Evaluation of deoxythymidine-based cationic amphiphiles as antimicrobial, antibiofilm, and anti-inflammatory agents.

OBJECTIVES: We recently designed a series of cationic deoxythymidine-based amphiphiles that mimic the cationic amphipathic structure of antimicrobial peptides (AMPs). Among these amphiphiles, ADG-2e and ADL-3e displayed the highest selectivity against bacterial cells. In this study, ADG-2e and ADL-3e were evaluated for their potential as novel classes of antimicrobial, antibiofilm, and anti-inflammatory agents. METHODS: Minimum inhibitory concentrations of ADG-2e and ADL-3e against bacteria were determined using the broth microdilution method. Proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K was determined by radial diffusion and HPLC analysis. Biofilm activity was investigated using the broth microdilution and confocal microscopy. The antimicrobial mechanism was investigated by membrane depolarization, cell membrane integrity analysis, scanning electron microscopy (SEM), genomic DNA influence and genomic DNA binding assay. Synergistic activity was evaluated using checkerboard method. Anti-inflammatory activity was investigated using ELISA and RT-PCR. RESULTS: ADG-2e and ADL-3e showed good resistance to physiological salts and human serum, and a low incidence of drug resistance. Moreover, they exhibit proteolytic resistance against pepsin, trypsin, α-chymotrypsin, and proteinase K. ADG-2e and ADL-3e were found to kill bacteria by an intracellular target mechanism and bacterial cell membrane-disrupting mechanism, respectively. Furthermore, ADG-2e and ADL-3e showed effective synergistic effects when combined with several conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Pseudomonas aeruginosa (MDRPA). Importantly, ADG-2e and ADL-3e not only suppressed MDRPA biofilm formation but also effectively eradicated mature MDRPA biofilms. Furthermore, ADG-2e and ADL-3e drastically decreased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) gene expression and protein secretion in lipopolysaccharide (LPS)-stimulated macrophages, implying potent anti-inflammatory activity in LPS-induced inflammation. CONCLUSION: Our findings suggest that ADG-2e and ADL-3e could be further developed as novel antimicrobial, antibiofilm, and anti-inflammatory agents to combat bacterial infections.

Cell selectivity and antibiofilm and anti-inflammatory activities and antibacterial mechanism of symmetric-end antimicrobial peptide centered on D-Pro-Pro.

This study aimed to develop a new symmetric-end antimicrobial peptide (AMP) with cell selectivity, antibiofilm, and anti-inflammatory activities. Two symmetric-end AMPs, Lf6-pP and Lf6-GG, were designed based on the sequence RRWQWRzzRWQWRR, which contains two symmetric repeat sequences connected by a ß-turn-promoting sequence (zz) that can be a rigid turn by D-Pro-Pro (pP) or a flexible turn by Gly-Gly (GG). Both Lf6-pP and Lf6-GG exhibited potent antibacterial activity without causing hemolysis, but Lf6-pP exhibited better cell selectivity, likely due to the more significant impact of the rigid pP turn. Compared to Lf6-GG, Lf6-pP demonstrated approximately three times higher antimicrobial activity against drug-resistant bacteria, had a low incidence of drug resistance, and maintained its activity in the presence of physiological salts and human serum. Additionally, Lf6-pP was more effective than Lf6-GG in inhibiting biofilm formation and eradicating mature biofilms. The BODIPY-cadaverine assay indicated that the potent anti-inflammatory activity of Lf6-pP may be attributed to its direct interaction with LPS, resulting in decreased TNF-α and IL-6 levels in LPS-stimulated macrophages. Mechanistic studies, including membrane depolarization, outer/inner membrane permeation, and membrane integrity change, demonstrated that Lf6-pP exerts its antibacterial action through an intracellular-target mechanism. Overall, we propose that Lf6-pP has potential as a novel antibacterial, antibiofilm, and anti-inflammatory agent against drug-resistant bacterial infections.

Cationic, amphipathic small molecules based on a triazine-piperazine-triazine scaffold as a new class of antimicrobial agents.

Poor proteolytic resistance, toxicity and salt/serum sensitivity of antimicrobial peptides (AMPs) limits their practical clinical application. Here, to overcome these drawbacks of AMPs and develop novel antimicrobial agents, a series of small molecules based on a triazine-piperazine-triazine scaffold that mimic the cationic amphipathic structure of AMPs were synthesized and evaluated their potential as a new class of antimicrobial agents. All designed compounds showed strong antimicrobial activity and negligible hemolytic activity. Particularly, five compounds (9, 11, 12, 15, and 16) presented excellent cell selectivity with proteolytic resistance, salt/serum stability and anti-inflammatory activity against lipopolysaccharide (LPS)-induced inflammation. These five compounds exhibited similar or 2-4 fold higher antimicrobial activity than melittin against six antibiotic-resistant bacteria tested. Similar to the intracellular-targeting AMP, buforin-2, these compounds displayed an intracellular mode of antimicrobial action. These compounds showed potent biofilm inhibitory and eradicating activities against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Additionally, these compounds displayed synergistic or additive effects when combined with selected clinically used antibiotics. Furthermore, these compounds have been proven to inhibit pro-inflammatory cytokine release by directly binding to LPS and blocking the interaction between LPS and CD14/TLR4 receptor in LPS-stimulated RAW264.7 macrophage cells. Overall, our results demonstrate the potential of the designed compounds as a novel class of multifunctional antimicrobial agents to combat bacterial infection.

A novel hybrid peptide composed of LfcinB6 and KR-12-a4 with enhanced antimicrobial, anti-inflammatory and anti-biofilm activities.

Hybridizing two known antimicrobial peptides (AMPs) is a simple and effective strategy for designing antimicrobial agents with enhanced cell selectivity against bacterial cells. Here, we generated a hybrid peptide Lf-KR in which LfcinB6 and KR-12-a4 were linked with a Pro hinge to obtain a novel AMP with potent antimicrobial, anti-inflammatory, and anti-biofilm activities. Lf-KR exerted superior cell selectivity for bacterial cells over sheep red blood cells. Lf-KR showed broad-spectrum antimicrobial activities (MIC: 4-8 µM) against tested 12 bacterial strains and retained its antimicrobial activity in the presence of salts at physiological concentrations. Membrane depolarization and dye leakage assays showed that the enhanced antimicrobial activity of Lf-KR was due to increased permeabilization and depolarization of microbial membranes. Lf-KR significantly inhibited the expression and production of pro-inflammatory cytokines (nitric oxide and tumor necrosis factor-α) in LPS-stimulated mouse macrophage RAW264.7 cells. In addition, Lf-KR showed a powerful eradication effect on preformed multidrug-resistant Pseudomonas aeruginosa (MDRPA) biofilms. We confirmed using confocal laser scanning microscopy that a large portion of the preformed MDRPA biofilm structure was perturbed by the addition of Lf-KR. Collectively, our results suggest that Lf-KR can be an antimicrobial, anti-inflammatory, and anti-biofilm candidate as a pharmaceutical agent.

Proadrenomedullin N-terminal 20 peptide (PAMP) and its C-terminal 12-residue peptide, PAMP(9-20): Cell selectivity and antimicrobial mechanism.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a regulatory peptide that is found in various cell types. It is involved in many biological activities and is rich in basic and hydrophobic amino acids, a common feature of antimicrobial peptides (AMPs). In this study, the cell selectivity and antimicrobial mechanism of PAMP and its C-terminal peptide, PAMP(9-20), were investigated. PAMP and PAMP(9-20) displayed potent antimicrobial activity (minimum inhibitory concentration: 4-32 µM) against standard bacterial strains, but showed no hemolytic activity even at the highest tested concentration of 256 µM. PAMP(9-20) showed 2- to 4-fold increase in antimicrobial activity against gram-negative bacteria compared to PAMP. Cytoplasmic membrane depolarization, leakage of calcein dye from membrane mimic liposomes, SYTOX Green uptake, membrane permeabilization, and flow cytometry studies indicated that the major target of PAMP and PAMP(9-20) is not the microbial cell membrane. Interestingly, laser-scanning confocal microscopy demonstrated that FITC-labeled PAMP and PAMP(9-20) enter the cytoplasm of Escherichia coli similar to buforin-2, and gel retardation assay indicated that PAMP and PAMP(9-20) effectively bind to bacterial DNA. These results suggest that the intracellular target mechanism is responsible for the antimicrobial action of PAMP and PAMP(9-20). Collectively, PAMP and PAMP(9-20) could be considered promising candidates for the development of new antimicrobial agents.