Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights From Experimental and Molecular Dynamics Simulations Studies

Abstract

Background/Aim: Host defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and the secondary structure and underlying mechanism of action of CaD23.Methods: Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard and time-kill assays. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FIC<0.5), additive (FIC between 0.5–1.0), indifferent (FIC between >1.0 and ≤4), or antagonistic (FIC>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes. Circular dichroism (CD) spectroscopy was also performed to examine the secondary structures of CaD23.Results: CaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI = 0.60–0.69) and MRSA43300 (FICI = 0.56–0.60) but an indifferent effect against P. aeruginosa (FIC = 1.03–1.15). CaD23 (at 25 μg/ml; 2xMIC) completely killed S. aureus within 30 min. When used at sub-MIC concentration (3.1 μg/ml; 0.25xMIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23. The secondary structures of CaD23 observed in MD simulations were validated by CD spectroscopy.Conclusion: CaD23 is a novel alpha-helical, membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulations serves as a powerful tool in revealing the peptide secondary structure, dissecting the mechanism of action, and guiding the design and optimisation of HDPs.

Pixantrone Sensitizes Gram-Negative Pathogens to Rifampin

“…Similarly, SYTOX Green (final concentration of 5 μM) ( 58 ) and propidium iodide (PI, final concentration of 10 μM) ( 8 ) were applied to evaluate the cell membrane integrity with excitation/emission wavelengths of 504 nm/523 nm and 535 nm/615 nm, respectively. In addition, the membrane potential was measured using 3,3-dipropylthiadicarbocyanine iodide (DiSC3[5], final concentration of 2 μM) ( 59 ) with an excitation/emission wavelength of 622 nm/670 nm with an interval of 5 min for 30 min.…”

Host Defence Peptides: A Potent Alternative to Combat Antimicrobial Resistance in the Era of the COVID-19 Pandemic

“…Continuing on, our group has recently developed a library of novel bactericidal peptides through hybridisation of two different HDPs sequences, i.e., combining the benefits of two classes of HDPs into one molecule. A hybrid derivative of LL-37 and HBD-3, termed CaD23, was shown to enhance the potency of amikacin against Staphylococcus aureus [145,146]. Overall, antimicrobials targeting resistant pathogens will have their effect enhanced by HDP adjuvants and so reducing the effect of AMR.…”

“…Our group has fully characterised a range of HDPs on human corneal surface during disease and health [50,[68][69][70][71][72][183][184][185][186]. Recent studies from our laboratory showed that synthetic HDPs can be utilised for the treatment of Gram-positive and Gram-negative corneal infections [144][145][146].…”

Krein support vector machine classification of antimicrobial peptides

“…11,12 Owing to the complex SAR and the costly and time-consuming process of wet-lab experiments associated with AMP investigations, many researchers have proposed computational approaches, including molecular dynamics (MD) simulations and machine learning (ML) algorithms, to accelerate the discovery and development of potential AMPs for clinical use. [13][14][15][16][17][18][19] Several studies have highlighted the promise of ML algorithms in predicting the antimicrobial activity, dissecting the complex SAR, and informing the drug design of AMPs. [13][14][15] A wide range of ML algorithms have been utilised, including random forests, 20 support vector machines (SVMs) [20][21][22][23][24] and articial neural networks.…”

“…[13][14][15][16][17][18][19] Several studies have highlighted the promise of ML algorithms in predicting the antimicrobial activity, dissecting the complex SAR, and informing the drug design of AMPs. [13][14][15] A wide range of ML algorithms have been utilised, including random forests, 20 support vector machines (SVMs) [20][21][22][23][24] and articial neural networks. [20][21][22]25,26 Many of these algorithms are used in combination with a carefully selected set of peptide features, which can be divided into two categories: compositional and physicochemical.…”