PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola odorata

Abstract

Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized VIa Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature's complexity.

Keywords: Antimicrobial peptides; Bioactive peptides; Cyclotides; Mass spectometry; Viola odorata, Violaceae.

Figure 1

Cycloviolacin O8 (cyO8), a known anthelmintic cyclotide from Viola odorata. A. CyO8 sequence, MW 3225.42 Da, showing disulfides. B. Predicted three-dimensional cyO8 structure, not showing disulfides. Magenta arrows and cyan domain represent anti-paralled β-sheets and a 3₁₀ α-helix, respectively (cybase.org (Mulvenna et al., 2006)); figure generated in Pymol).

Figure 2

Viola odorata SCX fraction library versus Fusarium graminearum bioactivity assay. Data are represented as mean ± SD of three replicates. Antifungal bioactivity is seen in V. odorata fractions 25–31.

Figure 3

Inhibitory concentration (IC₅₀) determination of cyO8 against human cancer cell lines. Purified cyO8 tested against human cancer cell lines, OVCAR-3 ovarian (A), PC-3 prostate (B), and MDA-MB-231 breast (C), and against non-cancerous human dermal fibroblasts (D). Vehicle indicates cells are cultured with water in place of cyO8. Data are represented as mean ± SD of three replicates.

Figure 4

Collision induced dissociation (CID) fragmentation of cyO8. CID MS² spectra of reduced, alkylated, gluC digested cyO8 (+4) (top) and reduced, alkylated, gluC digested cyclotide mass 3257.37 Da (+4) (bottom) showing 32 Da double-oxidation mass shift localized to the tryptophan residue of cyO8_nfk (cyO8 N-formylkynurenine). Fragment ions are colored the same in both spectra to aid in quick identification.

Figure 5

Ultraviolet Photodissociation (UVPD) fragmentation of cyO8. A. MS/MS of (+4, m/z 894.39) cyO8 (2 pulses, 2 mJ/pulse) shows abundant, low intensity fragment ions. Fragments are labeled by the fragment ion type and the numeric index (in parentheses) from the sequence map shown indicating the initial cleavage site (e.g. a15(13) indicates an initial cleavage between residues 12 and 13 and a second fragmentation event resulting in an a15 fragment ion). B. Representative cyO8 sequence iterations showing UVPD MS² coverage. Ion types are color-coded based on the number of ambiguous fragment ion matches.