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The work presented is a part of our continual study on the behavior of the polyene macrolide antibiotic amphotericin B (AmB) complexes with sterols on the molecular level. In contrast to the previously researched AmB-ergosterol binary complex, the AmB-ergosterol-AmB aggregates simulated of 2:1 stoichiometry retain significantly higher stability and relatively rigid, "sandwich" geometry. Van der Waals forces with a considerable share of the electrostatic interactions are responsible for such behavior. System of the intermolecular hydrogen bonds also seems to be of notable importance for the complex's structure preservation. The most energetically favored geometries match fairly close the geometric criteria and the network of interactions postulated in the contemporary hypothetical and computational models of antibiotic-sterol complexes. On the basis of works previously published and the present study novel hypotheses on the AmB selectivity towards sterols varying in chemical structure and on the possible mechanisms of channel structure formation were presented.

The novel group of amphotericin B (AmB) cationic derivatives has been examined in terms of relationship between self-association and selective toxicity. In all determinations AmB has been used as reference compound. In vitro toxic effects of the compounds on human erythrocytes were determined by measuring leakage of intracellular potassium ions and hemolysis. Antifungal effects were determined as MIC and intracellular potassium loss. The compounds self-association was followed by UV-Vis spectroscopy. The results suggested that: i) unlike AmB the monomer/self-associated species ratio is not an essential in governing the selective toxicity of the derivatives studied; ii) the presence of a bulky substituent in the AmB molecule, preferably located at the amino group of mycosamine moiety is the structural factor essential for the selective toxicity improvement.

The properties of amphotericin B-ergosterol primary complex have been studied with the use of the molecular dynamics simulation. Possible geometries of the complex were tested first in order to find the structures with the most favourable values of the intermolecular interactions energy. The molecules studied possessed a tendency to fit each other's shapes, which favours intermolecular van der Waals interactions. The main simulations were performed for the best structures found. Presence of hydrogen bonds between the sterol hydroxyl group and polar fragments of mycosamine (most frequently 2'OH) was coupled with a relatively high level of the intermolecular energy values. The structures obtained are hardly comparable to the hypothetical and 'computational' models of the antibiotic-sterol complex. The geometries found are not suitable to assemble the presupposed structure of the water channel, however, the existence of the complex in the shape anticipated is not in contradiction to the results of biophysical experiments on the complexation in water and in hydroalcoholic media.

In aqueous solutions N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME), a novel amphotericin B derivative with low animal toxicity, similar to its parent antibiotic, exists in three forms: monomeric, soluble and insoluble aggregates in equilibrium [1]. The aim of our work was to examine the influence of medium composition on the MFAME self-association and the relationship between MFAME self-association and its toxicity towards red blood cells. The toxicity of MFAME in aggregated state towards red blood cells was tested by measuring the induction of potassium leakage and extent of haemolysis. The proportions of antibiotic species present in various aqueous media were determined by analysis of the UV-Vis spectra as a function of the antibiotic concentration. Numeric decomposition of the spectra allowed identification of four spectral species present in MFAME solutions: monomeric and three aggregated forms. Our results indicate that these aggregates, named type I, type II and type III, are different in terms of spectral properties, as well as effectiveness towards red blood cells. Soluble aggregate types I and III are the active forms of MFAME towards erythrocytes. The medium composition seems to be the main factor determining which type of antibiotic aggregate prevails in solution.

Rational chemical modification of amphotericin B (AMB) led to the synthesis of sterically hindered AMB derivatives. The selected optimal compound, N-methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME) retains the broad spectrum of antifungal activity of the parent antibiotic, and exhibits a two orders of magnitude lower toxicity in vivo and in vitro against mammalian cells. Comparative studies of MF-AME and AMB comprising the determination of the spectroscopic properties of monomeric and self-associated forms of the antibiotics, the investigation of the influence of self-association on toxicity to human red blood cells, and of the antibiotic-sterol interaction were performed. On the basis of the results obtained it can be assumed that the improvement of the selective toxicity of MF-AME could in part be a consequence of the diminished concentration of water soluble oligomers in aqueous medium, and the better ability to differentiate between cholesterol and ergosterol.

Molecular dynamics simulations were performed for a dimer of the antifungal antibiotic, amphotericin B, in water. In the first step of the work three appropriately selected versions of the dimer structure were taken into consideration. In each version antibiotic molecules were placed antiparallel with polar and ionizable groups outside the hydrophobic core formed by polyene chromophores. During short dynamic simulations versions of the dimer structure were compared in respect of the energy of dimerization. The highest energy was observed for the structure in which polyene chromophores superimposed each other as much as possible and this version was subjected to the main simulation. The analysis of 66 snapshot geometries stored during 33 ps dynamic trajectory allowed us to draw three main conclusions: (i) the relative orientation of the amino-sugar moiety and chromophore as well as conformation of the antibiotic macrolide ring were different in both molecules and could exhibit dynamic changes, (ii) the dimer structure exhibited intrinsic asymmetry which could be responsible for characteristic circular dichroism spectra of the aggregated form of the antibiotic, (iii) relatively high stability of the dimer structure resulted not only from hydrophobic interactions between chromophores but also from hydrogen bonds networks that were observed around polar terminals of antibiotic molecules. Implications of these features of the dimer structure for its susceptibility on the ionic state of carboxyl and/or amino groups are also discussed.

The interaction of amphotericin B and some of its semisynthetic derivatives with cholesterol and ergosterol has been tested in 1:4 (v/v) ethanol/water mixture by circular dichroism and absorption spectroscopy. The effect of the chemical modification of the 'ionizable head' of the antibiotic, the pH of the medium, and the sterol/antibiotic ratio has been studied. The results obtained show that in the presence of the sterols, amphotericin B forms several spectroscopically different species. A high extent of polyene-sterol interaction is observed for: (i) amphotericin B in neutral or acidic media, (ii) esters and amides at neutral or alkaline media, (iii) N-acyl derivatives only in acidic medium. The extent of interaction at neutral pH is highly correlated with the biological activity of compounds tested. The implication of these findings on the nature of the forces responsible for the antibiotic-sterol interaction is discussed.

Synthesis and biological properties of amphotericin B O-methyl oxime are described. The presence of an intact hemiketal ring in the antibiotic molecule appeared to be essential for its biological activity.

The poor solubility of polyene antibiotics in aqueous media limits their application in the therapy of systemic fungal infections. In the present paper we have demonstrated that the ionic state (net electrical charge) of the antibiotic molecule is an important factor in determining the aggregation and solubility properties of amphotericin B and its derivatives. A multi-step model of polyene self-association in aqueous media has been proposed as an explanation for the fact that some major differences are observed when aggregation is monitored by different techniques.

The interactions of unilamellar lipid vesicles with vacidin A, an aromatic heptaene antibiotic, and with amphotericin B, a nonaromatic heptaene antibiotic were compared. Uptake of both antibiotics, monitored by circular dichroism, was found to be faster with small vesicles than with large ones. By combining permeability measurements (Gary-Bobo and Cybulska, J. Antibiotics 35, 1068 (1982)) and circular dichroism spectra, we found that for vacidin A, the same permeability inducing species is formed regardless of vesicles size. However, at a given concentration of antibiotic, less of the permeability inducing species is formed in the presence of small vesicles than in the presence of large vesicles. This may account for the differences between small and large vesicles in antibiotics-induced permeability. For amphotericin B, the permeability inducing species formed in the presence of small vesicles differs from that formed in the presence of large vesicles.

The quantitative structure-activity relationships studies of amphotericin B and its 16 semisynthetic derivatives obtained by modification at carboxyl and amino groups have been done. The results of five biological tests were subjected to principal component analysis, a numerical method useful in the investigation of large sets of data. For some compounds, also, interaction with lipidic vesicles was investigated by spectroscopic methods. The results obtained indicate that: (i) The presence of positively charged nitrogen atom (protonable or bearing fixed charge) is indispensable for biological activity and antibiotic-sterol interaction; (ii) The lack of free carboxyl group in the molecule favours the differentiation between cholesterol and ergosterol containing cells.