Recombinant Viola odorata Cycloviolacin-O21

What is the molecular structure of Cycloviolacin-O21 and how does it compare to other cyclotides?

Cycloviolacin-O21 is a 29-amino acid cyclotide with the sequence GLPVCGETCVTGSCYTPGCTCSWPVCTRN . Like other cyclotides, it possesses a head-to-tail cyclized backbone and contains cysteine residues that form a characteristic cyclic cystine knot (CCK) motif through disulfide bonding . This structure gives Cycloviolacin-O21 remarkable stability against thermal, chemical, and enzymatic degradation.

Comparing Cycloviolacin-O21 to related cyclotides such as Cycloviolacin O2 (CyO2), they share the fundamental cyclotide architecture but differ in specific amino acid composition. Both belong to the "Bracelet" subfamily of cyclotides, characterized by their specific arrangement of loops between cysteine residues .

What are the recommended storage conditions for maintaining Cycloviolacin-O21 stability in research settings?

For optimal stability of recombinant Cycloviolacin-O21, the following storage protocol is recommended:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C to -80°C

• Avoid repeated freeze-thaw cycles

• Working aliquots can be maintained at 4°C for up to one week

For reconstitution:

• Briefly centrifuge vial before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Aliquot before freezing to minimize freeze-thaw cycles

What are the recommended approaches for purifying recombinant Cycloviolacin-O21 for research applications?

Purification of recombinant Cycloviolacin-O21 typically involves:

• Expression in E. coli expression systems with appropriate tags for purification

• Initial capture using affinity chromatography based on the fusion tag

• Optional tag cleavage using specific proteases

• Secondary purification through reversed-phase HPLC

• Quality control using SDS-PAGE (aim for >85% purity) and mass spectrometry

When working with commercially available recombinant Cycloviolacin-O21, researchers should note that tag types may vary depending on the manufacturing process, which could affect experimental design .

What methods are most effective for studying the membrane-disrupting activity of Cycloviolacin-O21?

Based on research with related cyclotides like CyO2, the following methodologies are effective for studying membrane-disrupting activities:

• SYTOX-green dye-uptake assay: This fluorescence-based method measures pore formation in cell membranes. SYTOX-green is a cell-impermeable dye that fluoresces upon binding to nucleic acids, indicating membrane disruption .

• Radiolabeled drug uptake assays: Using radiolabeled compounds (like 3H-SQV in HIV studies) to measure increased cellular uptake facilitated by cyclotide-induced membrane permeabilization .

• Hemolysis assays: To determine the concentration threshold between therapeutic effect and cytotoxicity. Red blood cell hemolysis studies establish physiologically safe concentrations (typically below 0.5 μM for CyO2) .

• Artificial membrane models: Using liposomes with defined lipid composition to study binding affinity and the mechanism of membrane disruption.

What methodological considerations are important when designing antifungal studies with Cycloviolacin-O21?

When designing antifungal studies with Cycloviolacin-O21, consider the following methodological approaches based on research with related cyclotides:

• Fungal strain selection: Include both clinically relevant species and environmental isolates for comprehensive evaluation

• Assay selection:

  • Broth microdilution assays for MIC determination

  • Time-kill assays to determine fungicidal vs. fungistatic activity

  • Growth inhibition zone assays on solid media

• Mechanism investigation:

  • Membrane permeabilization assays using fluorescent dyes

  • Ergosterol binding assays to determine if the cyclotide targets fungal-specific membrane components

  • Electron microscopy to visualize membrane damage

• Resistance development: Long-term exposure studies to assess potential for resistance development

Cyclotides including Cycloviolacin O2, O8, and O19 have demonstrated antifungal activities , suggesting Cycloviolacin-O21 may also possess these properties due to structural similarities.

What are the structure-activity relationships determining the selective cytotoxicity of Cycloviolacin-O21 against cancer cells?

The selective cytotoxicity of cyclotides like Cycloviolacin-O21 against cancer cells appears to depend on several key structural features:

• Amphipathicity: The distribution of hydrophobic and hydrophilic residues is crucial for membrane interaction. The balance between these properties influences selectivity toward tumoral cell membranes .

• Cationicity: Studies on CyO2-derived peptides show that increasing cationic charge (through lysine substitutions) enhances anticancer activity, but only when balanced with appropriate hydrophobicity. Overly cationic peptides with extremely low hydrophobicity show reduced efficacy .

• Cyclization and disulfide bonds: The cyclic cystine knot motif provides structural stability and resistance to degradation, allowing cyclotides to maintain their active conformation in physiological environments .

For optimizing selective cytotoxicity, researchers working with Cycloviolacin-O21 should consider:

• Maintaining moderate hydrophobicity (-0.04 to -0.27) while increasing cationicity

• Preserving critical structural elements like the cyclic backbone and disulfide bonding pattern

• Targeting modifications to residues involved in membrane interaction but not essential for structural stability

How can computational approaches improve the design of Cycloviolacin-O21 derivatives with enhanced anticancer properties?

In silico techniques have proven valuable for designing improved cyclotide derivatives with enhanced anticancer properties, as demonstrated with CyO2:

• Fragment-based design: Identifying bioactive fragments (e.g., 15 amino acid length) from the full sequence that retain anticancer activity while reducing potential side effects .

• Physicochemical property optimization: Using computational tools to predict and optimize:

  • Amphipathicity

  • Cationicity

  • Hydrophobicity

  • Half-life

  • Molecular weight

• Predictive modeling: Employing specialized algorithms to predict:

  • Anticancer potential (using tools like AntiCP)

  • Cell penetrative ability (using CellPPD-MOD and C2Pred)

  • Hemolytic activity

A successful example is the T2.2 peptide (derived from CyO2 with double lysine substitution), which demonstrated optimized physicochemical properties while maintaining anticancer activity . Similar approaches could be applied to Cycloviolacin-O21 to enhance its anticancer potential.

What is the mechanism by which Cycloviolacin-O21 might enhance the efficacy of antiretroviral drugs?

Based on research with the related Cycloviolacin O2, cyclotides may enhance antiretroviral drug efficacy through multiple mechanisms:

• Selective membrane permeabilization: At concentrations below the hemolytic threshold (<0.5 μM), cyclotides can create pores in HIV-infected cells, increasing the uptake of antiretroviral drugs. This was demonstrated with saquinavir (3H-SQV) uptake assays .

• Direct viral particle disruption: Cyclotides can directly disrupt viral integrity, as shown through ultracentrifugation studies with CyO2, which decreased the p24 content of viral particles .

• Synergistic effect with entry inhibitors: The membrane-active properties of cyclotides complement the action of HIV entry inhibitors like enfuvirtide (T-20) by compromising viral envelope integrity .

For researchers investigating Cycloviolacin-O21 in this context, establishing the appropriate concentration range that maximizes drug uptake enhancement while minimizing cytotoxicity is critical (typically <0.5 μM for CyO2) .

What experimental design considerations are important when evaluating Cycloviolacin-O21 as an adjuvant for antiviral therapies?

When designing experiments to evaluate Cycloviolacin-O21 as an antiviral adjuvant, researchers should consider:

• Concentration optimization:

  • Perform RBC hemolysis assays to determine physiologically safe concentrations

  • Test multiple concentrations below the hemolytic threshold

• Drug uptake studies:

  • Use fluorescent or radiolabeled drugs to quantify enhanced cellular uptake

  • Compare uptake in infected versus uninfected cells to establish selectivity

• Combination therapy assessment:

  • Evaluate synergy with different classes of antivirals (protease inhibitors, fusion inhibitors, etc.)

  • Use appropriate controls including drug alone, cyclotide alone, and combination treatments

• Viral load quantification:

  • Employ p24 ELISA for HIV studies

  • Use reporter assays to distinguish between effects on viral entry versus replication

• Direct virucidal activity assessment:

  • Ultracentrifugation studies to evaluate viral particle integrity

  • Pre-exposure of virus to cyclotide before infection assays

How does Cycloviolacin-O21 compare structurally and functionally to other cyclotides from Viola odorata?

Viola odorata produces multiple cyclotides with varying structures and bioactivities. Here's a comparative analysis of Cycloviolacin-O21 with other V. odorata cyclotides:

Structurally, Cycloviolacin-O21 shares the fundamental cyclotide architecture of a cyclic backbone and cysteine-knot motif. The primary sequence differences between these cyclotides likely account for their varied bioactivities and potencies against different targets.

What methodological approaches would best identify novel applications for Cycloviolacin-O21 beyond those established for other cyclotides?

To identify novel applications for Cycloviolacin-O21 beyond established cyclotide functions, researchers should consider:

• Target-based screening approaches:

  • Protein-protein interaction disruption assays

  • Enzyme inhibition screens against disease-relevant targets

  • Receptor binding and modulation assays

• Phenotypic screening approaches:

  • Testing against neglected tropical disease pathogens

  • Screening against multidrug-resistant clinical isolates

  • Evaluating effects on cancer stem cells and therapy-resistant cancer cell lines

• Computational prediction methods:

  • Molecular docking against novel protein targets

  • Pharmacophore modeling based on known bioactive cyclotides

  • Simulation of membrane interactions in different lipid environments

• Structure-activity relationship studies:

  • Alanine scanning mutagenesis to identify critical residues

  • Hybrid cyclotides combining loops from different native cyclotides

  • Grafting of bioactive peptide sequences into the cyclotide scaffold

• Delivery system applications:

  • Evaluation as cell-penetrating peptides for drug delivery

  • Investigation as mucosal delivery enhancers

  • Assessment as blood-brain barrier penetration enhancers

What are the major challenges in expressing and purifying recombinant Cycloviolacin-O21, and how can they be overcome?

Recombinant production of cyclotides presents several challenges that researchers should address:

• Cyclization challenges:

  • Problem: Achieving native head-to-tail cyclization in E. coli.

  • Solution: Use specialized intein-based expression systems or enzymatic methods with sortase A or butelase ligase for post-expression cyclization.

• Disulfide bond formation:

  • Problem: Correct folding with three disulfide bridges.

  • Solution: Employ oxidative folding conditions post-purification or co-expression with disulfide isomerases; consider step-wise oxidation protocols.

• Protein yield:

  • Problem: Low expression due to potential toxicity to host cells.

  • Solution: Use fusion partners like thioredoxin or SUMO to improve solubility and reduce toxicity; optimize induction conditions (temperature, IPTG concentration) .

• Purification complexity:

  • Problem: Separating correctly folded cyclotides from misfolded variants.

  • Solution: Implement two-step chromatography processes with both affinity and reversed-phase HPLC; validate structure by mass spectrometry .

For working with commercially available recombinant Cycloviolacin-O21, researchers should perform quality control tests to verify proper folding and activity before experimental use .

What analytical methods are most effective for verifying the structural integrity of recombinant Cycloviolacin-O21?

To verify the structural integrity of recombinant Cycloviolacin-O21, researchers should employ a combination of analytical methods:

• Mass spectrometry:

  • MALDI-TOF MS for molecular weight confirmation

  • Tandem MS/MS for sequence verification

  • Disulfide mapping through partial reduction and alkylation

• Chromatographic analysis:

  • Reversed-phase HPLC for purity assessment (aim for >85%)

  • Size-exclusion chromatography to detect aggregates

  • Ion-exchange chromatography to separate charge variants

• Spectroscopic methods:

  • Circular dichroism (CD) spectroscopy for secondary structure evaluation

  • NMR spectroscopy for detailed 3D structural analysis

  • Fourier-transform infrared spectroscopy (FTIR) for confirmation of structural elements

• Biological activity assays:

  • Membrane permeabilization assays as functional verification

  • Comparison with native cyclotide activity profiles

  • Thermal and chemical stability tests to confirm CCK motif integrity

A comprehensive analytical approach combining these methods provides confidence in the correct folding and structural integrity of recombinant Cycloviolacin-O21 preparations.

What are the most promising applications for Cycloviolacin-O21 in drug delivery systems?

Based on the properties of cyclotides, Cycloviolacin-O21 shows promise for several drug delivery applications:

• Enhancement of antiretroviral drug efficacy:

  • Leveraging membrane-permeabilizing properties to increase cellular uptake of HIV protease inhibitors like saquinavir and nelfinavir

  • Potential to reduce required drug doses through synergistic effects

• Peptide-drug conjugates:

  • Using Cycloviolacin-O21 as a stable scaffold for attaching small molecule drugs

  • Exploiting its stability against enzymatic degradation for improved pharmacokinetics

• Targeted delivery systems:

  • Modification of Cycloviolacin-O21 with targeting moieties for specific cell types

  • Potential for selective delivery to cancer cells based on membrane composition differences

• Oral delivery enhancement:

  • Resistance to gastrointestinal proteolysis

  • Potential to improve bioavailability of poorly absorbed drugs

• Combination with nanomaterial-based delivery systems:

  • Integration into liposomes or nanoparticles for specialized delivery

  • Using membrane-active properties to enhance endosomal escape after cellular uptake

Future research should focus on optimizing Cycloviolacin-O21 derivatives with tailored physical properties for specific delivery applications while minimizing off-target effects.

How might genetic engineering approaches be used to create novel Cycloviolacin-O21 variants with enhanced therapeutic properties?

Genetic engineering offers powerful approaches to develop enhanced Cycloviolacin-O21 variants:

• Site-directed mutagenesis:

  • Targeted lysine substitutions to optimize charge and anticancer activity

  • Modifying hydrophobic residues to fine-tune membrane interactions

  • Altering surface-exposed residues while maintaining the CCK scaffold

• Loop grafting:

  • Replacing one or more loops between conserved cysteines with bioactive peptide sequences

  • Creating chimeric cyclotides combining structural elements from different cyclotide families

• De novo design:

  • Computational design of novel cyclotides based on structure-activity relationships

  • Machine learning approaches incorporating data from multiple cyclotide variants

• Incorporation of non-natural amino acids:

  • Adding fluorescent amino acids for tracking and imaging

  • Incorporating click-chemistry compatible residues for post-translational modifications

  • Using heavy isotope amino acids for specialized analytical applications

• Expression system optimization:

  • Developing plant-based expression systems that naturally produce cyclotides

  • Engineering bacterial systems with improved cyclization and folding machinery

These approaches could yield Cycloviolacin-O21 variants with enhanced potency, improved selectivity, reduced side effects, and tailored pharmacokinetic properties for specific therapeutic applications.