Structure and interaction of membrane active polyene antibiotics amphotericin B and filipin

Abstract

Polyene antibiotics are a group of highly unsaturated macrolide compounds. They alter the permeability of membranes of cells by displaying a selective action against organisms whose membranes contain sterols. The polyenes are therefore active against yeast and a wide variety of fungi but have no action on bacteria. They are produced mainly by members of the Streptomycetaceae and include some of the most effective antifungal agents known. While extensive studies are reported in the literature on the action of polyene antibiotics at the cellular and membrane level, the mechanism of their action at the molecular level is not fully understood.

In this thesis, detailed investigations on the structure, aggregation properties, and interaction with model membranes of two polyene antibiotics, namely, Amphotericin B and Filipin, are reported with the objective of gaining insight into the molecular mechanism of their action. While Amphotericin B is a very effective antifungal agent used in systemic mycotic therapy, Filipin, which exhibits weaker antifungal properties, is not used for clinical purposes and is more known as a sterol probe.

The methods employed in these investigations are principally nuclear magnetic resonance (NMR) and circular dichroism (CD). Molecular modeling techniques have been used as an extension to NMR studies wherever possible. Differential scanning calorimetry (DSC) and electron spin resonance (ESR), as well as fluorescence assays, have been used when required. The thesis is divided into three parts: Section I describes the studies on Amphotericin B, Section II deals with Filipin III, and Section III encompasses the modeling studies of both Amphotericin B and Filipin III.

Chapter 1 gives a brief introduction to polyene antibiotics, followed by an overview of Amphotericin B and Filipin III. The aspects reviewed include their discovery, structure, therapeutic effects, and models that try to explain their mode of action. The objectives of the present investigation are stated at the end of the chapter.

Chapter 2 deals with methodology, details of experimental techniques, and reagents used.

Structure and aggregation properties of Amphotericin B in solution and the effect of divalent cations on the structure are described in Chapter 3. The detailed CD and NMR results on Amphotericin B showed very interesting solvent-dependent aggregation properties of the molecule. The detailed solution structure of Amphotericin B in DMSO based on NMR data is a “head-to-tail” dimer, similar to that observed in the crystal structure (Rajini Balakrishnan and Easwaran, 1993b). The model suggests a strong hydrophobic interaction between the heptene conjugated stretch in stabilizing the structure. Studies on the Amphotericin B-cation complexes showed that the molecule preferentially binds to divalent cations. Detailed NMR studies on the Amphotericin B–Mg² complex showed that the most plausible model for the structure of the complex is a 2:1 Amphotericin B–Mg², interacting in a head-to-head fashion with Mg² coordinating with three oxygen atoms from each molecule.

In Chapter 4, the CD, NMR, DSC, and ESR studies of Amphotericin B incorporated into phospholipid vesicles are described. The major objectives of these studies are: (a) To understand the conformation and aggregation of Amphotericin B within sterol-free and sterol-containing model membrane systems, (b) Location of Amphotericin B when incorporated into liposomal membranes, (c) To find out the effect of Amphotericin B on the phase transition properties of lipid bilayers, and (d) To understand how the drug might modulate the microenvironment of the bilayer structure.

The studies clearly showed that Amphotericin B strongly interacts with membrane phospholipids and that sterol helps in inducing the aggregation of the drug molecules.

Chapter 5 deals with the studies on Amphotericin B in chloroform–phospholipid solution. The experimental evidence presented demonstrated for the first time the formation of a stoichiometric complex of phospholipid:Amphotericin B in chloroform (Rajini Balakrishnan and Easwaran, 1993a). The model proposed based on NMR results showed that the conjugated heptene stretch of Amphotericin B strongly interacts with the methylene groups of lipid acyl chains, while the sugar moiety of Amphotericin B interacts with the phosphate group of the lipid via hydrogen bonding. These results point to the fact that formation of the lipid:Amphotericin B complex is likely the first step in the aggregation process in cell membranes. Sterol interaction with Amphotericin B and Amphotericin B–phospholipid complex is also discussed in this chapter.

Chapter 6 describes the studies on: (a) The solvent-dependent interactions of Filipin III and determination of the solution structure for Filipin III, (b) The interaction of Filipin with liposomal membranes with and without sterol present, (c) The solution structure of Filipin III–cholesterol complex, and (d) A comparative analysis between Amphotericin B and Filipin III.

The complete assignments and the conformation of the Filipin III monomer have been worked out using 2D NMR experiments. The studies have also clearly highlighted some of the significant differences in the structure and aggregation properties of Amphotericin B and Filipin. While Amphotericin B forms a strong complex with phospholipids, Filipin forms a tight complex with sterol.

Chapter 7 describes the molecular modeling studies on Amphotericin B and Filipin. The first half concentrates on: (a) Modeling and refining Amphotericin B monomer structure derived from NMR data, (b) Details of the molecular simulation of the head-to-tail dimer structure of Amphotericin B, and (c) Details of the modeling studies on the lipid:Amphotericin B complex.

The second half deals with: (a) Modeling of Filipin III monomer structure derived from NMR data, and (b) Modeling of Filipin III–sterol complexes using NMR results.

The results obtained from these studies are discussed in relation to the experimental findings.