Antimicrobial peptides (AMPs) are promising next generation antibiotics that hold great potential for combating bacterial resistance. AMPs can be both bacteriostatic and bactericidal, induce rapid killing and display a lower propensity to develop resistance than do conventional antibiotics. Despite significant progress in the past 30 years, no peptide antibiotic has reached the clinic yet. Poor understanding of the action mechanisms and lack of rational design principles have been the two major obstacles that have slowed progress. Technological developments are now enabling multidisciplinary approaches including molecular dynamics simulations combined with biophysics and microbiology toward providing valuable insights into the interactions of AMPs with membranes at atomic level. This has led to increasingly robust models of the mechanisms of action of AMPs and has begun to contribute meaningfully toward the discovery of new AMPs. This review discusses the detailed action mechanisms that have been put forward, with detailed atomistic insights into how the AMPs interact with bacterial membranes. The review further discusses how this knowledge is exploited toward developing design principles for novel AMPs. Finally, the current status, associated challenges, and future directions for the development of AMP therapeutics are discussed.

The proteins found in tears have an important role in the maintenance of the ocular surface and changes in the quality and quantity of tear components reflect changes in the health of the ocular surface. In this study, we have used quantitative proteomics, iTRAQ technology coupled with 2D-nanoLC-nano-ESI-MS/MS and with a statistical model to uncover proteins that are significantly and reliably changed in the tears of dry eye patients in an effort to reveal potential biomarker candidates. Fifty-six patients with dry eye and 40 healthy subjects were recruited for this study. In total, 93 tear proteins were identified with a ProtScore >or=2 (>or=99% confidence). Associated with dry eye were 6 up-regulated proteins, alpha-enolase, alpha-1-acid glycoprotein 1, S100 A8 (calgranulin A), S100 A9 (calgranulin B), S100 A4 and S100 A11 (calgizzarin) and 4 down-regulated proteins, prolactin-inducible protein (PIP), lipocalin-1, lactoferrin and lysozyme. Receiver operating curves (ROC) were evaluated for individual biomarker candidates and a biomarker panel. With the use of a 4-protein biomarker panel, the diagnostic accuracy for dry eye was 96% (sensitivity, 91.0%; specificity, 90.0%). Two biomarker candidates (alpha-enolase and S100 A4) generated from iTRAQ experiments were successfully verified using an ELISA assay. The levels of these 10 tear proteins reflect aqueous secretion deficiency by lacrimal gland, inflammatory status of the ocular surface. The clinical classification of the severity of the dry eye condition was successfully correlated to the proteomics by using three proteins that are associated with inflammation, alpha1-acid glycoprotein 1, S100 A8 and S100 A9. The nine tear protein biomarker candidates (except alpha1-acid glycoprotein 1) were also verified using an independent age-matched patient sample set. This study demonstrated that iTRAQ technology combined with 2D-nanoLC-nanoESI-MS/MS quantitative proteomics is a powerful tool for biomarker discovery.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has created the need for better therapeutic options. In this study, five natural xanthones were extracted and purified from the fruit hull of Garcinia mangostana and their antimicrobial properties were investigated. α-Mangostin was identified as the most potent among them against Gram-positive pathogens (MIC=0.78-1.56 μg/mL) which included two MRSA isolates. α-Mangostin also exhibited rapid in vitro bactericidal activity (3-log reduction within 5 min). In a multistep (20 passage) resistance selection study using a MRSA isolated from the eye, no resistance against α-mangostin in the strains tested was observed. Biophysical studies using fluorescence probes for membrane potential and permeability, calcein encapsulated large unilamellar vesicles and scanning electron microscopy showed that α-mangostin rapidly disrupted the integrity of the cytoplasmic membrane leading to loss of intracellular components in a concentration-dependent manner. Molecular dynamic simulations revealed that isoprenyl groups were important to reduce the free energy for the burial of the hydrophobic phenyl ring of α-mangostin into the lipid bilayer of the membrane resulting in membrane breakdown and increased permeability. Thus, we suggest that direct interactions of α-mangostin with the bacterial membrane are responsible for the rapid concentration-dependent membrane disruption and bactericidal action.

Antibiotic resistance is a critical global health care crisis requiring urgent action to develop more effective antibiotics. Utilizing the hydrophobic scaffold of xanthone, we identified three components that mimicked the action of an antimicrobial cationic peptide to produce membrane-targeting antimicrobials. Compounds 5c and 6, which contain a hydrophobic xanthone core, lipophilic chains, and cationic amino acids, displayed very promising antimicrobial activity against multidrug-resistant Gram-positive bacteria, including MRSA and VRE, rapid time-kill, avoidance of antibiotic resistance, and low toxicity. The bacterial membrane selectivity of these molecules was comparable to that of several membrane-targeting antibiotics in clinical trials. 5c and 6 were effective in a mouse model of corneal infection by S. aureus and MRSA. Evidence is presented indicating that 5c and 6 target the negatively charged bacterial membrane via a combination of electrostatic and hydrophobic interactions. These results suggest that 5c and 6 have significant promise for combating life-threatening infections.

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This work describes how to tune the amphiphilic conformation of α-mangostin, a natural compound that contains a hydrophobic xanthone scaffold, to improve its antimicrobial activity and selectivity for Gram-positive bacteria. A series of xanthone derivatives was obtained by cationic modification of the free C3 and C6 hydroxyl groups of α-mangostin with amine groups of different pKa values. Modified structures using moieties with high pKa values, such as AM-0016 (3b), exhibited potent antimicrobial properties against Gram-positive bacteria. Compound 3b also killed bacteria rapidly without inducing drug resistance and was nontoxic when applied topically. Biophysical studies and molecular dynamics simulations revealed that 3b targets the bacterial inner membrane, forming an amphiphilic conformation at the hydrophobic-water interface. In contrast, moieties with low pKa values reduced the antimicrobial activity of the parent compound when conjugated to the xanthone scaffold. This strategy provides a new way to improve "hits" for the development of membrane-active antibiotics that target drug-resistant pathogens.

A new series of semisynthetic flavone-based small molecules mimicking antimicrobial peptides has been designed from natural icaritin to combat drug-resistant Gram-positive bacterial infections. Compound 6 containing two arginine residues exhibited excellent antibacterial activity against Gram-positive bacteria, including MRSA, and very low toxicity to mammalian cells, resulting in a high selectivity of more than 511, comparable to that of several membrane-active antibiotics in clinical trials. Our data show for the first time that icaritin derivatives effectively kill bacteria. Meanwhile, this is the first study deploying a biomimicking strategy to design potent flavone-based membrane targeting antimicrobials. 6 showed rapid bactericidal activity by disrupting the bacterial membrane and can circumvent the development of bacterial resistance. Importantly, 6 was highly efficacious in a mouse model of corneal infection caused by MRSA and Staphylococcus aureus.

A series of engineered linear analogues [coded as F6, W6, Y6, A6, S6 and C(Acm)6] were modeled, designed, synthesized and structurally characterized by mass spectra, circular dichroism, hydrophobicity analysis and molecular modeling. We have screened antimicrobial activity, hemolysis to rabbit erythrocytes, and cytotoxicity to human conjunctival epithelial cells. No significant hemolytic effect was observed for hBD3 or from five of the six analogues [F6, Y6, A6, S6 and C(Acm)6] over the range of 3-100 microg mL(-1). The six linear analogues have reduced cytotoxicity to human conjunctival epithelial cells over the range of 6-100 microg mL(-1) compared to hBD3. By tuning the overall hydrophobicity of linear hBD3 analogues, reduced cytotoxicity and hemolysis were obtained while preserving the antimicrobial properties. The decreased cytotoxicity of the linear analogues is suggested to be structurally related to the removal of disulfide bridges, and the flexible structure of the linear forms, which seem to be associated with loss of secondary structure. These results suggest a new approach for guiding the design of new linear analogues of defensin peptides with strong antibiotic properties and reduced cytotoxicity to mammalian cells.