Molecular Dynamics Simulation of the Interaction of Two Linear Battacin Analogs with Model Gram-Positive and Gram-Negative Bacterial Cell Membranes.

Antimicrobial peptides (AMPs) are a potential solution to the increasing threat of antibiotic resistance, but successful design of active but nontoxic AMPs requires understanding their mechanism of action. Molecular dynamics (MD) simulations can provide atomic-level information regarding how AMPs interact with the cell membrane. Here, we have used MD simulations to study two linear analogs of battacin, a naturally occurring cyclic, lipidated, nonribosomal AMP. Like battacin, these analogs are active against Gram-negative multidrug resistant and Gram-positive bacteria, but they are less toxic than battacin. Our simulations show that this activity depends upon a combination of positively charged and hydrophobic moieties. Favorable interactions with negatively charged membrane lipid head groups drive association with the membrane and insertion of hydrophobic residues, and the N-terminal lipid anchors the peptides to the membrane surface. Both effects are required for stable membrane binding.

Covalently Immobilized Battacin Lipopeptide Gels with Activity against Bacterial Biofilms.

Novel antibiotic treatments are in increasing demand to tackle life-threatening infections from bacterial pathogens. In this study, we report the use of a potent battacin lipopeptide as an antimicrobial gel to inhibit planktonic and mature biofilms of S. aureus and P. aeruginosa. The antimicrobial gels were made by covalently linking the N-terminal cysteine containing lipopeptide (GZ3.163) onto the polyethylene glycol polymer matrix and initiating gelation using thiol-ene click chemistry. The gels were prepared both in methanol and in water and were characterised using rheology, Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). Antibacterial and antibiofilm analyses revealed that the gels prepared in methanol have better antibacterial and antibiofilm activity. Additionally, a minimum peptide content of 0.5 wt% (relative to polymer content) is required to successfully inhibit the planktonic bacterial growth and disperse mature biofilms of P. aeruginosa and S. aureus. The antibacterial activity of these lipopeptide gels is mediated by a contact kill mechanism of action. The gels are non-haemolytic against mouse red blood cells and are non-cytotoxic against human dermal fibroblasts. Findings from this study show that battacin lipopeptide gels have the potential to be developed as novel topical antibacterial agents to combat skin infections, particularly caused by S. aureus.

Novel Cell-Penetrating Peptide Conjugated Proteasome Inhibitors: Anticancer and Antifungal Investigations.

Cell-penetrating peptide conjugated peptide aldehydes Tat-A and Tat-B showed low micromolar anticancer and antifungal activities and synergistic action in combination with cisplatin and amphotericin B against cancer and fungal cells, respectively. Tat-A and Tat-B were significantly more potent than Ixazomib in inhibiting the human 20S proteasomes with IC50 values in the low nanomolar range. Treatment with Tat-A and Tat-B caused membrane disruption and pore formation in HeLa and BE(2)-C cells and inhibition and eradication of C. albicans biofilms. Apoptotic cell death of the treated HeLa and BE(2)-C cells was demonstrated by Annexin V/PI staining. Flow cytometry analyses showed that more than 78% (HeLa) and 92% (BE(2)-C cells showed signs of apoptosis and necrosis upon treatment with Tat-A and Tat-B. This study forms the first report that documents the benefits of cell-penetrating peptide conjugation to enhance the potential of peptide aldehydes as therapeutics.

Solution structure of linear battacin lipopeptides - the effect of lengthening fatty acid chain.

In recent years, lipopeptides have received attention for their enhanced antimicrobial activity, especially against multi-drug resistant (MDR) pathogens. We have previously reported that the bacterial soil extracted, novel cyclic lipopeptide, battacin, and its synthetic analogues have enhanced antimicrobial activity against various Gram negative, Gram positive and fungal pathogens. In particular, the modification of the hydrophobic fatty acid chain and molecular structure has improved its activity. We have used small angle X-ray scattering (SAXS) and circular dichroism (CD) to characterise the low resolution structure of battacin lipopeptides containing covalently bonded fatty acid chains and the one without it. In the absence of fatty acids or with short fatty acid chain, the peptides adopted an extended random coil structure that is best described barbell-like shape, while fatty acids that are sufficiently long induced an aggregation into a ∼4.0 nm diameter core shell sphere. While the kinked structure found within this barbell shape may have a role in antimicrobial activities, the self-assembly of the battacin analogue with the longest fatty acid chain may have a correlation to the declined antibacterial activities.

Battacin-Inspired Ultrashort Peptides: Nanostructure Analysis and Antimicrobial Activity.

Peptides can serve as versatile therapeutics with a highly modular structure and tunable biophysical properties. In particular, the efficacy of peptide antibiotics against drug-resistant pathogens is of great promise, as few new classes of antibiotics are being developed to overcome the ever-increasing bacterial resistance to contemporary drugs. This work reports biophysical and antimicrobial studies of a designed library of ultrashort peptides that self-assemble into hydrogels at concentrations as low as 0.5% w/v in buffered saline, as confirmed by rheology. The hydrogels are constituted by ß-sheet-rich nanofibril networks, as determined by biophysical techniques including spectroscopy (attenuated total reflectance Fourier transform infrared spectroscopy and Congo red binding assay), short- and wide-angle X-ray scattering, and electron microscopy. Both peptide solutions and self-assembled hydrogels show potent antimicrobial activity against S. aureus and Pseudomonas aeruginosa by membrane lysis. These peptides also displayed selectivity toward bacterial cells over human dermal fibroblasts in vitro, as determined from Live/Dead, scanning electron microscopy, and coculture assays. This work reports an antimicrobial self-assembling motif of only three residues comprising an aromatically acylated cationic d-Dab/Lys amino acid, a second cationic residue, and naphthylalanine that heavily influences the self-assembly of these peptides into hydrogels. The variations in the antimicrobial activity and self-assembly properties between analogues may have implications in future studies on the correlation between self-assembly and biological activity in ultrashort peptides.

Unexplored antifungal activity of linear battacin lipopeptides against planktonic and mature biofilms of C. albicans.

Novel antifungal agents are required against pathogenic fungi such as Candida albicans. We report the anticandidal activity of battacin lipopeptide antibiotics with previously unexplored antifungal activity. From amongst sixteen battacin lipopeptides tested against C. alibicans (SC5314) the 4-methyl hexanoyl conjugated trimeric lipopeptide 13 emerged as the lead candidate with a MIC of 6.25 µM and negligible haemolysis of mouse red blood cells. The potency of this lipopeptide was maintained under acidic conditions. Additionally, antifungal activity was further enhanced with amphotericin B at its non-haemolytic concentrations. Herein we have demonstrated for the first time that battacin lipopeptides prevent C. albicans biofilm colonisation as well as inhibit pre-formed biofilms of this fungal pathogen. XTT biofilm assays revealed that 13 prevented colonisation of C. albicans biofilms at its MIC (6.25 µM) and, at a higher concentration, eradicated 24 h (25 µM) and 48 h (62.5 µM) old preformed biofilms. In comparison, we found that amphotericin at much lower concentrations prevented biofilm colonisation (0.78 µM) and inhibited 24 h old preformed biofilms (6.25 µM), however was completely inactive against 48 h old preformed biofilms. Thus, lipopeptide 13 is more effective than amphotericin at eradicating more mature C. albicans biofilms. The membrane lytic mechanism of action of compound 13 was validated by a colorimetric assay using lipid vesicles mimicking fungal membranes in which compound 13 effected an immediate dark purple to red colour transition of suspended vesicles upon peptide interaction. In addition, TEM images of C. albicans cells exposed to 13 showed clearly disrupted cellular membranes. Interestingly, compound 13 increased the endogenous generation of reactive oxygen species (ROS) in a concentration dependent manner. In the presence of an antioxidant, ascorbic acid, ROS production was diminished yet antifungal activity persisted, possibly indicating that ROS production is a secondary effect from membrane lysis caused by lipopeptide 13. The lipopeptide was non-haemolytic against mouse red blood cells at the highest tested concentration (1 mM).

Feasibility Study Exploring the Potential of Novel Battacin Lipopeptides as Antimicrobial Coatings.

Colonization of medical implant surfaces by pathogenic microorganisms causes implant failure and undermines their clinical applicability. Alarming increase in multidrug-resistant bacteria poses serious concerns with the use of medical implants. Antimicrobial peptides (AMPs) that form part of the innate immune system in all forms of life are attractive alternatives to conventional antibiotics to treat multidrug-resistant bacterial biofilms. The aim of this study was to assess the in vitro antibacterial potency of our recently discovered lipopeptides from the battacin family upon immobilization to various surfaces. To achieve this, glass, silicon, and titanium surfaces were functionalized through silanization followed by addition of the heterobifunctional cross-linker, succinimidyl-[N-maleimidopropionamido]-poly(ethylene glycol) ester to generate maleimide-functionalized surfaces. The lipopeptide, GZ3.27, with an added N-terminal cysteine was covalently coupled to the surfaces via a thioether bond through a Michael-type addition between the cysteine sulfhydryl group and the maleimide moiety. Success of surface immobilization and antimicrobial activity of the coated surfaces was assessed using water contact angle measurements, X-ray photoelectron spectroscopy, ellipsometry, scanning electron microscopy, colony forming unit assays and biofilm analysis. The lipopeptide-coated surfaces caused significant damage to the cellular envelop of Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) upon contact and prevented surface colonization by P. aeruginosa and E. coli biofilms. The lipopeptides investigated in this study were not hemolytic to mouse blood cells in solution. Findings from this study indicate that these lipopeptides have the potential to be developed as promising antimicrobial coatings on medical implants.

Synthesis and cytotoxicity of thieno[2,3-b]quinoline-2-carboxamide and cycloalkyl[b]thieno[3,2-e]pyridine-2-carboxamide derivatives.

Seventy nine derivatives of thieno[2,3-b]quinolines, tetrahydrothieno[2,3-b]quinoline, dihydrocyclopenta[b]thieno[3,2-e]pyridine, cyclohepta[b]thieno[3,2-e]pyridine and hexahydrocycloocta[b]thieno[3,2-e]pyridine were either synthesized or obtained commercially and tested for their antiproliferative activity against HCT116, MDA-MB-468 and MDA-MB-231 human cancer cell lines. The most potent eight compounds were active against all cell lines with IC50 values in the 80-250nM range. In general hexahydrocycloocta[b]thieno[3,2-e]pyridines were most active with increasing activity observed as larger cycloalkyl rings were fused to the pyridine ring.

Antimicrobial peptides with potential for biofilm eradication: synthesis and structure activity relationship studies of battacin peptides.

We report on the first chemical syntheses and structure-activity analyses of the cyclic lipopeptide battacin which revealed that conjugation of a shorter fatty acid, 4-methyl-hexanoic acid, and linearization of the peptide sequence improves antibacterial activity and reduces hemolysis of mouse blood cells. This surprising finding of higher potency in linear lipopeptides than their cyclic counterparts is economically beneficial. This novel lipopeptide was membrane lytic and exhibited antibiofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, and, for the first time, Pseudomonas syringe pv. actinidiae. The peptide was unstructured in aqueous buffer and dimyristoylphosphatidylcholine-polymerized diacetylene vesicles, with 12% helicity induced in 50% v/v of trifluoroethanol. Our results indicate that a well-defined secondary structure is not essential for the observed antibacterial activity of this novel lipopeptide. A truncated pentapeptide conjugated to 4-methyl hexanoic acid, having similar potency against Gram negative and Gram positive pathogens was identified through alanine scanning.

Antimicrobial peptides against Pseudomonas syringae pv. actinidiae and Erwinia amylovora: Chemical synthesis, secondary structure, efficacy, and mechanistic investigations.

We report on structurally modified dodecapeptide amides (KYKLFKKILKFL-NH2) and two analogs of a hexapeptide amide (WRWYCR-NH2) with antibacterial activity against the Gram negative pathogens Pseudomonas syringae pv. actinidiae (Psa) and Erwinia amylovora (Ea). Dodecapeptide minimal inhibitory concentrations (MICs) ranged from 3.2 to 15.4 µM, with the unmodified peptide being the most potent against both pathogens. The unmodified dodecapeptide also had 32-58% α-helicity in membrane mimetic environments (50% v/v trifluoroethanol and 30 mM SDS micelles). Structural modifications which included branching, acylation, and conjugation with 5-nitro-2-furaldehyde (NFA) proved detrimental to both antimicrobial activity and α-helicity. Scanning electron microscopy (SEM) revealed distinct morphological changes to bacterial cells treated with the different peptides, leading to blistering of the membrane and cell lysis. MICs of the hexapeptide amide were 3.9-7.7 µM against both pathogens. The hexapeptide acid did not show anti-bacterial activity against either pathogen. However, the NFA conjugated hexapeptide acid was more active than the parent peptide or NFA alone with MICs of 1.6-3.2 µM against the pathogens. SEM analysis revealed shriveling and collapse of bacterial cells treated with the hexapeptide, whereas shortening and compactness on exposure to streptomycin. A colorimetric assay demonstrated that the dodecapeptides were likely to act by targeting the bacterial membrane, whereas the hexapeptides, streptomycin, and NFA were not, thereby supporting the morphological changes observed during SEM. To the best of our knowledge, this appears to be the first report of antimicrobial peptide activity against Psa, a pathogen that is currently devastating the kiwifruit industry internationally.