 2020 ), immunosuppressive ( Grundemann et al, 2013 ) as well as protease inhibition ( Hellinger et al, 2015a ).…”