A New Gramicidin S Analogue with Potent Antibacterial Activity and Negligible Hemolytic Toxicity.

Antibiotic resistance is an urgent threat to global health, with the decreasing efficacy of conventional drugs underscoring the urgency for innovative therapeutic strategies. Antimicrobial peptides present as promising alternatives to conventional antibiotics. Gramicidin S is one such naturally occurring antimicrobial peptide that is effective against Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 4 µg/mL (3.6 µM). Despite this potent activity, its significant hemolytic toxicity restricts its clinical use to topical applications. Herein, we present rational modifications to the key ß-strand and ß-turn regions of gramicidin S to concurrently mitigate hemolytic effects, while maintaining potency. Critically, peptide 9 displayed negligible hemolytic toxicity, while possessing significant antibacterial potency against a panel of methicillin-sensitive and methicillin-resistant S. aureus clinical isolates (MIC of 8 µg/mL, 7.2 µM). Given the substantial antibacterial activity and near absence of cytotoxicity, 9 presents as a potential candidate for systemic administration in the treatment of S. aureus bacteremia/sepsis.

Intrinsic Lipid Curvature and Bilayer Elasticity as Regulators of Channel Function: A Comparative Single-Molecule Study.

Perturbations in bilayer material properties (thickness, lipid intrinsic curvature and elastic moduli) modulate the free energy difference between different membrane protein conformations, thereby leading to changes in the conformational preferences of bilayer-spanning proteins. To further explore the relative importance of curvature and elasticity in determining the changes in bilayer properties that underlie the modulation of channel function, we investigated how the micelle-forming amphiphiles Triton X-100, reduced Triton X-100 and the HII lipid phase promoter capsaicin modulate the function of alamethicin and gramicidin channels. Whether the amphiphile-induced changes in intrinsic curvature were negative or positive, amphiphile addition increased gramicidin channel appearance rates and lifetimes and stabilized the higher conductance states in alamethicin channels. When the intrinsic curvature was modulated by altering phospholipid head group interactions, however, maneuvers that promote a negative-going curvature stabilized the higher conductance states in alamethicin channels but destabilized gramicidin channels. Using gramicidin channels of different lengths to probe for changes in bilayer elasticity, we found that amphiphile adsorption increases bilayer elasticity, whereas altering head group interactions does not. We draw the following conclusions: first, confirming previous studies, both alamethicin and gramicidin channels are modulated by changes in lipid bilayer material properties, the changes occurring in parallel yet differing dependent on the property that is being changed; second, isolated, negative-going changes in curvature stabilize the higher current levels in alamethicin channels and destabilize gramicidin channels; third, increases in bilayer elasticity stabilize the higher current levels in alamethicin channels and stabilize gramicidin channels; and fourth, the energetic consequences of changes in elasticity tend to dominate over changes in curvature.

Gramicidin, a Bactericidal Antibiotic, Is an Antiproliferative Agent for Ovarian Cancer Cells.

Background and Objectives: Gramicidin, a bactericidal antibiotic used in dermatology and ophthalmology, has recently garnered attention for its inhibitory actions against cancer cell growth. However, the effects of gramicidin on ovarian cancer cells and the underlying mechanisms are still poorly understood. We aimed to elucidate the anticancer efficacy of gramicidin against ovarian cancer cells. Materials and Methods: The anticancer effect of gramicidin was investigated through an in vitro experiment. We analyzed cell proliferation, DNA fragmentation, cell cycle arrest and apoptosis in ovarian cancer cells using WST-1 assay, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL), DNA agarose gel electrophoresis, flow cytometry and western blot. Results: Gramicidin treatment induces dose- and time-dependent decreases in OVCAR8, SKOV3, and A2780 ovarian cancer cell proliferation. TUNEL assay and DNA agarose gel electrophoresis showed that gramicidin caused DNA fragmentation in ovarian cancer cells. Flow cytometry demonstrated that gramicidin induced cell cycle arrest. Furthermore, we confirmed via Western blot that gramicidin triggered apoptosis in ovarian cancer cells. Conclusions: Our results strongly suggest that gramicidin exerts its inhibitory effect on cancer cell growth by triggering apoptosis. Conclusively, this study provides new insights into the previously unexplored anticancer properties of gramicidin against ovarian cancer cells.

ß-Turn editing in Gramicidin S: Activity impact on replacing proline α-carbon with stereodynamic nitrogen.

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 ß-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.

Screening for bilayer-active and likely cytotoxic molecules reveals bilayer-mediated regulation of cell function.

A perennial problem encountered when using small molecules (drugs) to manipulate cell or protein function is to assess whether observed changes in function result from specific interactions with a desired target or from less specific off-target mechanisms. This is important in laboratory research as well as in drug development, where the goal is to identify molecules that are unlikely to be successful therapeutics early in the process, thereby avoiding costly mistakes. We pursued this challenge from the perspective that many bioactive molecules (drugs) are amphiphiles that alter lipid bilayer elastic properties, which may cause indiscriminate changes in membrane protein (and cell) function and, in turn, cytotoxicity. Such drug-induced changes in bilayer properties can be quantified as changes in the monomer↔dimer equilibrium for bilayer-spanning gramicidin channels. Using this approach, we tested whether molecules in the Pathogen Box (a library of 400 drugs and drug-like molecules with confirmed activity against tropical diseases released by Medicines for Malaria Venture to encourage the development of therapies for neglected tropical diseases) are bilayer modifiers. 32% of the molecules in the Pathogen Box were bilayer modifiers, defined as molecules that at 10 µM shifted the monomer↔dimer equilibrium toward the conducting dimers by at least 50%. Correlation analysis of the molecules' reported HepG2 cell cytotoxicity to bilayer-modifying potency, quantified as the shift in the gramicidin monomer↔dimer equilibrium, revealed that molecules producing <25% change in the equilibrium had significantly lower probability of being cytotoxic than molecules producing >50% change. Neither cytotoxicity nor bilayer-modifying potency (quantified as the shift in the gramicidin monomer↔dimer equilibrium) was well predicted by conventional physico-chemical descriptors (hydrophobicity, polar surface area, etc.). We conclude that drug-induced changes in lipid bilayer properties are robust predictors of the likelihood of membrane-mediated off-target effects, including cytotoxicity.

Photodynamic activity rather than drilling causes membrane damage by a light-powered molecular nanomotor.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.

Effects of Calcium Ions on the Antimicrobial Activity of Gramicidin A.

Gramicidin A (gA) is a linear antimicrobial peptide that can form a channel and specifically conduct monovalent cations such as H+ across the lipid membrane. The antimicrobial activity of gA is associated with the formation of hydroxyl free radicals and the imbalance of NADH metabolism, possibly a consequence caused by the conductance of cations. The ion conductivity of gramicidin A can be blocked by Ca2+ ions. However, the effect of Ca2+ ions on the antimicrobial activity of gA is unclear. To unveil the role of Ca2+ ions, we examined the effect of Ca2+ ions on the antimicrobial activity of gramicidin A against Staphylococcus aureus (S. aureus). Results showed that the antimicrobial mechanism of gA and antimicrobial activity by Ca2+ ions are concentration-dependent. At the low gA concentration (≤1 µM), the antimicrobial mechanism of gA is mainly associated with the hydroxyl free radical formation and NADH metabolic imbalance. Under this mode, Ca2+ ions can significantly inhibit the hydroxyl free radical formation and NADH metabolic imbalance. On the other hand, at high gA concentration (≥5 µM), gramicidin A acts more likely as a detergent. Gramicidin A not only causes an increase in hydroxyl free radical levels and NAD+/NADH ratios but also induces the destruction of the lipid membrane composition. At this condition, Ca2+ ions can no longer reduce the gA antimicrobial activity but rather enhance the bacterial killing ability of gramicidin A.

Chromone-Containing Allylmorpholines Influence Ion Channels in Lipid Membranes via Dipole Potential and Packing Stress.

Herein, we report that chromone-containing allylmorpholines can affect ion channels formed by pore-forming antibiotics in model lipid membranes, which correlates with their ability to influence membrane boundary potential and lipid-packing stress. At 100 µg/mL, allylmorpholines 1, 6, 7, and 8 decrease the boundary potential of the bilayers composed of palmitoyloleoylphosphocholine (POPC) by about 100 mV. At the same time, the compounds do not affect the zeta-potential of POPC liposomes, but reduce the membrane dipole potential by 80-120 mV. The allylmorpholine-induced drop in the dipole potential produce 10-30% enhancement in the conductance of gramicidin A channels. Chromone-containing allylmorpholines also affect the thermotropic behavior of dipalmytoylphosphocholine (DPPC), abolishing the pretransition, lowering melting cooperativity, and turning the main phase transition peak into a multicomponent profile. Compounds 4, 6, 7, and 8 are able to decrease DPPC's melting temperature by about 0.5-1.9 °C. Moreover, derivative 7 is shown to increase the temperature of transition of palmitoyloleoylphosphoethanolamine from lamellar to inverted hexagonal phase. The effects on lipid-phase transitions are attributed to the changes in the spontaneous curvature stress. Alterations in lipid packing induced by allylmorpholines are believed to potentiate the pore-forming ability of amphotericin B and gramicidin A by several times.

Cannabidiol increases gramicidin current in human embryonic kidney cells: An observational study.

Gramicidin is a monomeric protein that is thought to non-selectively conduct cationic currents and water. Linear gramicidin is considered an antibiotic. This function is considered to be mediated by the formation of pores within the lipid membrane, thereby killing bacterial cells. The main non-psychoactive active constituent of the cannabis plant, cannabidiol (CBD), has recently gained interest, and is proposed to possess various potential therapeutic properties, including being an antibiotic. We previously determined that CBD's activity on ion channels could be, in part, mediated by altering membrane biophysical properties, including elasticity. In this study, our goal was to determine the empirical effects of CBD on gramicidin currents in human embryonic kidney (HEK) cells, seeking to infer potential direct compound-protein interactions. Our results indicate that gramicidin, when applied to the extracellular HEK cell membrane, followed by CBD perfusion, increases the gramicidin current.

Mechanisms underlying drug-mediated regulation of membrane protein function.

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.

Gramicidin A-based unimolecular channel: cancer cell-targeting behavior and ion transport-induced apoptosis.

A series of glycoside-peptide conjugates were prepared by engineering at the N-terminus of the natural peptide gramicidin A. The conjugate containing galactose moiety formed a unimolecular transmembrane channel and mediated ion transport to induce apoptosis of cancer cells. More importantly, it exhibited liver cancer cell-targeting behavior due to the galactose-asialoglycoprotein receptor recognition.

Gramicidin-S-Inspired Cyclopeptidomimetics as Potent Membrane-Active Bactericidal Agents with Therapeutic Potential.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using ß,γ-diamino acids (ß,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.

Discovery of gramicidin A analogues with altered activities by multidimensional screening of a one-bead-one-compound library.

Gramicidin A (1) is a peptide antibiotic that disrupts the transmembrane ion concentration gradient by forming an ion channel in a lipid bilayer. Although long used clinically, it is limited to topical application because of its strong hemolytic activity and mammalian cytotoxicity, likely arising from the common ion transport mechanism. Here we report an integrated high-throughput strategy for discovering analogues of 1 with altered biological activity profiles. The 4096 analogue structures are designed to maintain the charge-neutral, hydrophobic, and channel forming properties of 1. Synthesis of the analogues, tandem mass spectrometry sequencing, and 3 microscale screenings enable us to identify 10 representative analogues. Re-synthesis and detailed functional evaluations find that all 10 analogues share a similar ion channel function, but have different cytotoxic, hemolytic, and antibacterial activities. Our large-scale structure-activity relationship studies reveal the feasibility of developing analogues of 1 that selectively induce toxicity toward target organisms.

Effect of charge status on the ion transport and antimicrobial activity of synthetic channels.

A class of unimolecular channels formed by pillararene-gramicidin hybrid molecules are presented. The charge status of the peptide domain in these channels has a significant impact on their ion transport and antimicrobial activity. These channels exhibited different membrane-association abilities between microbial cells and mammalian cells. One of the channels displayed a higher antimicrobial activity towards S. aureus (IC50 = 0.55 µM) and negligible hemolytic toxicity, showing potential to serve as a systemic antibiotic.

The Ionophores CCCP and Gramicidin but Not Nigericin Inhibit Trypanosoma brucei Aquaglyceroporins at Neutral pH.

Human African trypanosomiasis (HAT) is caused by Trypanosoma brucei parasites. The T. brucei aquaglyceroporin isoform 2, TbAQP2, has been linked to the uptake of pentamidine. Negative membrane potentials and transmembrane pH gradients were suggested to promote transport of the dicationic antitrypanosomal drug. Application of ionophores to trypanosomes further hinted at direct inhibition of TbAQP2 by carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Here, we tested for direct effects of three classical ionophores (CCCP, nigericin, gramicidin) on the functionality of TbAQP2 and the related TbAQP3 at conditions that are independent from the membrane potential or a proton gradient. We expressed TbAQP2 and TbAQP3 in yeast, and determined permeability of uncharged glycerol at neutral pH using stopped-flow light scattering. The mobile proton carrier CCCP directly inhibited TbAQP2 glycerol permeability at an IC50 of 2 µM, and TbAQP3 to a much lesser extent (IC50 around 1 mM) likely due to different selectivity filter layouts. Nigericin, another mobile carrier, left both isoforms unaffected. The membrane-integral pore-forming gramicidin evenly inhibited TbAQP2 and TbAQP2 in the double-digit micromolar range. Our data exemplify the need for suitable controls to detect unwanted ionophore side effects even when used at concentrations that are typically recommended to disturb the transmembrane ion distribution.

The Effect of Calcium and Halide Ions on the Gramicidin A Molecular State and Antimicrobial Activity.

Gramicidin A (gA) forms several convertible conformations in different environments. In this study, we investigated the effect of calcium halides on the molecular state and antimicrobial activity of gramicidin A. The molecular state of gramicidin A is highly affected by the concentration of calcium salt and the type of halide anion. Gramicidin A can exist in two states that can be characterized by circular dichroism (CD), mass, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. In State 1, the main molecular state of gramicidin A is as a dimer, and the addition of calcium salt can convert a mixture of four species into a single species, which is possibly a left-handed parallel double helix. In State 2, the addition of calcium halides drives gramicidin A dissociation and denaturation from a structured dimer into a rapid equilibrium of structured/unstructured monomer. We found that the abilities of dissociation and denaturation were highly dependent on the type of halide anion. The dissociation ability of calcium halides may play a vital role in the antimicrobial activity, as the structured monomeric form had the highest antimicrobial activity. Herein, our study demonstrated that the molecular state was correlated with the antimicrobial activity.

Antimicrobial Peptides as New Combination Agents in Cancer Therapeutics: A Promising Protocol against HT-29 Tumoral Spheroids.

Antimicrobial peptides are molecules synthetized by a large variety of organisms as an innate defense against pathogens. These natural compounds have been identified as promising alternatives to widely used molecules to treat infections and cancer cells. Antimicrobial peptides could be viewed as future chemotherapeutic alternatives, having the advantage of low propensity to drug resistance. In this study, we evaluated the efficiency of the antimicrobial peptide gramicidin A (GA) and the anticancer drug, doxorubicin (Doxo) against the spheroids from colorectal cancer cells (HT-29). The two drugs were applied separately against HT-29 spheroids as well as together to determine if they can act synergistically. The spheroid evolution, cell viability, and ATP levels were monitored at 24 and 48 h after the applied treatments. The results show significant drops in cell viability and cellular ATP levels for all the experimental treatments. The simultaneous use of the two compounds (GA and Doxo) seems to cause a synergistic effect against the spheroids.

Synthesis and biological evaluation of backbone-aminated analogues of gramicidin S.

We report the parallel synthesis of gramicidin S derivatives featuring backbone N-amino substituents. Analogues were prepared by incorporation of N-amino dipeptide subunits on solid support. Nine backbone-aminated macrocycles were evaluated for growth inhibitory activity against ESKAPE pathogens and hemolytic activity against human red blood cells. Diamination of the Orn residues in the ß-strand region of gramicidin S was found to enhance broad-spectrum antimicrobial activity without a corresponding increase in hemolytic activity.

Fluorometric Na+ Evaluation in Single Cells Using Flow Cytometry: Comparison with Flame Emission Assay.

BACKGROUND/AIMS: Sodium is a key player in the fundamental cell functions. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ indirectly using ionophores for equalizing external and intracellular ion concentration. Attempts to compare data obtained using fluorescent probes and by direct flame emission analysis are sparse and results are inaccurate. METHODS: We determined the intracellular sodium concentration in U937 cells by flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG), and by standard flame emission photometry combined with the cellular water determination by cell density in Percoll gradient. The intracellular Na+ concentrations was modified using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. RESULTS: It is revealed that both methods are comparable when intracellular sodium concentration was modified by ouabain-mediated blockage of the sodium pump or staurosporine-induced apoptosis. The ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. CONCLUSION: The sodium sensitive dye ANG-2 is a sensitive and useful probe for determination changes in Na+ content and concentration both in single cells and subcellular microparticles. The ANG fluorescence determined in the studied cells in the absence of ionophores, cannot be used as a measure of the real intracellular concentration of Na+ if calibration was carried out in the presence of ionophores.