Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Abstract

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, 23Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na+ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.

Structural Features of Dinoflagellate Toxins Underlying Biological Activity as Viewed by NMR

“…3). All these findings together with previous results 116 may lead to a hypothetical molecular mode of action for AMs: (a) binding to the bi- layer membrane containing sterols chiefly with the polyolefin region (the C52 moiety) and the membrane affinity is heavily dependent on the length of the polyolefinic chain; (b) the center region (the C20-C51 moiety) takes a hairpin-shaped conformation, which is stabilized by a hydrogen-bond network in amphipathic environments, and (c) the flexible polyhydroxy region (the C1-C20 moiety) acts as a lining of ion-permeable ion channels.…”

“…[113][114][115] On the other hand, the size of pores/lesions in erythrocyte membranes were estimated to be 2.0-2.9 nm in diameter, which is significantly larger than that of amphotericin B, 0.8 nm. 116 SDS micelles are reported to adequately mimic the amphipathic nature of a bilayer membrane even for a conformationally sensitive peptide such as gramicidin A. 114 Thus, the conformation of membrane-bound AM was deduced on the basis of NMR data obtained in SDS micelles.…”

Precise Size Determination of Amphotericin B and Nystatin Channels Formed in Erythrocyte and Liposomal Membranes Based on Osmotic Protection Experiments

“…In erythrocyte membranes, the sizes of channels were evaluated by osmotic protection experiments. [4][5][6] In these experiments, glucose passed through channels formed by amphotericin B and nystatin, [4][5][6] which were significantly different from the results of planar membranes showing impermeability to glucose. In the present study, we also observed that amphotericin B and nystatin made channels, allowing glucose to be permeable.…”

“…To test this possibility, we determined the channel sizes of polyene antibiotics, amphotericin B and nystatin, because these sizes formed in erythrocyte membranes have been reported to be roughly between radii of 0.36 and 0.46 nm, using sugars as protectants. [4][5][6] To verify the osmotic cell lysis process in this study, we simultaneously monitored the time courses of hemolysis and K + efflux from sheep erythrocytes induced by amphotericin B, by combining a fiber-optic spectrometer with a K + -selective electrode.…”

Isolation of activity and partial characterization of large non-proteinaceous lytic allelochemicals produced by the marine dinoflagellate Alexandrium tamarense

“…The only exception is maitotoxin with a molecular mass of 3422 g mol -1 , the largest non-biopolymer natural product known (Murata et al, 1994). Karlotoxins from the dinoflagellate Karlodinium veneficum (Bachvaroff et al, 2008;Van Wagoner et al, 2008) as well as amphidinols from Amphidium klebsii (Houdai et al, 2004;Houdai et al, 2005) have a similar mass range of about 1300 Da and membrane disruptive properties. These compounds form hairpin structures suggested to be important for the interaction with lipid bilayers of biomembranes.…”