Daniel M. Kamiński

Recent progress in the study of the interactions of amphotericin B with cholesterol and ergosterol in lipid environments.

Abstract In the past decade substantial progress has been made in understanding the organization and biological activity of amphotericin B (AmB) in the presence of sterols in lipid environments. This review concentrates mainly on interactions of AmB with lipids and sterols, AmB channel formation in membranes, AmB aggregation, AmB modifications important for understanding its biological activity, and AmB models explaining its mechanism of action. Most of the reviewed studies concern monolayers at the water–gas interface, monolayers deposited on a solid substrate by use of the Langmuir–Blodgett technique, micelles, vesicles, and multi-bilayers. Liposomal AmB formulations and drug delivery are intentionally omitted, because several reviews dedicated to this subject are already available.

On polymorphism of 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole (FABT) DMSO solvates

Structural and computational studies of two polymorphic (triclinic P and monoclinic P21/n) DMSO solvates of the biologically active molecule 2-(4-fluorophenylamino)-5-(2,4-dihydroxybenzeno)-1,3,4-thiadiazole (FABT) show that their structures are stabilized mainly by hydrogen bonds between the FABT and DMSO molecules. The geometry of both polymorphic molecules is very similar with a few significant differences in the corresponding valence angles. The only exceptions are the valence angles associated with the terminal para-hydroxyl group in both polymorphs. This group is differently H-bonded to the neighbouring solvent molecules. Additionally, the molecule in the second polymorph is slightly bent compared to the molecule in the first one. Both polymorphs also have very similar packing with layers of FABT molecules separated by DMSO moieties. The Hirshfeld surface analysis shows the most significant differences in the relative contributions of intermolecular interactions to the total Hirshfeld surface area for the FABT polymorphs. They are found for the C⋯H, C⋯C and H⋯H interactions. The triclinic polymorph crystallises as the first one and is thermodynamically less stable, while the monoclinic one is thermodynamically more stable but occurs in the crystallization mixture after a much longer time. According to the computational results, the monoclinic polymorph is ca. −9.93 kJ mol−1 more stable than the triclinic one, and dispersive interactions are dominant in these polymorphic crystals. It appears that the FABT⋯FABT interactions in the crystal lattice are stronger (ca. −75 kJ mol−1) for both polymorphs than the interactions of the central FABT moiety with the neighbouring DMSO molecules (ca. −52 kJ mol−1). Interlayer interaction energies calculated for the most characteristic slabs defined in the crystal lattices of the both polymorphs can be related to the stability of crystals.

The molecular organization of prenylated flavonoid xanthohumol in DPPC multibilayers: X-ray diffraction and FTIR spectroscopic studies

Xanthohumol (XN) is the major prenylated flavonoid found in hop resin. It has attracted considerable attention in recent years due to its wide spectrum of biological activities and the beneficial effect on human health. Since lipid membrane is first target for biologically active compounds, we decided to investigate the influence of XN on the dipalmitoylphosphatidylcholine (DPPC) multibilayers. Interactions of XN with DPPC were investigated as a function of temperature and its concentration by using X-ray diffraction and the ATR-FTIR spectroscopy techniques. The aim of understanding the mechanisms of molecular interactions between XN and DPPC was to indicate the localization of the XN with respect to the membrane and the type of interaction with phospholipids. The results revealed that XN changes the physical properties of the DPPC multibilayers in the form of dry film. A new complex formation between XN and DPPC is reported. The detailed analysis of refraction effect indicates the changes in electron density ratio between hydrophobic and hydrophilic zones of lipid at phase transition. This is in compliance with reported changes in FTIR spectra where at pretransition XN moves from interface region between polar heads to the neighborhood of phosphate groups.

Spectroscopic studies of amphotericin B–Cu2+ complexes

The aim of this research is to investigate amphotericin B (AmB)–Cu2+ complexes in aqueous solution at different pH values. Electronic absorption, circular dichroism (CD), Raman and FTIR spectroscopies were used in this study. We found that different concentrations of AmB and Cu2+ ions in solution leads to formation of complexes with stoichiometry of 2:1 and 1:1. The formation of AmB–Cu2+ complexes at physiological pH values is accompanied by changes of the molecular organization of AmB especially disaggregation. These observed effects might be significant from a medical point of view.

Influence of Solvent Polarizability on the Keto-Enol Equilibrium in 4-(5-(naphthalen-1-ylmethyl) -1,3,4-thiadiazol-2-yl)benzene-1,3-diol

This work presents spectroscopic studies of the keto–enol equilibrium induced by solvent polarizability in 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl]benzene-1,3-diol a strong antiproliferative and anticancer thiadiazol derivative. Electronic absorption, steady state and time resolved fluorescence, and infrared spectroscopies were applied to investigate the keto and enol forms of this compound in a series of polar and non-polar solvents. The enol form dominates in polar solvents while, surprisingly, the keto form dominates in non-polar solvents with high average electric dipole polarizability e.g. n-alkenes. The electronic absorption spectrum of this derivative is more dependent on spatially averaged electric dipole polarizability of the solvent than on Kirkwood’s correlation or on Lorenz-Lorenz electric polarizability. By analogy of n-alkanes to the alkyl parts of lipids, one can expect that the transformation of 1,3,4-thiadiazoles to the keto form may be facilitated in the hydrophobic core of the lipid membrane. Such a transition may be of great practical importance for the design of biologically active pharmaceutics, which are able to interact with the hydrophobic regions of cell membranes in a specific manner.

Effect of cholesterol and ergosterol on the antibiotic amphotericin B interactions with dipalmitoylphosphatidylcholine monolayers: X-ray reflectivity study.

Amphotericin B is a Streptomyces nodosus metabolite and one of the oldest polyene antibiotics used in the treatment of invasive systemic fungal infections. Despite its over 50-year existence in clinical practice and the recognition of amphotericin B as the gold standard in the treatment of serious systemic mycosis, it still remains one of the most toxic pharmaceuticals. Understanding of the processes at the molecular levels and the interactions between amphotericin B with lipid membranes containing sterols should elucidate the mechanisms of the action and toxicity of this widely used antibiotic. In this work, we use X-ray reflectivity to study the structural changes on a molecular scale after amphotericin B incorporation. These changes are accompanied by an increase in monolayer surface pressure which is more pronounced for ergosterol - rather than cholesterol-rich membranes. The data indicate that this difference is not due to the higher affinity of amphotericin B towards ergosterol-containing membranes but is rather due to a ~3Angstrom corrugation of the monolayer. Furthermore, the total quantity of amphotericin B incorporated into lipid monolayers containing cholesterol and ergosterol is the same.

Spectroscopic evidence for self-organization of N-iodoacetylamphotericin B in crystalline and amorphous phases.

In this paper, we propose a new way of thinking about molecular self-organization of the antibiotic amphotericin B (AmB) by examination of its N-iodoacetyl derivative (AmB-I). This choice was dictated by the simplicity of AmB-I crystallization as compared to pure AmB. The studies focus on spectroscopic investigations of the monocrystal and the amorphous state of AmB-I. The results of vibrational, FTIR, and Raman spectroscopy show differences between the crystalline and amorphous forms, in particular for bands attributed to C═O (1700-1730 cm(-1)) and C-C-H groups, as well as C═C-C (ca. 1010 cm(-1)) stretching vibrations. The process of crystallization is identified by strong differences in the intensities and locations of these characteristic bands. For the AmB-I crystals, the carbonyl band is shifted toward lower frequencies as a result of intensified hydrogen bonding in the crystalline form. Detailed analysis indicates that bands in the region characteristic for the C═C-C bending distortion in the chromophore are particularly intense for AmB-I in the crystalline form as compared to the intensity of this band in the amorphous state. These findings are corroborated by the results of fluorescence spectroscopy. We observe a much faster decay of the emission for the AmB-I monocrystal as compared to the DMSO solution of AmB-I. Interestingly, the fluorescence decay in the amorphous form requires three decay times for simulating the observed behavior; two of these decay constants are sufficient for estimating the decay measured for the AmB-I crystals. The proof of the molecular organization of AmB-I molecules is obtained from polarization-resolved fluorescence spectroscopy on a single AmB-I crystal. Strong anisotropy of the emission intensity correlates with the axes of the crystal, providing insight into actual alignment of the molecules in the AmB-I crystals. These findings related to molecular organization in AmB-I crystals are crucial for understanding toxicity mechanisms of the clinically used drug, amphotericin B.

Structural and energetic studies of FABT and FABTH+solvates

2 ( 4 f l u o r o p h e n y l a m i n o ) 5 ( 2 , 4 dihydroxybenze no)-1,3,4-thiadiazole (FABT) is a biologically active compound. It forms planar molecules (FABT) and cations (FABTH + ). Details of structural and computational studies of two polymorphs of FABT DMSO solvates [1] and three alcohol solvates of FABTH + chloride [2] are presented. Structures of both polymorphs of FABT DMSO solvates (the triclinic P-1 abbreviated FBDM I and the monoclinic P21/n abbreviated FBDM II) are stabilized by hydrogen bonds between the FABT and DMSO moieties. The geometry of both polymorphic molecules is very much alike with almost no significant difference in the corresponding bond lengths and valence angles. The only exceptions are the valence angles associated with the terminal parahydroxyl group which is differently H-bonded to the solvent molecules in both polymorphs. Additionally, the FBDM II molecule is slightly bent comparing to FBDM I. Both polymorphs have also a very similar packing with layers of FABT molecules separated by DMSO moieties. As can be seen from the Hirshfeld surface analysis [3], the most significant differences in the relative contributions of intermolecular interactions to the Hirshfeld surface area for FBDM polymorphs are found for the C..H, C...C and H...H interactions. It appears that the FBDM I polymorph crystallises first and is thermodynamically less stable, while FBDM II is thermodynamically more stable but occurs in the crystallization mixture after much longer time. The computational results allow to identify the more stable polymorph (FBDM II ca. -9.93 kJ∙mol –1 ). Single crystals of the FABTH +