Recombinant Viola odorata Cycloviolacin-O20

What are the structural characteristics of cycloviolacins from Viola odorata?

Cycloviolacins belong to the cyclotide family, which are small cyclic peptides containing approximately 30 amino acids with a characteristic knotted arrangement of three disulfide bonds (called a cyclic cystine knot). These peptides from Viola odorata feature a highly conserved structural motif with six cysteine residues forming three disulfide bridges, creating an exceptionally stable structure resistant to thermal, chemical, and enzymatic degradation. The cycloviolacins specifically belong to the "bracelet" subfamily of cyclotides, distinguished by their backbone topology from the "Möbius" subfamily. Their structure includes several charged residues (Glu, Lys, Arg) that are critical for their biological activity .

How can cyclotides be efficiently identified and characterized in complex plant extracts?

The PepSAVI-MS (Statistically-guided bioActive Peptides prioritized Via Mass Spectrometry) pipeline offers an adaptable method for rapid identification of bioactive peptides in complex natural product libraries. The process involves:

• Fractionation of plant extracts using strong cation exchange (SCX) chromatography

• Bioactivity screening of fractions against relevant targets

• Mass spectrometry analysis of active fractions

• Statistical correlation of mass spectral features with bioactivity data

• Reduction and alkylation of disulfide bonds (creating mass shifts of 348.16 ± 0.05 Da for cyclotides)

• MS/MS sequencing for peptide identification

This approach has been validated for identifying bioactive cyclotides from Viola odorata, including cycloviolacin O2 and cycloviolacin O8 .

What post-translational modifications are commonly observed in Viola odorata cyclotides?

Cyclotides from Viola odorata can undergo several post-translational modifications that affect their structure and potentially their activity. Common modifications include:

These modifications can be identified through mass spectrometric analysis, with characteristic mass shifts providing clues to the specific modifications present .

What are the most effective methods for isolating cycloviolacins from Viola odorata plant material?

The isolation of cycloviolacins from Viola odorata typically follows a multi-step process designed to maintain peptide integrity while achieving high purity:

• Plant material preparation: Collection of plant parts (aerial parts typically contain different cyclotide profiles compared to underground parts)

• Extraction: Maceration in organic solvents (typically methanol/dichloromethane)

• Initial fractionation: Solid-phase extraction (C18) to remove large proteins and hydrophilic compounds

• Chromatographic separation:

  • Strong cation exchange (SCX) chromatography for initial fractionation

  • Reversed-phase HPLC for final purification

For analytical identification, a combination of liquid chromatography with mass spectrometry (LC-MS) provides the most precise characterization. The differential expression patterns between aerial and underground parts should be considered when targeting specific cyclotides .

What are the current challenges in developing recombinant expression systems for cycloviolacins?

Recombinant production of cycloviolacins presents several challenges:

• Post-translational processing: Achieving correct disulfide bond formation and head-to-tail cyclization requires specialized cellular machinery often absent in common expression systems.

• Toxicity to host cells: The membrane-active properties of cyclotides can disrupt host cell membranes, limiting production yields.

• Folding complexity: The knotted disulfide arrangement creates kinetic folding traps that can lead to misfolded products.

• Expression construct design: Engineering efficient systems requires careful consideration of:

  • Fusion partners to prevent toxicity

  • Cleavage sites for precision excision

  • Cyclization domains for head-to-tail closure

  • Oxidative environments for disulfide formation

• Purification challenges: Separating correctly folded cyclotides from misfolded species requires sophisticated analytical techniques.

Current approaches focus on using specialized strains with enhanced disulfide isomerase activity, controlled induction systems to minimize toxicity, and fusion protein strategies that can be enzymatically processed post-expression.

How does the antimicrobial activity of cycloviolacin O2 compare with other cyclotides from Viola odorata?

Cycloviolacin O2 (cyO2) demonstrates superior antimicrobial activity compared to other cyclotides from Viola odorata, particularly against Gram-negative bacteria. Comparative studies revealed:

CyO2's selective activity against Gram-negative bacteria is attributed to its unique arrangement of charged residues. Experimental modification of these charged residues demonstrated their critical importance: masking of Glu and Lys residues caused almost complete loss of activity, while Arg modification had a less pronounced but still significant effect .

What is the mechanism of cycloviolacin's anticancer activity and how does it overcome drug resistance in cancer cells?

Cycloviolacins exhibit anticancer activity primarily through membrane permeabilization mechanisms, with additional chemosensitizing abilities that enhance the effectiveness of conventional chemotherapeutics. The mechanism involves:

• Membrane disruption: Cycloviolacins, particularly cycloviolacin O2, directly disrupt cancer cell membranes, as demonstrated by SYTOX Green assays where cellular disruption correlates with cyclotide exposure.

• Selective toxicity: Interestingly, cyO2 shows selective membrane disruption in rapidly proliferating cancer cells while causing minimal disruption in primary human brain endothelial cells, suggesting specificity toward tumor cells.

• Chemosensitization: CyO2 significantly enhances the effectiveness of doxorubicin in drug-resistant breast cancer cells (MCF-7/ADR). When co-exposed to cyO2 (3 μM) and doxorubicin (5 μM), drug-resistant cells show 57-64% increased uptake of doxorubicin compared to only 19% with doxorubicin alone.

• Overcoming drug resistance: Fluorescence microscopy studies demonstrate that cyO2 treatment allows increased cellular internalization of doxorubicin in drug-resistant cells, effectively circumventing resistance mechanisms related to drug efflux .

This membrane-active mechanism represents a novel approach to overcoming multidrug resistance in cancer, as it bypasses conventional resistance mechanisms like drug efflux pumps.

How does the antifungal activity of cycloviolacins compare to their anticancer and antibacterial properties?

Cycloviolacins demonstrate multifaceted bioactivity profiles across different organisms, with notable differences in potency and specificity:

The antifungal activity appears to involve multiple cyclotide species, with statistical modeling identifying both cycloviolacin O8 and potentially cycloviolacin O3/O7 (mass 3152.41 Da) as contributors to activity against F. graminearum. The mechanisms likely share commonalities with antibacterial activity, primarily involving membrane disruption, but with structural features that confer specificity toward fungal cell membranes .

What bioassay systems are most appropriate for evaluating the different bioactivities of cycloviolacins?

For comprehensive characterization of cycloviolacin bioactivities, multiple specialized assay systems are required:

• Antimicrobial activity assessment:

  • Radial diffusion assays (RDAs): Initial screening against bacterial strains

  • Minimum inhibitory concentration (MIC) assays: Quantitative measurement of inhibitory concentrations

  • Time-kill kinetics: Assessment of bactericidal versus bacteriostatic effects in buffer systems

  • SYTOX Green membrane permeabilization assays: Mechanistic evaluation of membrane disruption

• Anticancer activity evaluation:

  • Cell proliferation assays (MTT/thiazolyl blue tetrazolium bromide): Quantification of cytotoxicity

  • Combination studies with conventional chemotherapeutics: Evaluation of synergistic effects

  • Fluorescence microscopy: Direct visualization of membrane permeabilization and drug uptake

• Antifungal testing:

  • Growth inhibition assays against filamentous fungi

  • Statistical correlation of bioactivity with mass spectrometry data

The selection of appropriate positive controls, reference standards, and careful optimization of assay conditions (buffer composition, incubation time, cell density) is critical for obtaining reliable and reproducible results .

What are the critical parameters for successful chemical modification studies of cycloviolacins?

Chemical modification studies are essential for structure-activity relationship analysis of cycloviolacins. Critical parameters include:

• Selective modification strategies:

  • Targeting specific amino acid residues (e.g., charged residues like Glu, Lys, and Arg)

  • Maintaining structural integrity during modification

  • Confirming modification specificity through mass spectrometry

• Modification validation:

  • Mass spectrometric confirmation of expected mass shifts

  • Structural verification through circular dichroism or NMR to ensure global fold is maintained

  • Activity assays to correlate structural changes with functional impacts

• Experimental controls:

  • Parallel processing of unmodified peptides as controls

  • Sequential modification of different residues to isolate individual contributions

  • Concentration matching between modified and unmodified peptides in activity assays

• Data interpretation considerations:

  • Distinguishing between direct effects on target interaction versus indirect effects on structural stability

  • Accounting for potential aggregation behaviors in modified peptides

  • Correlating activity changes with 3D structural positioning of modified residues

These approaches have revealed that charged residues in cycloviolacin O2 are essential for antimicrobial activity, with Glu and Lys modifications causing near-complete activity loss, while Arg modification has lesser effects .

How can high-throughput screening approaches be optimized for discovery of novel cycloviolacins with specific bioactivities?

Optimizing high-throughput screening for novel cycloviolacins requires integration of multiple technological approaches:

• Library preparation optimization:

  • Strategic taxonomic sampling across Violaceae family plants

  • Differential extraction from various plant tissues (aerial vs. underground parts)

  • Creation of semi-purified fraction libraries to reduce interference

• Multi-dimensional screening approach:

  • Primary screening against diverse targets (cancer cell lines, bacterial panels, fungal pathogens)

  • Secondary confirmation assays with dose-response analysis

  • Counter-screening against non-target cells for selectivity assessment

• Advanced analytical integration:

  • Implementation of the PepSAVI-MS pipeline for statistical correlation of bioactivity with MS features

  • Automation of disulfide reduction/alkylation for cyclotide identification

  • Machine learning algorithms to predict bioactivity from MS/MS fragmentation patterns

• Validation strategy:

  • Isolation of hit compounds at sufficient scale for full characterization

  • Confirmatory bioassays with isolated material

  • Structure determination through combined MS/MS and NMR approaches

This integrated approach has successfully identified novel bioactive cyclotides like cycloviolacin O8, demonstrating potent anticancer and antifungal activities that were not previously characterized .

What is the current understanding of the structure-activity relationships of cycloviolacins in their different bioactivities?

The structure-activity relationships of cycloviolacins reveal distinct structural features critical for different bioactivities:

How can researchers address the challenge of potential off-target effects in cycloviolacin research?

Addressing off-target effects in cycloviolacin research requires systematic approaches:

• Comprehensive toxicity profiling:

  • Parallel screening against target and non-target cells

  • Hemolysis assays to assess red blood cell toxicity

  • Primary cell cultures representing different tissues (e.g., hepatocytes, endothelial cells)

  • In vivo models for systemic toxicity evaluation

• Mechanistic clarification:

  • Membrane permeabilization assays with fluorescent dyes (e.g., SYTOX Green)

  • Investigation of potential intracellular targets through pull-down assays

  • Evaluation of impact on cellular signaling pathways

• Structural modification strategies:

  • Rational design of analogs with reduced off-target activity

  • PEGylation or other conjugation approaches to modify biodistribution

  • Targeted delivery systems to limit systemic exposure

• Therapeutic window assessment:

  • Determination of IC₅₀ values across multiple cell types

  • Calculation of selectivity indices to quantify therapeutic potential

  • Dose-limiting toxicity identification in pre-clinical models

Evidence suggests cycloviolacins may offer inherent selectivity toward certain targets. For example, cycloviolacin O2 demonstrates significant membrane disruption in cancer cells while showing minimal effects on primary human brain endothelial cells, suggesting natural selectivity that can be further optimized .

What approaches can address the analytical challenges in distinguishing closely related cycloviolacins?

Distinguishing structurally similar cycloviolacins presents significant analytical challenges requiring advanced methodological approaches:

• Enhanced chromatographic resolution:

  • Ultra-high performance liquid chromatography (UHPLC) with sub-2μm particles

  • Extended gradient profiles optimized for closely eluting species

  • Multi-dimensional chromatography combining orthogonal separation mechanisms (e.g., SCX followed by reversed-phase)

• Advanced mass spectrometric techniques:

  • High-resolution accurate mass measurements (<2 ppm error)

  • MS/MS sequencing with electron transfer dissociation (ETD) for improved coverage

  • Ion mobility spectrometry for separation based on 3D structure

• Chemical derivatization approaches:

  • Selective modification of specific residues to create diagnostic mass shifts

  • Differential reduction/alkylation strategies to map disulfide connectivity

  • Enzymatic digestions with site-specific proteases

• Quantitative analysis considerations:

  • Internal standards for relative quantification

  • Isotopically labeled reference standards for absolute quantification

  • Statistical modeling to deconvolute co-eluting species

This comprehensive approach has enabled researchers to distinguish between cyclotides with mass differences as small as 1-2 Da, such as the identification of oxidized tryptophan modifications in cycloviolacins that result in a 16 Da mass shift per oxidation .

What are the most promising approaches for enhancing the therapeutic potential of cycloviolacins?

Enhancing the therapeutic potential of cycloviolacins involves several innovative approaches:

• Targeted delivery systems:

  • Nanoparticle encapsulation to improve pharmacokinetics

  • Cancer-targeting ligand conjugation for selective delivery

  • Stimuli-responsive release mechanisms (pH, enzyme, redox)

• Rational design strategies:

  • Grafting approach: Incorporation of bioactive sequences into cycloviolacin scaffolds

  • Charge distribution optimization for enhanced selectivity

  • Strategic modification of key residues to reduce off-target effects

• Combination therapy development:

  • Building on the demonstrated chemosensitizing abilities of cycloviolacin O2

  • Development of optimized dosing schedules with conventional therapeutics

  • Identification of synergistic drug combinations for maximizing efficacy

• Production optimization:

  • Development of efficient recombinant expression systems

  • Semi-synthetic approaches combining chemical synthesis with enzymatic cyclization

  • Sustainable plant cultivation and extraction methods

The chemosensitizing abilities of cycloviolacin O2 in drug-resistant cancer cells represent a particularly promising direction, potentially addressing one of the most significant challenges in cancer therapy .

How might emerging computational approaches advance our understanding of cycloviolacin mechanisms and development?

Computational approaches offer powerful tools for advancing cycloviolacin research:

• Molecular dynamics simulations:

  • Membrane interaction modeling at atomic resolution

  • Free energy calculations for membrane binding and disruption

  • Conformational sampling to identify bioactive states

• Machine learning applications:

  • Prediction of bioactivity from primary sequence

  • Classification of cyclotides into functional subgroups

  • Automated annotation of MS/MS spectra for rapid identification

• Quantitative structure-activity relationship (QSAR) modeling:

  • Development of predictive models for antimicrobial activity

  • Identification of key physicochemical parameters driving anticancer activity

  • Virtual screening of theoretical cycloviolacin variants

• Systems biology integration:

  • Network analysis of affected pathways in target organisms

  • Resistance mechanism prediction through evolutionary algorithms

  • Multi-scale modeling linking molecular interactions to cellular responses

These computational approaches can guide experimental design, reducing the need for extensive screening and accelerating the development of optimized cycloviolacin variants with enhanced therapeutic properties.

What quality control parameters are essential when working with recombinant or synthetic cycloviolacins?

Essential quality control parameters for recombinant or synthetic cycloviolacins include:

Each batch should be characterized using multiple orthogonal methods, with particular attention to correct disulfide bond formation and head-to-tail cyclization, as these features are critical for the stability and activity of cycloviolacins.

How should researchers design experiments to effectively compare natural and recombinant cycloviolacins?

Effective comparative studies between natural and recombinant cycloviolacins require careful experimental design:

• Sample preparation standardization:

  • Matched purification protocols for natural and recombinant material

  • Equivalent storage conditions to prevent differential degradation

  • Concentration determination using multiple methods (UV absorbance, amino acid analysis)

• Structural equivalence assessment:

  • High-resolution mass spectrometry for intact mass comparison

  • Circular dichroism spectroscopy for secondary structure comparison

  • NMR analysis for tertiary structure confirmation when possible

  • Disulfide connectivity mapping

• Functional comparison framework:

  • Parallel testing in multiple bioassay systems

  • Full dose-response curves rather than single-point comparisons

  • Statistical analysis of potency differences (EC50/IC50 values)

  • Evaluation across multiple biological targets

• Reference standards and controls:

  • Well-characterized reference cyclotide as internal control

  • Inclusion of positive controls specific to each assay

  • Same-day testing to minimize inter-assay variability