Nonactin

Erythrocyte Shrinkage and Cell Membrane Scrambling after Exposure to the Ionophore Nonactin.[Pubmed:26280658]

Basic Clin Pharmacol Toxicol. 2016 Feb;118(2):107-12.

The ionophore antibiotic Nonactin permeabilizes cell membranes to NH4+ and K(+) . Treatment of erythrocytes with Nonactin is expected to trigger cellular K(+) loss with subsequent cell shrinkage, which in turn is known to trigger suicidal death of a wide variety of cells including erythrocytes. This study explored whether Nonactin exposure induces eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signalling of eryptosis includes increase in cytosolic Ca(2+) activity [(Ca(2+) )i ] and stimulation of protein kinase C (PKC) as well as p38 mitogen-activated protein kinase. Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter (FSC) and (Ca(2+) )i from Fluo3-fluorescence. A 48-hr treatment of human erythrocytes with Nonactin significantly decreased FSC (>/=10 ng/ml) and significantly increased the percentage of annexin-V-binding cells (>/=10 ng/ml), effects paralleled by increase in (Ca(2+) )i (>/=50 ng/ml) and virtually abrogated by increase in extracellular K(+) concentration to 120 mM at the expense of Na(+) . The up-regulation of annexin-V-binding after Nonactin treatment was significantly blunted but not abolished by the removal of extracellular Ca(2+) and by addition of either PKC inhibitor staurosporine (0.4 muM) or p38 kinase inhibitor SB203580 (2 muM). In conclusion, exposure of erythrocytes to the K(+) ionophore Nonactin induces erythrocyte shrinkage and subsequent erythrocyte membrane scrambling, effects involving cellular K(+) loss, Ca(2+) entry and activation of staurosporine as well as SB203580-sensitive kinases.

A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a Strong Inducer of Autophagosomal Turnover.[Pubmed:28297606]

SLAS Discov. 2017 Jun;22(5):558-570.

Dysregulation of the basal autophagic flux has been linked to several pathological conditions, including neurodegenerative diseases and cancer. In addition, autophagy has profound effects on the response of tumor cells to therapy. Hence, the search for pharmacological modulators of autophagy is of great clinical relevance. We established a drug screening assay in which the autophagic flux is measured by recording the fluorescence emission of the tandem fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. We optimized the assay for the identification of autophagy modulators in three dimensions with U343 glioma cell spheroids, which represent a more realistic cancer model than conventional 2D cell cultures. We validated the assay by screening a library of known autophagy modulators. As the first application, a small library of 94 natural compounds was screened for its impact on autophagy. We discovered the cyclic ionophore Nonactin as a new and potent autophagy inducer. This novel autophagy screening assay based on 3D tumor spheroids is robust, reproducible, and scalable. It provides a valuable tool for both basic research and drug screening campaigns.

On the ionophoric selectivity of nonactin and related macrotetrolide derivatives.[Pubmed:27966685]

Phys Chem Chem Phys. 2017 Jan 4;19(2):1288-1297.

Nonactin and its analogs constitute a central class of macrocycles with an antibiotic activity closely related to their selective ionophoric behavior. In this study, we apply experimental and computational methods to revisit the specificity of cation binding and transport by three nactin variants differing in structural properties, such as the position of the ester linkages, the nature of the side groups, or the flexibility of the backbone. On the one hand, electrospray ionization mass spectrometry and infrared spectroscopy are employed to expose the selectivity of the liquid-liquid (water-chloroform) extraction of alkali cations by Nonactin and to demonstrate that the cation complexes are partially hydrated in the organic phase. Furthermore, laser desorption mass spectrometry is employed to determine the intrinsic cation affinities of Nonactin under solvent-free conditions. On the other hand, density functional theory calculations are performed to characterize the conformations of the alkali cation complexes of the three nactins, and to assess the role of intermolecular and solvent interactions in determining their relative stability. Depending on the structure of the macrocycle, the cation complexes adopt either a cage-like conformation or a tweezer-like conformation. The computations show that the partial hydration of those different conformations in the organic phase, determine the distinct cation extraction selectivities that are observed experimentally.

Efficient production of nonactin by Streptomyces griseus subsp. griseus.[Pubmed:27405846]

Can J Microbiol. 2016 Aug;62(8):711-4.

Here we report the production of the cyclic macrotetrolide Nonactin from the fermentation culture of Streptomyces griseus subsp. griseus. Nonactin is a member of a family of naturally occurring cyclic ionophores known as the macrotetrolide antibiotics. Our fermentation procedure of Streptomyces griseus was performed at 30 degrees C and 200 rev.min(-1) for 5 days on a rotary shaker. Diaion HP-20 and Amberlite XAD-16 were added to the fermentation medium. Isolated yield of Nonactin was up to 80 mg.L(-1) using our methodology. Nonactin is commonly known as an ammonium ionophore and also exhibits antibacterial, antiviral, and antitumor activities. It is also widely used for the preparation of ion-selective electrodes and sensors. Chemical synthesis of Nonactin has been achieved by some groups; however, overall yields are very low, making efficient biosynthesis an attractive means of production.

Determination of potassium ions in pharmaceutical samples by FIA using a potentiometric electrode based on ionophore nonactin occluded in EVA membrane.[Pubmed:12560044]

J Pharm Biomed Anal. 2003 Feb 5;31(1):11-8.

A simple and rapid method was developed for the K(+) ions determination employing a flow injection system using a flow-through electrode based on the naturally-occurring antibiotic ionophore Nonactin occluded in a polymeric membrane. The Nonactin ionophore was trapped in poly(ethylene-co-vinyl acetate) (EVA) matrix (40% w/w in vinyl acetate) and dispersed on the surface of a graphite-epoxy tubular electrode. The plasticizer-free all-solid-state potassium-selective electrode showed a linear response for K(+) concentrations between 5.0 x 10(-5) and 5.0x10(-2) M (r=0.9995) with a near-Nernstian slope of 51.5 mV per decade, when Tris-HCl buffer (pH 7.0;0,1 M) was employed as a carrier. The potentiometric-FIA system allows an analytical frequency of 120 samples per hour with a precision of 3.6%. The relative standard deviations (R.S.D.) for K(+) determination in pharmaceuticals samples, without any previous treatment, were lower than 4.0%, comparable to those obtained by flame photometry. Ammonium is the main analytical interference and the electrode response time was 5 s at 25 degrees C. The useful lifetime of the tubular sensor is longer than 3 months in continuous use.

Nonactin biosynthesis: the product of nonS catalyzes the formation of the furan ring of nonactic acid.[Pubmed:10390219]

Antimicrob Agents Chemother. 1999 Jul;43(7):1662-8.

Nonactin is the parent compound of a group of ionophore antibiotics, known as the macrotetrolides, produced by Streptomyces griseus subsp. griseus ETH A7796. Nonactin is a significant compound because of its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells. Nonactin is also significant in that it is a highly atypical polyketide. Very little is presently known about the genes of the Nonactin biosynthesis cluster. In this paper we describe our efforts to establish a connection between the product of a gene from the Nonactin biosynthesis cluster and a known biochemical transformation in Nonactin biosynthesis. Nonactate synthase is the enzyme which catalyzes the formation of nonactic acid from an acyclic precursor in Nonactin biosynthesis. We have synthesized the substrate for this enzyme and have detected the in vitro cyclization activity of the substrate in cell-free preparations of S. griseus subsp. griseus ETH A7796. Previous studies by R. Plater and J. A. Robinson (Gene 112:117-122, 1992) had suggested, based on sequence homology, that the product of a partial open reading frame found close to the tetranactin resistance gene of S. griseus could be the nonactate synthase. We have therefore cloned, sequenced, and heterologously expressed this full gene (nonS), and we have shown that the gene product, NonS, does indeed catalyze the formation of the furan ring of nonactic acid as hypothesized.

Mobile ionophores are a novel class of P-glycoprotein inhibitors. The effects of ionophores on 4'-O-tetrahydropyranyl-adriamycin incorporation in K562 drug-resistant cells.[Pubmed:7518390]

Eur J Biochem. 1994 Jul 1;223(1):125-33.

The decrease of the intracellular concentration of drug in resistant cells compared to sensitive cells is, in most cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein responsible for an active efflux of the drug. In an attempt to identify mechanism(s) by which multidrug resistance can be circumvented, we have examined the cellular accumulation of 4'-O-tetrahydropyranyl-adriamycin, alone and in conjunction with various ionophores on the one hand and with cyclosporin A on the other hand. The present study was performed using a spectrofluorometric method with which it is possible to follow continuously the uptake and release of fluorescent molecules by living cells, as the incubation of the cells with the drug proceeds. Erythroleukemia K562 cell lines were used. Using experimental conditions in which these ionophores were unable to modify either the intracellular pH, or the transmembrane potential, or to induce an intracellular ATP depletion, we have shown that mobile ionophores as well as cyclosporin inhibit the P-glycoprotein-mediated efflux of 4'-O-tetrahydropyranyl-adriamycin in K562 resistant cells, whereas gramicidin, a channel-forming ionophore, does not. The concentration that must be used to inhibit 50% of the efflux was 0.7 microM for valinomycin, 0.4 microM for Nonactin, 0.2 microM for nigericin, 1.1 microM for monensin, 0.4 microM for lasalocid, 1.2 microM for calcimycin and 0.4 microM for cyclosporin. Due to the high toxicity of the ionophores, the observation that they increased 4'-O-tetrahydropyranyl-adriamycin accumulation in the multidrug-resistant cells is not correlated with an effect of these compounds on drug resistance. However, the correlation exists in the case of cyclosporin. From our data showing that lipophilic neutral complexes, formed between carboxylic ionophores and metal ions, are both able to inhibit the P-glycoprotein-mediated efflux of anthracycline we can infer that the lipophilicity but not the cationic charge is an important physical property.

Nonactin is a naturally occurring macrotetrolide antibiotic from Streptomyces griseus. Nonactin acts as an ionophore for monovalent cations, including K+, and NH4+. Nonactin is able to uncouple the oxidative phosphorylation of mitochondria. Nonactin selectively induces apoptosis in cell lines harboring active mutant β-catenin. Nonactin inhibits the surface expression of endogenous HSP60.

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