Jacek Czub

Amphotericin B and its new derivatives - mode of action.

Amphotericin B (AmB) is a well known antifungal and antiprotozoal antibiotic used in the clinic for several decades. Clinical applications of AmB, however, are limited by its nephrotoxicity and many other acute side effects which are not acceptable by patients when their life is not threaten. In order to improve the therapeutic index of this drug, lipid formulations have been introduced and many efforts have been made to obtain less toxic AmB derivatives by chemical modifications of the parent drug. This review presents concise knowledge about this fascinating compound and a critical review of the data published within last few years about the mechanism of action of this antibiotic. In particular, in the present work we discuss: i) structure and properties of AmB and its recently synthesized new derivatives; ii) antifungal and antileishmanial activity and toxicity of these compounds; and iii) mode of action of AmB and its derivatives at cellular and molecular levels, with particular attention paid to interactions of AmB and different components of cellular membranes.

On the possibility of the amphotericin B-sterol complex formation in cholesterol- and ergosterol-containing lipid bilayers: a molecular dynamics study.

Amphotericin B (AmB) is a well-known membrane-active antibiotic that has been used to treat systemic fungal infections for more than 45 years. Therapeutic application of AmB is based on the fact that it is more active against ergosterol-containing membranes of fungal cells than against mammalian membranes with cholesterol. In this paper, we examine the hypothesis according to which the selectivity of the AmBs membrane action originates from its different ability to form the binary complexes with the relevant sterols. To this end, molecular dynamics simulations were performed for systems containing the preformed models of AmB/sterol complexes embedded in lipid bilayers containing either cholesterol or ergosterol. The initial structures of the studied binary associates were selected on the basis of a systematic scan of all possible mutual positions and orientations of the two molecules. The results obtained demonstrate that in general the complexes with ergosterol are more stable on the 100 ns time scale. Furthermore, on the basis of motional correlation analysis, taking into account the effects of lipid environment, we propose that, within the sterol-enriched liquid-ordered membrane phases, AmB molecules exhibit a greater tendency to bind ergosterol than cholesterol. The analysis of the interactions suggests that this affinity difference is of enthalpic origin and may arise from the considerable difference in the energy of the van der Waals interactions between AmB and the two types of sterols. Thus, our current results: (i) support the hypothesis that binary AmB/sterol complexes form within a lipid membrane and (ii) suggest that the higher toxicity may at least partly be attributed to the higher affinity of AmB for ergosterol than for cholesterol within a lipid membrane environment.

The effect of sterols on amphotericin B self-aggregation in a lipid bilayer as revealed by free energy simulations.

Amphotericin B (AmB) is an effective but toxic antifungal drug, known to increase the permeability of the cell membrane, presumably by assembling into transmembrane pores in a sterol-dependent manner. The aggregation of AmB molecules in a phospholipid bilayer is, thus, crucial for the drugs activity. To provide an insight into the molecular nature of this process, here, we report an atomistic molecular dynamics simulation study of AmB head-to-head dimerization in a phospholipid bilayer, a possible early stage of aggregation. To compare the effect of sterols on the thermodynamics of aggregation and the architecture of the resulting AmB-AmB complexes, free energy profiles for the dimerization in ergosterol- or cholesterol-containing and sterol-free membranes are derived from the simulations. These profiles demonstrate that although AmB dimers are formed in all the systems studied, they are significantly less favorable in the bilayer with ergosterol than in the cholesterol-containing or sterol-free ones. We investigate the structural and energetic determinants of this difference and discuss its consequences for the AmB mechanism of action.

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.

Specific Binding of Cholesterol to the Amyloid Precursor Protein: Structure of the Complex and Driving Forces Characterized in Molecular Detail.

C99 is the C-terminal membrane-bound fragment of the amyloid precursor protein that is cleaved by γ-secretase to release Aβ peptides, the hallmark of Alzheimers disease (AD). Specific interactions of C99 with cholesterol have been proposed to underlie the recognized role of cholesterol in promoting amyloidogenesis. By using molecular dynamics simulations, we studied cholesterol binding to C99 in a lipid bilayer. We determined the free-energy profile of binding and analyzed the structure of C99/cholesterol complexes in two low-energy binding modes. We also examined the complexation driving forces and found, unexpectedly, that the interactions between the GxxxG dimerization motif and the cholesterol ring system are not sufficient for binding and that further stabilization mediated by the C99 N-terminal domain is essential. Taken together, our results strongly support the view that C99 specifically binds cholesterol in the cell membrane; the detailed information on the structure and energetics of the complex may assist in the design of new anti-AD drugs.

Molecular dynamics simulations reveal the balance of forces governing the formation of a guanine tetrad—a common structural unit of G-quadruplex DNA

G-quadruplexes (G4) are nucleic acid conformations of guanine-rich sequences, in which guanines are arranged in the square-planar G-tetrads, stacked on one another. G4 motifs form in vivo and are implicated in regulation of such processes as gene expression and chromosome maintenance. The structure and stability of various G4 topologies were determined experimentally; however, the driving forces for their formation are not fully understood at the molecular level. Here, we used all-atom molecular dynamics to probe the microscopic origin of the G4 motif stability. By computing the free energy profiles governing the dissociation of the 3′-terminal G-tetrad in the telomeric parallel-stranded G4, we examined the thermodynamic and kinetic stability of a single G-tetrad, as a common structural unit of G4 DNA. Our results indicate that the energetics of guanine association alone does not explain the overall stability of the G-tetrad and that interactions involving sugar–phosphate backbone, in particular, the constrained minimization of the phosphate–phosphate repulsion energy, are crucial in providing the observed enthalpic stabilization. This enthalpic gain is largely compensated by the unfavorable entropy change due to guanine association and optimization of the backbone topology.

Membrane Sterols Modulate the Binding Mode of Amphotericin B without Affecting Its Affinity for a Lipid Bilayer

Membrane-active antibiotics are known to selectively target certain pathogens based on cell membrane properties, such as fluidity, lipid ordering, and phase behavior. These are in turn modulated by the composition of a lipid bilayer and in particular by the presence and type of membrane sterols. Amphotericin B (AmB), the golden standard of antifungal treatment, exhibits higher activity toward ergosterol-rich fungal membranes, which permits its use against systemic mycoses; however, the selectivity for fungal membranes is far from satisfactory leading to severe side effects. Despite decades of research, no consensus has emerged on the origin of AmB specificity for fungal cells and its actual mode of action at the molecular level. Previously, it has been proposed that the specific action of AmB is related to differences in its affinity for membranes of different composition. In this work, we investigate this relationship by employing molecular dynamics simulations to compare the free energy of insertion of AmB into three types of membranes: a pure DMPC bilayer and DMPC bilayers containing 30% of cholesterol or ergosterol. We analyze the orientation of AmB molecules within the bilayer in order to unambiguously establish their membrane binding mode and relate the orientational freedom to the sterol-dependent tightness of lipid packing. Our results strongly indicate that the membrane insertion of AmB proceeds virtually to completion independent of membrane type, and hence the higher toxicity against fungal membranes may rather result from differences in subsequent oligomerization in the membrane and assembly of monomers into functional transmembrane pores. In particular, the latter could be facilitated by sterol-induced ordering of AmB molecules along the membrane normal, revealed by our free energy profiles. Moreover--in contrast to certain claims--we find no stable binding mode corresponding to the horizontal adsorption of AmB on the membrane surface.

Hydration of amino acids: FTIR spectra and molecular dynamics studies

The hydration of selected amino acids, alanine, glycine, proline, valine, isoleucine and phenylalanine, has been studied in aqueous solutions by means of FTIR spectra of HDO isotopically diluted in H2O. The difference spectra procedure and the chemometric method have been applied to remove the contribution of bulk water and thus to separate the spectra of solute-affected HDO. To support interpretation of obtained spectral results, molecular dynamics simulations of amino acids were performed. The structural-energetic characteristic of these solute-affected water molecules shows that, on average, water affected by amino acids forms stronger and shorter H-bonds than those in pure water. Differences in the influence of amino acids on water structure have been noticed. The effect of the hydrophobic side chain of an amino acid on the solvent interactions seems to be enhanced because of the specific cooperative coupling of water strong H-bond chain, connecting the carboxyl and amino groups, with the clathrate-like H-bond network surrounding the hydrocarbon side chain. The parameter derived from the spectral data, which corresponds to the contributions of the population of weak hydrogen bonds of water molecules which have been substituted by the stronger ones in the hydration sphere of amino acids, correlated well with the amino acid hydrophobicity indexes.

Molecular basis of the osmolyte effect on protein stability: a lesson from the mechanical unfolding of lysozyme

Osmolytes are a class of small organic molecules that shift the protein folding equilibrium. For this reason, they are accumulated by organisms under environmental stress and find applications in biotechnology where proteins need to be stabilized or dissolved. However, despite years of research, debate continues over the exact mechanisms underpinning the stabilizing and denaturing effect of osmolytes. Here, we simulated the mechanical denaturation of lysozyme in different solvent conditions to study the molecular mechanism by which two biologically relevant osmolytes, denaturing (urea) and stabilizing (betaine), affect the folding equilibrium. We found that urea interacts favorably with all types of residues via both hydrogen bonds and dispersion forces, and therefore accumulates in a diffuse solvation shell around the protein. This not only provides an enthalpic stabilization of the unfolded state, but also weakens the hydrophobic effect, as hydrophobic forces promote the association of urea with nonpolar residues, facilitating the unfolding. In contrast, we observed that betaine is excluded from the protein backbone and nonpolar side chains, but is accumulated near the basic residues, yielding a nonuniform distribution of betaine molecules at the protein surface. Spatially resolved solvent-protein interaction energies further suggested that betaine behaves in a ligand- rather than solvent-like manner and its exclusion from the protein surface arises mostly from the scarcity of favorable binding sites. Finally, we found that, in the presence of betaine, the reduced ability of water molecules to solvate the protein results in an additional enthalpic contribution to the betaine-induced stabilization.

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation.