Recombinant Viola odorata Cycloviolacin-O25

What is Cycloviolacin O2 and how does it differ from other cyclotides?

Cycloviolacin O2 (cyO2) is a bracelet cyclotide isolated from Viola odorata (sweet violet) that belongs to the larger family of plant-derived circular peptides containing 27-38 amino acids. Unlike Möbius cyclotides such as kalata B1, cyO2 has a bracelet cystine knot topology without a twist in the backbone. It contains three disulfide bonds characteristic of cyclotides, but has a distinct surface-exposed pattern of charged residues that contributes to its biological activity profile .

The distinguishing features of cyO2 include:

• Higher membrane-binding affinity compared to Möbius cyclotides

• Superior activity against Gram-negative bacteria

• More potent cytotoxic effects against cancer cells

• Unique glycosylation patterns observed in MS analysis

These differences make cyO2 particularly valuable for antimicrobial and anticancer research applications, where it shows greater potency than many other cyclotides in the same family .

How stable is Cycloviolacin O2 and what contributes to its stability?

Cycloviolacin O2 demonstrates exceptional structural stability due to several key features:

• Circular backbone (head-to-tail cyclization)

• Three disulfide bonds forming a cystine knot motif

• Compact folding with hydrogen bond networks

Research has confirmed the remarkable stability of cyO2, with studies detecting intact cyclotides in Viola odorata specimens collected as early as 1820. Analysis of historical samples from 1820, 1849, 1886, 1948, and modern specimens (2006) revealed that major cyclotide components including cycloviolacin O2 remained detectable and preserved their structure over nearly 200 years .

This exceptional stability makes cyO2 particularly valuable for applications requiring prolonged storage or resistance to harsh conditions, including high temperatures, extreme pH environments, and proteolytic degradation.

What are the most effective methods for isolating native Cycloviolacin O2 from plant material?

The established methodology for isolating native cyO2 from Viola odorata includes:

• Plant material preparation:

  • Collection of fresh or dried Viola odorata material

  • Grinding to increase surface area for extraction

• Extraction procedure:

  • Maceration in methanol/dichloromethane mixtures (typically 1:1)

  • Multiple extraction cycles with solvent replacement

  • Filtration and concentration of extracts

• Initial fractionation:

  • Liquid-liquid partitioning between water and organic solvents

  • Solid-phase extraction using C18 cartridges

• Purification:

  • Reversed-phase HPLC with acetonitrile/water gradients containing 0.05% TFA

  • Sequential purification using different column chemistries (C18, C8, phenyl-hexyl)

  • Final polishing using analytical HPLC columns

• Identification:

  • Mass spectrometry to confirm molecular weight

  • Reduction and alkylation of disulfide bonds to confirm the presence of six cysteine residues

  • Enzymatic digestion and MS/MS sequencing for sequence verification

For optimal yield, extraction parameters should be optimized regarding solvent composition, temperature, duration, and plant material-to-solvent ratio.

What analytical techniques are most informative for characterizing Cycloviolacin O2 structure and modifications?

Multiple complementary analytical techniques are required for comprehensive characterization:

• Mass Spectrometry (MS):

  • MALDI-TOF MS for intact mass determination

  • LC-MS/MS for sequence confirmation

  • Detection of post-translational modifications, including glycosylation

  • Identification of disulfide bond patterns after partial reduction

• Nuclear Magnetic Resonance (NMR):

  • 2D techniques (TOCSY, NOESY, HSQC) for structural elucidation

  • Determination of three-dimensional structure

  • Analysis of dynamics in solution

• Chromatographic techniques:

  • RP-HPLC for purity assessment

  • Size-exclusion chromatography for aggregation studies

  • Ion-exchange chromatography for charge variant analysis

• Post-translational modification analysis:

  • Detection of glycosylation (additional 162 Da mass units)

  • MS data has shown that cyO2 can undergo both single and double glycosylation

When analyzing cyO2, researchers should note that the doubly and triply charged ions (1570.62+/1047.73+) are characteristic for this cyclotide in MS analysis, and glycosylated derivatives show predictable mass shifts of 162 Da per sugar moiety .

What expression systems are most suitable for recombinant production of Cycloviolacin O2?

Several expression systems have been evaluated for recombinant production of cyclotides, with the following considerations specific to cyO2:

• Bacterial expression systems (E. coli):

  • Fusion protein approaches with self-cleaving inteins

  • Thioredoxin or SUMO fusion partners to enhance solubility

  • Challenges include correct disulfide bond formation and cyclization

  • Requires in vitro processing for final cyclization

• Plant cell culture systems:

  • Viola odorata cell suspension cultures in bioreactors

  • Maintains native cellular machinery for correct folding and post-translational modifications

  • Model-based fed-batch cultivation has been demonstrated for V. odorata cells

  • Both stirred tank reactors and airlift bioreactors have been successful

• Synthetic approaches:

  • Solid-phase peptide synthesis followed by chemical cyclization

  • Native chemical ligation for assembly of larger sequences

  • Enzymatic cyclization using modified sortase or asparaginyl endopeptidases

For the highest yield of correctly folded cyO2, plant cell suspension cultures offer significant advantages as they contain the complete cellular machinery for proper folding and post-translational modifications. Recent research has demonstrated successful cultivation of V. odorata cells in bioreactors with confirmed production of bioactive cyclotides .

How can the correct folding and post-translational modifications of recombinant Cycloviolacin O2 be verified?

Verification of proper folding and post-translational modifications requires a multi-faceted analytical approach:

• Structural verification:

  • Comparison of chromatographic profiles with native standards

  • Mass spectrometry to confirm exact mass

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • NMR fingerprinting compared to authentic standards

• Disulfide bond analysis:

  • Partial reduction and MS mapping

  • Enzymatic digestion followed by MS/MS analysis

  • Comparison of disulfide bonding patterns with native cyO2

• Functional assays:

  • Membrane permeabilization assays (SYTOX-green dye uptake)

  • Hemolytic activity tests (should match native cyO2 profile)

  • Antimicrobial activity against Gram-negative bacteria

  • Cytotoxicity against cancer cell lines (e.g., MCF-7, A549, Caco-2)

• Glycosylation analysis:

  • MS detection of +162 Da mass shifts characteristic of glycosylation

  • Enzymatic deglycosylation followed by MS analysis

  • Glycan structure characterization by MS/MS and glycosidase digestion

When validating recombinant cyO2, it's critical to compare multiple parameters simultaneously with authentic standards, as correct primary structure does not necessarily indicate proper folding or biological activity.

How does Cycloviolacin O2 interact with and disrupt cellular membranes?

The membrane-active properties of cyO2 have been extensively studied, revealing the following mechanism:

• Initial membrane binding:

  • Electrostatic interactions between charged residues (particularly Glu, Lys, and Arg) and membrane phospholipids

  • Preference for negatively charged membranes (explaining selectivity for bacterial and cancer cell membranes)

  • Higher membrane affinity compared to Möbius cyclotides like kalata B1

• Membrane insertion and pore formation:

  • Hydrophobic patches insert into the lipid bilayer

  • Oligomerization creates transmembrane pores

  • SYTOX-green dye uptake assays confirm rapid pore formation in cell membranes

• Membrane disruption effects:

  • Concentration-dependent membrane permeabilization

  • At lower concentrations: selective pore formation

  • At higher concentrations: membrane lysis and cell death

  • Disruption of viral particle integrity when applied to HIV virions

Chemical modification studies have shown that charged residues (Glu, Lys, Arg) in cyO2 are essential for its membrane activity. Masking the Glu and Lys residues causes near-total loss of antibacterial activity, while masking Arg produces a less pronounced reduction in activity .

What bioassays best demonstrate the mechanism of action of Cycloviolacin O2?

Several bioassays have proven useful for elucidating cyO2's mechanism of action:

• Membrane permeabilization assays:

  • SYTOX-green dye uptake assay to quantify pore formation kinetics

  • Calcein release from liposomes to assess membrane disruption

  • Propidium iodide uptake for cell permeabilization studies

  • These assays have demonstrated rapid pore formation in HIV-infected T-cells and monocytes at concentrations below 0.5 μM

• Hemolytic activity tests:

  • Red blood cell hemolysis assays to determine membrane selectivity

  • Concentration-dependent studies show hemolytic effects above 0.5 μM

  • Critical for establishing safety window for therapeutic applications

• Electrophysiology studies:

  • Patch-clamp recordings to characterize pore properties

  • Measurement of ion conductance across membranes

• Fluorescently labeled cyclotide tracking:

  • Confocal microscopy to visualize membrane localization

  • FRET-based interaction studies with membrane components

• Drug uptake enhancement measurement:

  • Radiolabeled saquinavir (³H-SQV) uptake assays have demonstrated that cyO2 enhances drug uptake into cells

These assays collectively provide a comprehensive understanding of how cyO2 interacts with membranes, forms pores, and facilitates drug uptake in various cell types.

How do specific residues in Cycloviolacin O2 contribute to its biological activities?

Structure-function studies have identified key residues that determine cyO2's biological profile:

• Charged residues:

  • Glutamic acid (Glu): Essential for initial membrane binding; chemical masking causes near-total loss of antibacterial activity

  • Lysine (Lys): Critical for electrostatic interactions with membranes; modification significantly reduces activity

  • Arginine (Arg): Contributes to membrane binding but is less critical than Glu or Lys; masking causes less pronounced activity reduction

• Hydrophobic residues:

  • Form a continuous hydrophobic patch that inserts into membranes

  • Critical for the formation of membrane pores

  • Modifications that alter hydrophobicity affect membrane insertion capacity

• Cysteine residues:

  • Six cysteines forming three disulfide bonds are essential for structural integrity

  • The cystine knot motif provides exceptional stability

  • Reduction of disulfides results in loss of biological activity

• Glycosylation sites:

  • Glycosylation of certain residues affects pharmacokinetic properties

  • MS analysis shows cyO2 can undergo both single and double glycosylation

The spatial arrangement of these residues creates an amphipathic structure with clear hydrophobic and hydrophilic faces, which is optimal for membrane interaction and disruption.

How can structure-activity relationships be used to design optimized Cycloviolacin O2 variants?

Rational design of optimized cyO2 variants can be approached through:

• Selective residue substitution:

  • Modulation of charged residues to enhance selectivity

  • Tuning the hydrophobic/hydrophilic balance

  • Introduction of non-natural amino acids at key positions

  • Based on the importance of charged residues (Glu, Lys, Arg), targeted substitutions can tune antimicrobial specificity

• Surface engineering:

  • Modifications to enhance specific targeting (e.g., cancer cell selectivity)

  • Addition of cell-specific recognition motifs

  • PEGylation to modify pharmacokinetic properties

• Hybrid cyclotide design:

  • Grafting of bioactive sequences into cyO2 scaffold

  • Creation of chimeric cyclotides combining features of different cyclotide subfamilies

  • Integration of features from both bracelet and Möbius cyclotides

• Glycoengineering:

  • Control of glycosylation patterns for optimized stability and activity

  • Site-directed mutagenesis to introduce or remove glycosylation sites

  • MS analysis has shown that natural cyO2 can undergo both single and double glycosylations

When designing variants, researchers should consider the structure-activity data showing that cyO2's antimicrobial activity against Gram-negative bacteria is highly dependent on charged residues, while its membrane-permeabilizing properties are essential for both antimicrobial and anticancer activities .

How effective is Cycloviolacin O2 against different bacterial pathogens?

Research has demonstrated a distinct antimicrobial profile for cyO2:

• Activity against Gram-negative bacteria:

  • Potent bactericidal effects against:

    • Escherichia coli

    • Salmonella enterica serovar Typhimurium

    • Klebsiella pneumoniae

    • Pseudomonas aeruginosa

  • Rapid killing kinetics observed in time-kill assays

  • Activity maintained in both radial diffusion assays (RDAs) and MIC determinations

• Activity against Gram-positive bacteria:

  • Limited activity against Staphylococcus aureus

  • Generally less effective against Gram-positive species

  • This selectivity differs from many other antimicrobial peptides

• Comparative potency:

  • More potent against Gram-negative bacteria than Möbius cyclotides (e.g., kalata B1)

  • Activity significantly higher than other bracelet cyclotides tested

  • Concentration-dependent bactericidal effects observed within the 0.5-10 μM range

The selectivity of cyO2 for Gram-negative over Gram-positive bacteria makes it particularly interesting for addressing infections caused by multidrug-resistant Gram-negative pathogens, which represent a significant clinical challenge.

What experimental approaches best demonstrate the potential of Cycloviolacin O2 as an antimicrobial agent?

The following experimental approaches provide comprehensive evaluation of cyO2's antimicrobial potential:

• Susceptibility testing:

  • Minimum inhibitory concentration (MIC) determination

  • Minimum bactericidal concentration (MBC) assessment

  • Time-kill kinetics studies (showed rapid bactericidal activity against multiple Gram-negative species)

  • Radial diffusion assays to visualize growth inhibition zones

• Resistance development assessment:

  • Serial passage experiments to evaluate resistance emergence

  • Comparison with conventional antibiotics

  • Mechanisms of potential resistance (if any)

• Combination studies:

  • Synergy testing with conventional antibiotics (checkerboard assays)

  • Fractional inhibitory concentration index (FICI) determination

  • Time-kill studies with drug combinations

• Mechanism investigations:

  • Membrane permeabilization assays (using fluorescent dyes)

  • Transmission electron microscopy to visualize membrane effects

  • Chemical modification of charged residues (Glu, Lys, Arg) revealed their essential role in activity

• In vivo efficacy models:

  • Infection models in appropriate animal systems

  • Pharmacokinetic and biodistribution studies

  • Toxicity and safety assessments

When conducting these experiments, it's important to include appropriate controls, including other cyclotides with different structural features (e.g., Möbius cyclotides) to contextualize results within the larger cyclotide family.

How does Cycloviolacin O2 affect different cancer cell lines?

Cycloviolacin O2 demonstrates significant anticancer activities against multiple cancer cell types:

• Breast cancer:

  • Cytotoxic against MCF-7 cells

  • Active against drug-resistant MCF-7/ADR subline

  • Effective at concentrations of 0.2-10 μM

• Lung cancer:

  • Active against A549 lung adenocarcinoma cells

  • IC50 values in the range of 2.7-3.0 mg/mL for extracts containing cyO2

• Colorectal cancer:

  • Cytotoxic against Caco-2 human colorectal adenocarcinoma

  • IC50 values ranging from 1.5-3.4 mg/mL for extracts containing cyO2

• Selectivity profile:

  • Demonstrates selectivity for cancer cells over normal cells

  • Non-cytotoxic to L929 fibroblast cells at concentrations up to 6 mg/mL

  • Creates pores in cancer cell membranes while showing minimal effects on normal cells

The mechanism involves membrane permeabilization, leading to disruption of cancer cell membranes and subsequent cell death, with notable activity against drug-resistant cancer cell lines.

How can Cycloviolacin O2 be used to enhance cancer drug efficacy and overcome resistance?

CyO2 offers several strategies for enhancing cancer therapy efficacy:

• Chemosensitizing effects:

  • Enhances uptake of anticancer drugs by creating membrane pores

  • Overcomes drug resistance mechanisms based on decreased uptake

  • Demonstrated chemosensitizing abilities with doxorubicin in the drug-resistant MCF-7/ADR cell line

• Combination therapy approaches:

  • Synergistic effects with conventional chemotherapeutics

  • Sub-cytotoxic concentrations of cyO2 (0.2-1 μM) can enhance drug efficacy

  • Creates pores in cancer cell membranes that support drug penetration

• Resistance-breaking mechanisms:

  • Bypasses efflux pump-mediated resistance (common in MCF-7/ADR)

  • Creates alternative routes for drug entry

  • Membrane-based mechanism differs from typical drug resistance pathways

• Experimental design considerations:

  • Pre-treatment with cyO2 followed by chemotherapeutic administration

  • Simultaneous administration protocols

  • Development of co-delivery systems (e.g., nanoparticles)

Cyclotides like cyO2 support the penetration of cancer drugs into cancer cells by creating pores in the cancer cell membrane, thereby enhancing drug accumulation inside resistant cells . This membrane-active mechanism provides a physical rather than biochemical approach to overcoming resistance.

What evidence supports the anti-HIV activity of Cycloviolacin O2?

Multiple lines of evidence demonstrate cyO2's anti-HIV activities:

• Direct antiviral effects:

  • Decreased HIV-1 p24 production at concentrations below 0.5 μM

  • Disruption of viral particle integrity as demonstrated in ultracentrifugation studies

  • Reduction of p24 content in viral particles

• Drug enhancement effects:

  • Enhanced antiviral efficacy of protease inhibitors (saquinavir and nelfinavir)

  • Increased efficacy of the fusion inhibitor enfuvirtide (T-20)

  • Demonstrated in multiple T-lymphocyte models (HuT78*, HIV IIIB H9, J-Lat 9.2)

• Mechanism studies:

  • Rapid pore-formation in HIV-infected T-cells and monocytes

  • Increased drug uptake demonstrated with radiolabeled saquinavir (³H-SQV)

  • Direct HIV-1 inactivation through disruption of viral envelope integrity

• Safety window:

  • Concentrations below 0.5 μM show minimal hemolytic activity

  • Effective antiviral activity achieved at non-hemolytic concentrations

  • Selectivity for infected cells over healthy cells

The ability of cyO2 to both directly affect viral particles and enhance antiretroviral drug efficacy presents a dual mechanism for HIV suppression.

How should experiments be designed to evaluate Cycloviolacin O2's potential as an antiviral agent or adjuvant?

Comprehensive evaluation of cyO2's antiviral potential requires:

• Viral replication assays:

  • ELISA for HIV-1 p24 to measure viral production (demonstrated cyO2 enhances efficacy of SQV and NFV)

  • Reporter assays to evaluate impact on viral transcription

  • Assessment of direct effects on viral particles through ultracentrifugation studies

• Mechanism studies:

  • SYTOX-green dye-uptake assays to measure pore-formation

  • Radiolabeled drug uptake assays (³H-SQV) to quantify enhanced drug penetration

  • Viral integrity assessment after cyO2 exposure

• Combination studies with antiretrovirals:

  • Co-exposure experiments with protease inhibitors (shown to enhance SQV and NFV)

  • Combination with entry/fusion inhibitors (enhanced efficacy of enfuvirtide)

  • Determination of optimal timing and concentration relationships

• Safety assessment:

  • Red blood cell hemolysis assays (showed safety below 0.5 μM)

  • Cytotoxicity evaluation in non-infected cells

  • Therapeutic window determination

• Resistance assessment:

  • Long-term exposure studies to evaluate resistance development

  • Efficacy against drug-resistant HIV strains

  • Mechanistic studies of how membrane disruption affects viral escape

Experiments should compare cyO2 alone versus combination with antiretrovirals, as research has shown that while cyO2 alone decreases HIV-1 p24 production, its combination with antiretrovirals produces enhanced suppression of viral replication .

How can bioreactor cultivation of Viola odorata cells be optimized for Cycloviolacin O2 production?

Bioreactor cultivation of V. odorata cells requires optimization of several parameters:

• Cultivation strategy optimization:

  • Model-based fed-batch cultivation has been demonstrated to enhance biomass productivity

  • Mathematical modeling of growth and substrate utilization kinetics helps develop optimal feeding strategies

  • Both stirred tank reactors and airlift bioreactors have been successfully employed

• Media composition factors:

  • Carbon source type and concentration

  • Nitrogen source optimization

  • Hormone supplementation

  • Precursor feeding strategies

• Physical parameters:

  • Dissolved oxygen levels

  • Agitation speed in stirred tank reactors

  • pH control strategies

  • Temperature optimization

• Feeding strategy development:

  • Nutrient-feeding strategies based on mathematical modeling

  • Implementation of in silico models to minimize trial-and-error experiments

  • Real-time monitoring of key cultivation parameters

• Downstream processing:

  • Extraction protocols for cyclotide recovery

  • Purification strategies for isolating cyO2

  • Quality control of final product

Recent research has established model-based fed-batch cultivation as an effective approach for V. odorata cell suspension cultures, allowing for enhanced biomass productivity with minimal empirical testing at the reactor level .

What considerations are important when designing structure-activity relationship studies for Cycloviolacin O2 variants?

When designing SAR studies for cyO2 variants, researchers should consider:

• Strategic residue modifications:

  • Focus on charged residues (Glu, Lys, Arg) that have been demonstrated to be critical for activity

  • Sequential alanine scanning to identify essential positions

  • Conservative vs. non-conservative substitutions

  • Chemical modification studies have shown the importance of charged residues for antibacterial activity

• Preservation of structural integrity:

  • Maintaining the cystine knot framework

  • Ensuring correct disulfide bond formation

  • Structural verification using NMR and circular dichroism

• Activity spectrum evaluation:

  • Testing against multiple bacterial strains (especially Gram-negative)

  • Cancer cell line panel testing

  • HIV models for antiviral activity

  • Hemolytic activity assessment for safety profiling

• Mechanistic studies:

  • Membrane binding experiments

  • Pore formation assays

  • Drug uptake enhancement evaluation

• Comparative analysis:

  • Benchmarking against wild-type cyO2

  • Comparison with other cyclotide subtypes

  • Relative activity against different target cells

Studies should include appropriate controls and standardized assays to ensure comparability across variants. The critical importance of charged residues (particularly Glu and Lys) should be considered when designing variants, as chemical masking of these residues has been shown to cause near-total loss of antibacterial activity .

What are the main challenges in ensuring consistent quality of recombinant Cycloviolacin O2?

Researchers face several challenges in producing consistent recombinant cyO2:

• Folding and cyclization issues:

  • Incorrect disulfide bond formation

  • Incomplete cyclization

  • Formation of oligomers or aggregates

  • Monitoring requires comprehensive analytical methods including MS and NMR

• Post-translational modification variability:

  • Inconsistent glycosylation patterns

  • Natural cyO2 can undergo both single and double glycosylations

  • Differences between expression systems in glycosylation machinery

• Purification challenges:

  • Co-purification of closely related cyclotide variants

  • Separation of correctly folded from misfolded species

  • Removal of endotoxins from bacterial expression systems

• Activity variation:

  • Batch-to-batch variations in biological activity

  • Establishment of reliable potency assays

  • Correlation between structural parameters and activity

• Stability during processing and storage:

  • Oxidation of methionine residues

  • Degradation during purification

  • Long-term storage conditions optimization

To address these challenges, a comprehensive quality control system incorporating multiple analytical techniques (HPLC, MS, NMR, bioassays) is essential for ensuring consistent quality across batches.

How can researchers troubleshoot inconsistent results in Cycloviolacin O2 bioactivity assays?

When encountering inconsistent bioactivity results, consider the following troubleshooting approaches:

Research has shown that the charged residues (Glu, Lys, Arg) in cyO2 are critical for activity, so conditions that affect their protonation state or availability for interaction with target membranes can significantly impact assay results .

Data Table 1: Comparative Activity of Cycloviolacin O2 Against Different Bacterial Species

Data Table 2: Effect of Chemical Modification of Charged Residues on Cycloviolacin O2 Activity

Data Table 3: Anticancer Activity of Cycloviolacin O2 Against Various Cell Lines