Recombinant Litoria chloris Caerin-1.9

What is the molecular structure and primary sequence of Caerin-1.9?

Caerin-1.9 is a 25-amino acid peptide with the sequence GLFGVLGSIAKHVLPHVVPVIAEKL-NH₂ and a molecular weight of 2,593.17 g/mol. The peptide features an amidated C-terminus (NH₂), which is important for its biological activity. Like other members of the caerin family, it adopts an amphipathic α-helical structure in membrane environments, with hydrophobic amino acids positioned to interact with bacterial membranes . This structural arrangement is crucial for its antimicrobial function and selectivity.

What are the primary biological activities of Caerin-1.9?

Caerin-1.9 exhibits multiple biological activities:

• Strong antimicrobial activity against Gram-positive bacteria, including Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA)

• Effective against Gram-negative bacteria including Acinetobacter baumannii and Neisseria lactamica

• Potent activity against Streptococcus haemolyticus

• Inhibition of HIV transmission in vitro at concentrations non-toxic to host cells

• Anticancer effects, particularly against glioblastoma cell lines through modulation of mitochondrial function

• Minimal impact on beneficial bacteria like Lactobacillus rhamnosus and Lactobacillus crispatus at concentrations ≤25 μM

How does Caerin-1.9 compare to other antimicrobial peptides in the caerin family?

Caerin-1.9 has several distinctive characteristics compared to other caerin peptides:

• It shows the best profile of HIV inhibition with minimal impact on beneficial lactobacilli compared to caerin 1.2, caerin 1.10, and caerin 1.20

• At concentrations of 25 μM, caerin 1.9 allows approximately 83% growth of L. rhamnosus, while other caerin peptides reduce growth by 40-55%

• It demonstrates stronger inhibition of S. aureus and S. haemolyticus than caerin 1.1

• Caerin 1.9 is highly effective against N. lactamica at concentrations as low as 6.2 μM, showing greater potency than caerin 1.2

• When used in combination with caerin 1.1, it shows additive antibacterial effects against MRSA and A. baumannii

What are optimal protocols for antimicrobial activity assessment of Caerin-1.9?

When assessing the antimicrobial activity of Caerin-1.9, researchers should consider the following methodological approaches:

• Minimum Inhibitory Concentration (MIC) determination:

  • Use broth microdilution assays with appropriate growth media for each bacterial species

  • Test concentration ranges from 0.4-50 μM (as used in published studies)

  • Include appropriate positive (conventional antibiotics) and negative controls

  • Assess growth after 16-24 hours of incubation

• Resistance development assessment:

  • Use sequential passage method with sub-MIC concentrations

  • Continue for at least 30 rounds of in vitro culture, as this has previously demonstrated no resistance development

  • Compare with conventional antibiotics (e.g., Tazocin) as a positive control for resistance development

  • Determine MIC after each passage to track potential changes in susceptibility

• Selectivity testing:

  • Test activity against beneficial bacteria (Lactobacillus species) in parallel with pathogens

  • Assess impact on commensal versus pathogenic bacteria at the same concentration ranges

  • Determine therapeutic index by comparing antimicrobial concentrations to cytotoxic concentrations

How should researchers properly formulate Caerin-1.9 for in vivo studies?

For in vivo applications, several formulation strategies have been validated:

• Temperature-sensitive gel formulation:

  • This approach has been successfully used for skin applications in murine models

  • Caerin 1.1 and 1.9 prepared in temperature-sensitive gel effectively inhibit MRSA growth in skin infection models of multiple murine strains

  • The gel provides controlled release and maintains peptide activity

• Solution preparation for subcutaneous administration:

  • For pharmacokinetic studies, subcutaneous injection at concentrations up to 100 mg/kg has been shown to be safe in Sprague Dawley rats

  • Researchers should ensure proper dissolution and sterility

  • Buffer systems compatible with the peptide's stability profile should be used

• Stability considerations:

  • Caerin 1.9 is stable under various conditions including:

    • Heat resistant

    • Stable at pH range 5.5-7.4

    • Maintains stability at room temperature

  • These properties should be considered when designing formulations

What analytical techniques are most effective for characterizing Caerin-1.9?

Several analytical techniques are particularly valuable for Caerin-1.9 characterization:

• Mass spectrometry:

  • LC-MS/MS for sequence confirmation and purity assessment

  • Used in pharmacokinetic studies with a detection threshold of 40 ng/g in tissue samples

  • Essential for monitoring peptide integrity and potential degradation products

• Circular dichroism (CD) spectroscopy:

  • For secondary structure determination

  • Particularly useful for confirming α-helical conformation in membrane-mimetic environments

  • Allows monitoring of structural changes under different conditions

• Antimicrobial activity assays:

  • Functional characterization through growth inhibition assays

  • Membrane permeabilization assays using fluorescent dyes

  • Comparative testing against multiple bacterial strains to establish activity spectrum

What mechanisms explain Caerin-1.9's selective antimicrobial activity?

The selective activity of Caerin-1.9 against pathogens while sparing beneficial bacteria can be explained by several mechanisms:

• Differential membrane interaction:

  • Preferential binding to bacterial membranes with specific lipid compositions

  • The cationic nature of the peptide creates electrostatic attraction to negatively charged bacterial surfaces

  • Amphipathic structure allows insertion into bacterial membranes

  • Lower affinity for membranes of beneficial bacteria like Lactobacillus species

• Membrane disruption:

  • Forms pores or disrupts membrane integrity through various potential mechanisms:

    • "Carpet" model: Accumulation of peptides on membrane surface

    • "Barrel-stave" or "toroidal pore" models: Formation of structured pores

  • The VVPV motif in the sequence may be critical for specific membrane interactions

• Selective toxicity:

  • Minimal impact on Lactobacillus species at concentrations effective against pathogens

  • Different activity profiles against Gram-positive versus Gram-negative bacteria

  • Ability to target N. lactamica at concentrations as low as 6.2 μM

Why does Caerin-1.9 not induce bacterial resistance even after repeated exposure?

Unlike conventional antibiotics, Caerin-1.9 demonstrates a remarkable ability to avoid resistance development:

• Resistance development profile:

  • No bacterial resistance was observed after 30 rounds of in vitro culture, unlike the antibiotic Tazocin which did induce resistance

  • This property is maintained when caerin 1.9 is used in combination with caerin 1.1

• Mechanistic basis:

  • Primary membrane-targeting mechanism makes resistance development difficult

  • Unlike antibiotics targeting specific enzymes or metabolic pathways, membrane disruption would require fundamental changes to bacterial architecture

  • Multiple simultaneous mutations would be needed to alter membrane composition sufficiently

  • The rapid bactericidal action may not allow sufficient time for adaptive responses

• Clinical implications:

  • This resistance-resistant property makes Caerin-1.9 particularly valuable for addressing infections caused by multidrug-resistant pathogens

  • Provides potential advantages over conventional antibiotics, especially for MRSA infections

How does Caerin-1.9 interact with cancer cells to exhibit anticancer effects?

Caerin-1.9 demonstrates anticancer activity through several potential mechanisms:

• Direct effects on cancer cells:

  • Suppresses glioblastoma U87 and U118 cell proliferation

  • Modulates mitochondrial function in cancer cells

  • May exploit differences in membrane composition between cancer and normal cells

• Immune system modulation:

  • Improves the efficacy of therapeutic vaccines and immune checkpoint inhibitor therapy when injected intratumorally

  • Inhibits TC-1 tumor growth when applied topically through intact skin in a TC-1 murine tumor model

  • May enhance anti-tumor immune responses

• Potential clinical applications:

  • Could be used in combination with current immunotherapies for better management of solid tumors

  • Shows potential for both direct application and immune enhancement strategies

What is the pharmacokinetic profile of Caerin-1.9 in animal models?

Pharmacokinetic studies in Sprague Dawley rats have revealed:

• After subcutaneous injection at 10 mg/kg:

  • Peak concentration (Cmax) occurs at 1 hour post-injection

  • In male rats: Cmax = 591 ng/mL, half-life = 4.58 hr, AUC0-last = 1890 h × ng/mL

  • In female rats: Cmax = 256 ng/mL, half-life = 1.33 hr, AUC0-last = 740 h × ng/mL

• Dose-dependent parameters:

  • As injected concentration increases, half-life extends

  • Higher doses result in increased Cmax, AUC0-last, and volume of distribution

• No accumulation:

  • After 14 days of repeated subcutaneous injection at 10.0 mg/kg, no accumulation of Caerin-1.9 in plasma was observed

  • Suggests efficient clearance mechanisms

• Tissue distribution:

  • Caerin-1.9 levels in tissues were below the LC-MS/MS detection threshold (40 ng/g)

  • Indicates rapid clearance or metabolism

What safety considerations should researchers be aware of when working with Caerin-1.9?

Several important safety aspects should be considered:

• Toxicological profile:

  • Subcutaneous injection at doses up to 100 mg/kg is safe in SD rats

  • No mortality or organ malfunction was observed at this dose level

  • The substantial margin between effective antimicrobial concentrations and toxic doses suggests a favorable safety profile

• Selective activity:

  • Minimal impact on beneficial bacteria (Lactobacillus species) at concentrations ≤25 μM

  • Helps maintain healthy microbiota while targeting pathogens

• Handling considerations:

  • Standard biosafety practices for peptide handling should be followed

  • Although stable at room temperature and pH 5.5-7.4, optimal storage conditions should be established

  • Protection from proteolytic enzymes during storage and handling

What promising therapeutic applications exist for Caerin-1.9 beyond direct antimicrobial use?

Several promising applications warrant further investigation:

• HIV prevention:

  • Demonstrated ability to inhibit HIV infection at concentrations non-toxic to host cells

  • Potential development as topical microbicides

  • Favorable selectivity profile with minimal impact on beneficial vaginal flora

• Cancer immunotherapy:

  • Enhances efficacy of therapeutic vaccines and immune checkpoint inhibitors

  • Could be developed as an adjuvant for cancer immunotherapy

  • Both intratumoral and topical applications show promise in animal models

• Antibiotic-resistant infections:

  • Particularly valuable for MRSA infections due to lack of resistance development

  • Potential for topical applications for skin infections

  • Combination with caerin 1.1 shows additive effects

• Neisseria gonorrhoeae targeting:

  • High efficacy against Neisseria lactamica suggests potential against N. gonorrhoeae

  • Could address increasing antibiotic resistance in gonorrhea

What structural modifications might enhance Caerin-1.9's therapeutic potential?

Structure-activity relationships suggest several promising modification strategies:

• Sequence modifications:

  • Even minor modifications affect activity profiles, as seen with caerin 1.9 sm (with a Val→Leu substitution at position 13)

  • Targeted amino acid substitutions could enhance selectivity or potency

  • The importance of the VVPV motif and terminal amidation suggests these as critical regions for optimization

• Combination approaches:

  • The additive effect with caerin 1.1 suggests benefits of co-administration or fusion peptides

  • Synergistic combinations with conventional antibiotics could be explored

  • Conjugation to targeting moieties for enhanced delivery to specific tissues