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The cyclic peptide cyclo(Val-Leu-Leu-D-Phe-Pro)2 (peptide 1) was specifically designed for structural chemistry investigations, drawing inspiration from Gramicidin S (GS). Previous studies have shown that Pro residues within 1 adopt a down-puckering conformation of the pyrrolidine ring. By incorporating fluoride-Pro with 4-trans/cis-isomers into 1, an up-puckering conformation was successfully induced. In the current investigation, introducing hydroxyprolines with 4-trans/cis-isomer configurations (tHyp/cHyp) into 1 gave cyclo(Val-Leu-Leu-D-Phe-tHyp)2 methanol disolvate monohydrate, C62H94N10O12·2CH4O·H2O (4), and cyclo(Val-Leu-Leu-D-Phe-cHyp)2 monohydrate, C62H94N10O12·H2O (5), respectively. However, the puckering of 4 and 5 remained in the down conformation, regardless of the geometric position of the hydroxyl group. Although the backbone structure of 4 with trans-substitution was asymmetric, the asymmetric backbone of 5 with cis-substitution was unexpected. It is speculated that the anticipated influence of stress from the geometric positioning, which was expected to affect the puckering, may have been mitigated by interactions between the hydroxyl groups of hydroxyproline, the solvent molecules, and peptides.

Gramicidin S (GS), one of the first discovered antimicrobial peptides, still shows strong antibiotic activity after decades of clinical use, with no evidence of resistance. The relatively high hemolytic activity and narrow therapeutic window of GS limit its use in topical applications. Encapsulation and targeted delivery may be the way to develop the internal administration of this drug. The lipid composition of membranes and non-covalent interactions affect GS's affinity for and partitioning into lipid bilayers as monomers or oligomers, which are crucial for GS activity. Using both differential scanning calorimetry (DSC) and FTIR methods, the impact of GS on dipalmitoylphosphatidylcholine (DPPC) membranes was tested. Additionally, the combined effect of GS and cholesterol on membrane characteristics was observed; while dipalmitoylphosphatydylglycerol (DPPG) and cerebrosides did not affect GS binding to DPPC membranes, cholesterol significantly altered the membrane, with 30% mol concentration being most effective in enhancing GS binding. The effect of star-like dextran-polyacrylamide D-g-PAA(PE) on GS binding to the membrane was tested, revealing that it interacted with GS in the membrane and significantly increased the proportion of GS oligomers. Instead, calcium ions affected GS binding to the membrane differently, with independent binding of calcium and GS and no interaction between them. This study shows how GS interactions with lipid membranes can be effectively modulated, potentially leading to new formulations for internal GS administration. Modified liposomes or polymer nanocarriers for targeted GS delivery could be used to treat protein misfolding disorders and inflammatory conditions associated with free-radical processes in cell membranes. [ABSTRACT FROM AUTHOR]

Background: Natural antimicrobial peptides are used in the fight against pathogens resistant to existing synthetic antibiotics. The non-specific mechanism of cytostatic action of antimicrobial peptides, in particular gramicidin S, against bacteria is also effective for damaging the cells of neoplasms. The existence of such a property in a registered antibiotic will indicate its antineoplastic potential and can be used to expand the spectrum of its therapeutic application. Aim of work is to clarify the possible antitumor effect of the antimicrobial peptide gramicidin S. Materials and Methods: Using the methods of confocal laser microscopy and light microscopy, the morphological and functional features of connective tissue cells under the influence of gramicidin S in the concentration range 0.5–50 μg/ml were studied using L929 fibroblasts cell culture. The cell area, nucleus area, and nucleus-to-cytoplasm ratio were determined. To study the migratory and proliferative activity of cells in vitro, the “scratch assay” was used, the confluency of the monolayer of cells was evaluated, morphometric studies were performed, and the relative area of the scratch was measured after 24, 48, and 72 hours. Results: The lytic effect of gramicidin S in a concentration of 50 μg/ml on L929 cells was established, in concentrations of 0.5 μg/ml and 5.0 μg/ml, the antibiotic increases the synthetic activity of cells and stimulates the proliferation of fibroblasts in a monolayer. Cell anisomorphism is more pronounced in the presence of 5.0 μg/ml gramicidin S added to the culturing medium during monolayer formation, while a one-third of the cells in the sample form a population that is morphologically different from other cells in the culture. The addition of gramicidin S at non-lytic concentrations of 0.5 and 5.0 μg/ml to unattached fibroblasts reliably inhibits monolayer formation. Under the influence of 5.0 μg/ml gramicidin S, the rate of monolayer formation is low, even despite the significant content of cells with a high nuclear-cytoplasmic ratio. The kinetics of filling the cell monolayer defect using the “scratch assay” shows that GS in concentrations of 0.5 and 5.0 μg/ml can control the migratory and proliferative properties of L929 cells. Conclusions: The effect of gramicidin S on the morphometric parameters of cells depends on the concentration of the peptide and the cell status in the culture. GS corrupts the adhesive properties of L929 fibroblasts in monolayer cell culture and the rate of cell monolayer formation. Cells at the stage of attachment and monolayer formation were most sensitive to non-lytic concentrations of GS. Inhibition of the adhesive properties of connective tissue cells by gramicidin S is a new non-canonical effect of a known antimicrobial drug, which may indicate the possibility of using gramicidin S as an anti-neoplasm agent.

Engineering of biosynthetic enzymes is increasingly employed to synthesize structural analogues of antibiotics. Of special interest are nonribosomal peptide synthetases (NRPSs) responsible for the production of important antimicrobial peptides. Here, directed evolution of an adenylation domain of a Pro‐specific NRPS module completely switched substrate specificity to the non‐standard amino acid piperazic acid (Piz) bearing a labile N−N bond. This success was achieved by UPLC‐MS/MS‐based screening of small, rationally designed mutant libraries and can presumably be replicated with a larger number of substrates and NRPS modules. The evolved NRPS produces a Piz‐derived gramicidin S analogue. Thus, we give new impetus to the too‐early dismissed idea that widely accessible low‐throughput methods can switch the specificity of NRPSs in a biosynthetically useful fashion. [ABSTRACT FROM AUTHOR]

Das Engineering biosynthetischer Enzyme wird zunehmend eingesetzt, um strukturelle Analoga von Antibiotika zu synthetisieren. Von besonderem Interesse sind nichtribosomale Peptidsynthetasen (NRPS), die für die Produktion wichtiger antimikrobieller Peptide verantwortlich sind. Hier beschreiben wir, wie durch gerichtete Evolution einer Adenylierungsdomäne eines Pro‐spezifischen NRPS‐Moduls dessen Substratspezifität vollständig auf die Nicht‐Standard‐Aminosäure Piperazinsäure (Piz) geändert wurde, welche eine labile N−N‐Bindung enthält. Dieser Erfolg wurde durch UPLC‐MS/MS‐basiertes Screening kleiner, rational konzipierter Mutantenbibliotheken erzielt und kann vermutlich mit einer größeren Anzahl von Substraten und NRPS‐Modulen repliziert werden. Die evolvierte NRPS ist in der Lage, ein Piz‐beinhaltendes Gramicidin S‐Analogon zu produzieren. Damit geben wir der zu früh verworfenen Idee neuen Auftrieb, dass weithin zugängliche Methoden mit niedrigem Durchsatz die Spezifität von NRPS in einer biosynthetisch nützlichen Weise verändern können. [ABSTRACT FROM AUTHOR]

Background. Sore throat of infectious and inflammatory origin is one of the most common reasons for the prescription of antimicrobial drugs. The use of antimicrobial peptides (AMPs) for topical therapy may be a promising solution, due to the peculiarities of AMPs’ mechanism of action, taking into account the high importance of microbial resistance spread problem in relation to systemic etiotropic drugs.The aim of the study was to assess the formation of resistance to AMP Gramicidin S (GS) of clinical isolates of Streptococcus pneumoniae and Staphylococcus aureus, including strains resistant to antimicrobial drugs during repeated exposure, in the setting close to their actual use in clinical practice.Materials and methods. The object under study is AMP GS. The test microorganisms are reference strains of S.pneumoniae (ATCC 6303), S.aureus (ATCC 6538–P), and 10 strains of clinical isolates of each type of microorganisms, types with antimicrobial resistance (including MRSA). At the first stage, the antimicrobial activity of GS was evaluated by the method of microdilutions in plates by estimating the value of the minimum inhibitory and the minimum bactericidal concentrations (MIC and MBC, respectively). At the second stage, using reference strains and 4 clinical isolates of each microorganism, including types with antimicrobial resistance, resistance was formed in two ways simultaneously (with liquid and solid media) for 7 consecutive passages over 7 days with GS at concentrations equal to 0.5 and 1.0 MIC. The number of surviving microorganisms was estimated and the MIC and MBC were determined at each passage.Results. At the first stage, the MIC of GS in relation to S.pneumoniae was 8 µg/ml for 5 strains, 16 µg/ml for 5 strains, and 4 µg/ml for one. For 10 out of 11 strains of S.aureus, including MRSA, the MIC of GS was 4 µg/ml (for 1 strain — 8 µg/ml). For 4 strains of S.pneumoniae, MBC of GS was equal to MIC (16 µg/ml), for 7 strains MBC was 4–8 times higher than MIC, and mostly amounted to 32 µg/ml (for 1 strain — 64 µg/ml). MBC for 6 strains of S.aureus was equal to MIC (4 µg/ml), for the 5 remaining strains MBC was 2–8 times higher than MIC, but did not exceed 64 µg/ml. At the second stage, it was found that the estimated values of GS MIC and MBC with repeated exposure to GS remained at the same level for 7 passages in all strains, including those resistant to antibiotics in liquid and solid media.Conclusions. Gramicidin S demonstrated high bactericidal activity against reference strains and clinical isolates of leading pathogenic microorganisms S.pneumoniae and S.aureus, including strains resistant to systemic antimicrobial drugs, while MIC and MBC did not exceed concentrations contained in single doses of drugs used in clinical practice. During repeated exposure (7 passages in 7 days), there were no signs of the formation of resistance of test strains of pathogens, including those resistant to systemic antimicrobial medicines from the groups of beta-lactams, macrolides, fluoroquinolones, tetracyclines, aminoglycosides, etc.

Introduction. Gramicidin S is a peptide antibiotic that has been widely used for more than 70 years. Gramicidin S is available in the form of tablets with a low dosage, which leads to possible deviations in the "Uniformity of dosage" parameter during manufacturing. Another limitation is the presence of lactose and sucrose in the formulation, which limits the drug application by patients demonstrating intolerance. As an alternative, we propose inclusion complexes of gramicidin S with β-cyclodextrin.Aim. The work aims to describe the influence of the methodology to prepare the inclusion complex on the characteristics and properties of the final product.Materials and methods. The gramicidin S – β-cyclodextrin inclusion complex has been prepared by dry mixing, paste complexation, co-precipitation and fluid-bed complexation. The complex formation has been confirmed by 1H NMR spectroscopy, differential scanning calorimetry and thermogravimetry while the morphology and size by scanning electron microscopy for the solid and dynamic light scattering for the solution. The flowability, slope angle, bulk density of the obtained powders were estimated using the methods described in Russian Pharmacopoeia of the XIV edition.Results and discussion. In the present work, we prepared a set of gramicidin S and β-cyclodextrin inclusion complexes by various approaches. The thermal analysis demonstrated a significant change in the peak referring to phase transition of the substances, indicating the interaction between the components. The 1H NMR spectroscopy reveals that the L-ornithine amino group is the part of gramicidin S involved in the complexation. Evaluating the technological properties of gramicidin S and β-cyclodextrin inclusion complexes significant variability, which is associated with the particle morphology. Complexes obtained using co-precipitation and fluid-bed complexation methods are more suitable for producing gramicidin S tablet production by direct compression technology.Conclusion. Herein, we demonstrate that the formation of the gramicidin S and β-cyclodextrin inclusion complex occurs through the L-ornithine amino group in the gramicidin S. In addition, depending on the method significant differences in the particle size and shape have been observed. The obtained results could provide valuable information for the development of new gramicidin S buccal formulations, which are more consistent in the "Uniformity of dosage" and allow to avoid the use of lactose and sucrose as excipients.

The rise of multidrug-resistant bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA), necessitates the development of new antibacterial therapies. Antimicrobial peptides offer a promising alternative to conventional antibiotics due to their unique mechanisms of action. Gramicidin S exhibits potent bactericidal activity against S. aureus, however, high haemolytic toxicity currently limits its application to topical use. A new series of gramicidin S analogues is presented with rational modifications to the β-turn and β-strand regions, to reduce haemolytic and nephrotoxic effects, while preserving antibacterial potency. The minimum inhibitory concentration (MIC) for each analogue was determined against benchmark methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, with toxicity characterised in vitro using human red blood cells and human embryonic kidney cells (HEK-293). Peptide 12 demonstrated a significant two-fold increase in antibacterial activity against both MSSA and MRSA (MIC: 2 μg/mL) compared to gramicidin S (MIC: 4 μg/mL), albeit with increased cytotoxicity. Similarly, peptide 15 showed exceptional efficacy (MIC: 3 μg/mL), but with reduced cytotoxicity, culminating in a two-fold improvement to the therapeutic index (TI) of gramicidin S. Peptides 14 (HC 50 : 50.48 ± 1.15 μg/mL, IC 50 : 38.09 μg/mL) and 16 (HC 50 : 84.09 ± 1.02 μg/mL, IC 50 : 12.60 μg/mL) also significantly reduced haemolytic toxicity and nephrotoxicity, compared to gramicidin S (HC 50 : 12.34 ± 0.27 μg/mL, IC 50 : 6.45 μg/mL). Detailed NMR, CD and computational modelling were used to provide critical insights into how molecular conformation influences both antibacterial potency and cytotoxicity. Collectively, these results expand the therapeutic window of gramicidin S by up to 12-fold, with negligible cytotoxicity observed at concentrations well beyond the acceptable safety threshold, which indicates the potential for safe systemic administration in the treatment of infection caused by resistant pathogens., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Prof. Andrew D. Abell has patent pending to The University of Adelaide. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Introduction. Gramicidin S has been conventionally manufactured as buccal tablets. However, in the past decade, the interest in the development of spray formulations has been growing. Those formulations contain excipients that enhance the solubility of the antibiotic in water solutions. However, the real structure of gramicidin S containing sprays remains unrevealed.Aim. Investigation of colloidal structure and biopharmaceutical properties of new gramicidin S antibacterial composition.Materials and methods. The composition sample was obtained using gramicidin S dihydrochloride, propylene glycol, polysorbate-80, ethanol and purified water. Raman spectroscopy has been performed to determine the composition of the phases. Dynamic light scattering analysis was performed to characterize the composition particles. Release of gramicidin S was performed by dialysis method and the concentration was determined by HPLC. The antimicrobial properties were investigated in accordance with the requirements of the XIV edition of the Russian pharmacopoeia.Results and discussion. Dynamic light scattering analysis results show gramicidin S formulation particles having an average size in solution 5–50 nm and ζ-potential (–1.1: +7.9 mV). Based on the obtained data on the composition properties and formulation parameters it was classified as colloidal solution. The kinetic stability evaluation was performed. We compared the solubility in water and release parameters of the active pharmaceutical ingredient in the native state and in the micelles. The enhancement of the antimicrobial activity of the peptide in the colloidal solution was confirmed and ascribed to the synergic effect gramicidin S – surfactant.Conclusion. We reported the colloidal type of the composition, that aggregate gramicidin S at a concentration of 8 mg/mL. We found that gramicidin S inclusion into the colloidal solution led to significant efficiency increase, which reveals the potential to reduce the drug dose and side effects level.

The soil-borne species Aneurinibacillus migulanus (A. migulanus) strains Nagano and NCTC 7096 were shown to be potent biocontrol agents active against several plant diseases in agricultural and forest ecosystems. Both strains produce the cyclic peptide gramicidin S (GS) that was described as the main weapon inhibiting some gram-negative and gram-positive bacteria and fungus-like organisms along with the production of biosurfactant and hemolysis activities. However, the contribution of the cyclic peptide gramicidin S (GS) to the biocontrol ability of A. migulanus has never been studied experimentally. In this paper, using a mutant of the A. migulanus Nagano strain (E1 mutant) impaired in GS biosynthesis we evaluated the contribution of GS in the biocontrol potential of A. migulanus against Phytophthora spp. The two strains of A. migulanus, Nagano and NCTC 7096, were tested in a pilot study for the inhibition of the growth of 13 Phytophthora species in dual culture assays. A. migulanus Nagano was significantly more inhibitory than NCTC 7096 to all species. Additionally, using apple infection assays, P. rosacearum MKDF-148 and P. cryptogea E2 were shown to be the most aggressive on apple fruits displaying clear infection halos. Therefore, the three A. migulanus strains, Nagano, NCTC 7096, and E1, were used in apple infection experiments to check their effect on infection ability of these two Phytophthora species. Treatment with A. migulanus Nagano significantly reduced the severity of symptoms in apple fruits compared with NCTC 7096. A. migulanus E1 mutant showed total loss of biocontrol ability suggesting that GS is a major actor in the biocontrol ability of A. migulanus Nagano strain. [ABSTRACT FROM AUTHOR]

This study evaluates the embryotoxicity of dithienylethene-modified peptides upon photoswitching, using 19 analogues based on the β-hairpin scaffold of the natural membranolytic peptide gramicidin S. We established an in vivo assay in two variations (with ex vivo and in situ photoisomerization), using larvae of the model organism Danio rerio, and determined the toxicities of the peptides in terms of 50% lethal doses (LD50). This study allowed us to: (i) demonstrate the feasibility of evaluating peptide toxicity with D. rerio larvae at 3–4 days post fertilization, (ii) determine the phototherapeutic safety windows for all peptides, (iii) demonstrate photoswitching of the whole-body toxicity for the dithienylethene-modified peptides in vivo, (iv) re-analyze previous structure–toxicity relationship data, and (v) select promising candidates for potential clinical development.

The cyclic peptide cyclo (Val-Leu-Leu-d-Phe-Pro) 2 (peptide 1 ) was specifically designed for structural chemistry investigations, drawing inspiration from Gramicidin S (GS). Previous studies have shown that Pro residues within 1 adopt a down-puckering conformation of the pyrrolidine ring. By incorporating fluoride-Pro with 4- trans / cis -isomers into 1 , an up-puckering conformation was successfully induced. In the current investigation, introducing hy-droxy-prolines with 4- trans / cis -isomer configurations (tHyp/cHyp) into 1 gave cyclo (Val-Leu-Leu-d-Phe-tHyp) 2 methanol disolvate monohydrate, C 62 H 94 N 10 O 12 ·2CH 4 O·H 2 O ( 4 ), and cyclo (Val-Leu-Leu-d-Phe-cHyp) 2 monohydrate, C 62 H 94 N 10 O 12 ·H 2 O ( 5 ), respectively. However, the puckering of 4 and 5 remained in the down conformation, regardless of the geometric position of the hydroxyl group. Although the backbone structure of 4 with trans -substitution was asymmetric, the asymmetric backbone of 5 with cis -substitution was unexpected. It is speculated that the anti-cipated influence of stress from the geometric positioning, which was expected to affect the puckering, may have been mitigated by inter-actions between the hydroxyl groups of hy-droxy-proline, the solvent mol-ecules, and peptides., (© Asano et al. 2024.)

Gramicidin S (GraS) is an amphiphilic peptide that has emerged as an effective alternative antibiotic. However, its applicability is restricted for clinical uses due to its effect on eukaryotic cells and low aqueous solubility. In this work, a novel water‐soluble peptide formulation with bactericidal activity is developed by the incorporation of Gramicidin S in the lipid bilayer of liposomes (L‐GraS). As a result, GraS included in the lipid vesicles is stabilized in aqueous medium and showed antimicrobial activity against Staphylococcus aureus. In addition, L‐GraS reveals enhanced biocompatibility with mammalian cells in comparison with the free peptide. Molecular dynamics simulations shed light on the collective behavior between GraS peptides and liposomes from a molecular approach. The molecular dynamic simulations are in agreement with experimental results and further confirm the effective incorporation of GraS in the liposomes, letting understand the best formulation procedure of the vesicles. Therefore, the L‐GraS nanosystem presented here is an interesting alternative to conventional antibiotics, with less restrictions and promising features to improve current therapies. Practical Applications: Incorporation of GraS in liposomes is an interesting approach to increase the solubility of the peptide, thus expanding its therapeutic horizon as a promising alternative to combat bacterial colonization especially in wound infections. [ABSTRACT FROM AUTHOR]

Emerging issues of Antibiotic drug resistance have increased the mortality cases all around the world. To deal with the situation natural antimicrobial peptide (AMP) replaces the classic antibiotics. Antimicrobial peptides are short, natural peptides that are active against most bacteria's, viruses, and fungi etc., The AMP acts as a potent therapeutic agent and finds a new way in the field of drug discovery and pharmaceuticals. One such antimicrobial peptide is gramicidin (gramicidin D and gramicidin S) has high potential of almost 100% killing mechanisms against most of the gram-positive and few gram-negative bacteria's and fungi which can be used as an alternative antibiotic in the near future. There are reviews focusing only on individual types of gramicidin or particular about their function. Hence, this review focus on providing an overview of antimicrobial peptide gramicidin and its types, structures, its interaction with the membranes/lipid bilayers, functions, stability, and uses. This review also focuses on the challenges to enhance the gramicidin peptide for further use as an efficient effective therapeutic peptide in the pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Polypeptide antibiotics abuse can lead to antibiotic residues in food products and have unwanted effects on human health. A selective, accurate, and sensitive analytical method for the simultaneous determination of four polypeptide antibiotics (gramicidin S, bacitracin, polymyxin B, and polymyxin E) in infant formula powder was developed using solid-phase extraction (SPE) combined with high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS). The samples were extracted with acidified methanol, deproteinized with acetonitrile, and degreased with n-hexane before SPE. After cleanup with 0.1% formic acid and methanol (3:1, v/v) in Oasis HLB cartridges, the extracts were analyzed by HPLC–MS/MS with electrospray ionization (ESI) source and time-scheduled multiple reaction monitoring (MRM). Linearity was assessed by using matrix-matched standard calibration and good determination coefficients (r2 > 0.995) were obtained. The average recoveries for blank sample at three spiked concentration levels were in the range of 82.8–101.2%. The limits of detection (LODs) and limits of quantitation (LOQs) of all analytes were in the range of 5–15 μg kg−1 and 20–50 μg kg−1, respectively. The intra-day and inter-day precisions were lower than 10%. The results of method validation demonstrated that the developed method is accurate and reliable, and it can be applied for screening and quantitation of target polypeptide antibiotics in food. [ABSTRACT FROM AUTHOR]

The functioning of many enzymes is metal ion dependent. In this work, deprotonation/sodiation of gramicidin S (GS) was studied by electrospray ionization mass spectrometry. Electrospray mass spectra for 10−5 M of GS in water/methanol with and without the addition of 1 mM NaOH, AcONa, NaCl, NH 4 Cl, AcONH 4 and 1% AcOH were measured. It was found that one and two amide N–H bonds for [GS + H]+ and [GS + 2H]2+, respectively, were preferably deprotonated to form [GS + H − H + Na]+ and [GS + 2H − 2H + 2Na]2+. The extent of sodiation was found to be in the order of NaOH > AcONa > NaCl. With the addition of 1 mM NaOH, [GS + 2H − nH + nNa]2+ with n up to 8 were observed indicating that all of the eight amide N–H bonds in GS were deprotonated. Sodiation sites are likely to be the negatively charged amide enolate C–O− to form salt-like C–O−⋅⋅⋅⋅Na+ bonds. In the negative mode, one and two amide N–H bonds are preferably deprotonated to form [GS − H]−, [GS − 2H]2− and [GS − 2H + Na]− for the solution with the addition of 1 mM NaOH. [Display omitted] • Deprotonation/sodiation of gramicidin S (GS) was studied by electrospray ionization. • With the addition of NaOH in solution, [GS + 2H − nH + nNa]2+ with n up to 8 were detected. • Amide N–H bonds are deprotonated and resultant amide enolates are sodiated to form salt-like C–O−⋅⋅⋅⋅Na+ bonds. [ABSTRACT FROM AUTHOR]

Rapid, direct, on-site and noninvasive food analysis is strongly needed for quality control of food. To satisfy this demand, the technique of dipping probe electrospray ionization/mass spectrometry (dPESI/MS) was developed. The sample surface was pricked with a fine acupuncture needle and a sample of ∼200 pL was captured at the needle tip. After drying the sample, the needle tip was dipped into the solvent for ∼50 ms and was moved upward. A high-voltage was applied to the needle to generate electrospray when the needle reached the highest position, and mass spectra were measured with a time-of-flight mass spectrometer. For evaluation of the method, the technique was used to analyze foods such as vegetables, salmon flesh, cow’s milk, yogurt, and soy-bean milk. The detected major ions for cow’s milk and yogurt were [(Lac) n  + Ca] 2+ with n = 1–6 (where (Lac) is lactose), indicating that Ca 2+ is tightly bound by Lac molecules. [ABSTRACT FROM AUTHOR]

The biological activity impact on editing proline α -carbon with stereodynamic nitrogen in Gramicidin S is investigated. Analysis of data reveals that the proline edited peptide 13 demonstrated equipotent activity against drug sensitive and resistant S. aureus and Enterococcus spp. with lesser cytotoxicity compared the counterpart Gramicidin S. [Display omitted] • β -Turn in Gramicidin S was edited with stereodynamic nitrogen to examine the direct impact on biological activity and cytotoxicity with respect to counterpart. • Investigation of antimicrobial activity revealed that proline edited analogous peptide 13 demonstrated equipotent antimicrobial activity against MDR S. aureus and vancomycin-resistant Enterococcus spp. • Cytotoxicity against Vero cells and red blood cells showed peptide 13 two-fivefold lesser toxicity than the counterpart Gramicidin S. • Our study suggests that peptide 13 is a promising substituent of Gramicidin S for further development as antibiotic. Gramicidin S, natural antimicrobial peptide is used commercially in medicinal lozenges for sore throat and Gram-negative and Gram-positive bacterial infections. However, its clinical potential is limited to topical applications because of its high red blood cells (RBC) cytotoxicity. Given the importance of developing potential antibiotics and inspired by the cyclic structure and druggable features of Gramicidin S, we edited proline α-carbon with stereodynamic nitrogen to examine the direct impact on biological activity and cytotoxicity with respect to prolyl counterpart. Natural Gramicidin S (12) , proline-edited peptides 13 – 16 and wild-type d -Phe- d -Pro β -turn mimetics (17 and 18) were synthesized using solid phase peptide synthesis and investigated their activity against clinically relevant bacterial pathogens. Interestingly, mono-proline edited analogous peptide 13 showed moderate improvement in antimicrobial activity against E. coli ATCC 25922 and K.pneumoniae BAA 1705 as compared to Gramicidin S. Furthermore, proline edited peptide 13 exhibited equipotent antimicrobial effect against MDR S. aureus and Enterococcus spp. Analysis of cytotoxicity against VERO cells and RBC, reveals that proline edited peptides showed two-fivefold lesser cytotoxicity than the counterpart Gramicidin S. Our study suggests that introducing single azPro/Pro mutation in Gramicidin S marginally improved the activity and lessens the cytotoxicity as compared with the parent peptide. [ABSTRACT FROM AUTHOR]

Metal ions play significant roles in biological systems. Although sodium ions play a key role in biological systems, site-specific interactions between sodium ions and protonated peptides and proteins have not been thoroughly explored. In this work, the time-dependent sodiation of protonated peptides was investigated by pulsed nanoelectrospray ionization. Mass spectra for aqueous mixtures of 5×10−6 M angiotensin II (A), bradykinin (B), and gramicidin S (G) were measured as a function of the high voltage (4000 V) pulse width (PW) with and without the application of a bias voltage (BV) to the solution. With BV = 0 V, only [B + 2H − H + Na]2+ was detected with an increase in PW from 220 μs (threshold for the signal appearance) to 1 ms. In contrast, [A + 2H − nH + nNa]2+ (n = 1–3) started to be detected with PW = 500 μs and up to n = 4 with PW = 1 ms. By the application of BV = 900 V, [B + 2H − nH + nNa]2+ with n = 0 and 1 started to be detected with PW = 40 μs. With further increase in PW up to 1 ms, [B + 2H − nH + nNa]2+ with n = 0–3 and [A + 2H − nH + nNa]2+ (n = 1–5) were detected. The observation of [B + 2H − 3H + 3Na]2+ and [A + 2H − 5H + 5Na]2+ with BV = 900 V suggests that carboxyl (−COOH), imidazole > N–H, hydroxyl (−OH) as well as amide > N–H bonds are deprotonated to form respective sodiated forms of carboxylate –COO−⋅⋅⋅⋅Na+, imidazole > N−⋅⋅⋅⋅Na+, hydroxyl –O−⋅⋅⋅⋅Na+ and amide –N C–O−⋅⋅⋅⋅Na+ bonds. Ions of G were totally suppressed by the presence of A and B in aqueous solution. [Display omitted] • Time-dependent sodiation of peptides was investigated by pulsed nanoelectrospray ionization. • Progressive sodiation of carboxyl –COOH, hydroxyl –OH, imidazole > N–H, and amide > N–H occurred with the pulse width. • Amide > N–H is deprotonated by the cooperative interaction of Na+ with C=O and Cl− with >N–H in the amide group. [ABSTRACT FROM AUTHOR]

It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~Srf↔GS+Srf correlated to apparent K values of 6-9 μM in gas-phase and 1 μM in aqueous solution. The apparent K values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low μM K values of GS~Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na nor Ca had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca and Na from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. [ABSTRACT FROM AUTHOR]

The soil-borne gram-positive bacteria Aneurinibacillus migulanus strain Nagano shows considerable potential as a biocontrol agent against plant diseases. In contrast, A. migulanus NCTC 7096 proved less effective for inhibition of plant pathogens. Nagano strain exerts biocontrol activity against some gram-positive and gram-negative bacteria, fungi and oomycetes through the production of gramicidin S (GS). Apart from the antibiotic effects, GS increases the rate of evaporation from the plant surface, reducing periods of surface wetness and thereby indirectly inhibiting spore germination. To elucidate the molecular basis of differential biocontrol abilities of Nagano and NCTC 7096, we compared GS production and biosurfactant secretion in addition to genome mining of the genomes. Our results proved that: (i) Using oil spreading, blood agar lysis, surface tension and tomato leaves wetness assays, Nagano showed increased biosurfactant secretion in comparison with NCTC 7096, (ii) Genome mining indicated the presence of GS genes in both Nagano and NCTC 7096 with two amino acid units difference between the strains: T342I and P419S. Using 3D models and the DUET server, T342I and P419S were predicted to decrease the stability of the NCTC 7096 GS synthase, (iii) Nagano produced two additional GS-like molecules GS-1155 (molecular weight 1155) and GS-1169 (molecular weight 1169), where one or two ornithine residues replace lysine in the peptide. There was also a negative correlation between surface tension and the quantity of GS-1169 present in Nagano, and (iv) the Nagano genome had a full protein network of exopolysaccharide biosynthesis in contrast to NCTC 7096 which lacked the first enzyme of the network. NCTC 7096 is unable to form biofilms as observed for Nagano. Different molecular layers, mainly gramicidin secondary metabolite production, account for differential biocontrol abilities of Nagano and NCTC 7096. This work highlighted the basis of differential biological control abilities between strains belonging to the same species and demonstrates techniques useful to the screening of effective biocontrol strains for environmentally friendly secondary metabolites that can be used to manage plant pathogens in the field. [ABSTRACT FROM AUTHOR]

An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix® paste, which contains demeclocycline, or the new alternative endodontic paste Odontopaste, which contains clindamycin; however, these treatments are often ineffective. We studied the killing activity of the cyclic antimicrobial peptide gramicidin S (GS) against planktonic and biofilm cells of tetracycline-resistant clinical isolates of E. faecalis. The high therapeutic potential of GS for the topical treatment of problematic teeth is based on the rapid bactericidal effect toward the biofilm-forming, tetracycline-resistant E. faecalis. GS reduces the cell number of planktonic cells within 20-40 min at a concentration of 40-80 μg/mL. It kills the cells of pre-grown biofilms at concentrations of 100-200 μg/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad GS activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication. [ABSTRACT FROM AUTHOR]

From the evolutionary melting pot of natural product synthetase genes, microorganisms elicit antibiotics, communication tools, and iron scavengers. Chemical biologists manipulate these genes to recreate similarly diverse and potent biological activities not on evolutionary time scales but within months. Enzyme engineering has progressed considerably in recent years and offers new screening, modelling, and design tools for natural product designers. Here, recent advances in enzyme engineering and their application to nonribosomal peptide synthetases are reviewed. Among the nonribosomal peptides that have been subjected to biosynthetic engineering are the antibiotics daptomycin, calcium-dependent antibiotic, and gramicidin S. With these peptides, incorporation of unnatural building blocks and modulation of bioactivities via various structural modifications have been successfully demonstrated. Natural product engineering on the biosynthetic level is not a reliable method yet. However, progress in the understanding and manipulation of biosynthetic pathways may enable the routine production of optimized peptide drugs in the near future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

An intrinsic clindamycin-resistant Enterococcus faecalis, the most common single species present in teeth after failed root canal therapy, often possesses acquired tetracycline resistance. In these cases, root canal infections are commonly treated with Ledermix® paste, which contains demeclocycline, or the new alternative endodontic paste Odontopaste, which contains clindamycin; however, these treatments are often ineffective. We studied the killing activity of the cyclic antimicrobial peptide gramicidin S (GS) against planktonic and biofilm cells of tetracycline-resistant clinical isolates of E. faecalis. The high therapeutic potential of GS for the topical treatment of problematic teeth is based on the rapid bactericidal effect toward the biofilm-forming, tetracycline-resistant E. faecalis. GS reduces the cell number of planktonic cells within 20–40 min at a concentration of 40–80 μg/mL. It kills the cells of pre-grown biofilms at concentrations of 100–200 μg/mL, such that no re-growth is possible. The translocation of the peptide into the cell interior and its complexation with intracellular nucleotides, including the alarmon ppGpp, can explain its anti-biofilm effect. The successful treatment of persistently infected root canals of two volunteers confirms the high effectiveness of GS. The broad GS activity towards resistant, biofilm-forming E. faecalis suggests its applications for approval in root canal medication.

The objective of this work is the study of the ion mobility dependence of electrospray Gramicidin S ions [M + 2H] on the ion bunch drift velocity under variation of the specific electrical field strength at certain gas number density in the range from 0 to 300 Td. The ion mobility measurements were carried out under rotational excitation of ions using custom built gas filled segmented radio frequency quadrupole with the nitrogen buffer gas pressure of 1.9 Pa. The resulting ion mobility dependence of [M + 2H] Gramicidin S ions upon the bunch drift velocity is essentially weaker, than it could be expected from the hard sphere collision model. By comparing the acquired ion mobility dependencies with the theoretical ones derived from the hard sphere collision model we suppose that the attractive polarization potential takes an essential part into the ion-molecular interaction even for such big organic ions as Gramicidin S. [ABSTRACT FROM AUTHOR]

This paper describes the ability of a new class of heterocyclic γ-amino acids named ATCs (4-amino(methyl)-1,3-thiazole-5-carboxylic acids) to induce turns when included in a tetrapeptide template. Both hybrid Ac-Val-( R or S)-ATC-Ile-Ala-NH2 sequences were synthesized and their conformations were studied by circular dichroism, NMR spectroscopy, MD simulations, and DFT calculations. It was demonstrated that the ATCs induced highly stable C9 pseudocycles in both compounds promoting a twist turn and a reverse turn conformation depending on their absolute configurations. As a proof of concept, a bioactive analogue of gramicidin S was successfully designed using an ATC building block as a turn inducer. The NMR solution structure of the analogue adopted an antiparallel β-pleated sheet conformation similar to that of the natural compound. The hybrid α,γ-cyclopeptide exhibited significant reduced haemotoxicity compared to gramicidin S, while maintaining strong antibacterial activity. [ABSTRACT FROM AUTHOR]

Photobiological processes in nature are usually triggered by nonpeptidic chromophores or by modified side chains. A system is presented in which the polypeptide backbone itself can be conformationally switched by light. An amino acid analogue was designed and synthesized based on a reversibly photoisomerizable diarylethene scaffold. This analogue was incorporated into the cyclic backbone of the antimicrobial peptide gramicidin S at several sites. The biological activity of the resulting peptidomimetics could then be effectively controlled by ultraviolet/visible light within strictly defined spatial and temporal limits. [ABSTRACT FROM AUTHOR]

Abstract: We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5′-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated l-Phe-AMS (l-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates l-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes. [Copyright &y& Elsevier]