How do sterols determine the antifungal activity of amphotericin B? Free energy of binding between the drug and its membrane targets

Amphotericin B (AmB) is a well-known polyene antibiotic used to treat systemic fungal infections. It is commonly accepted that the presence of sterols in the membrane is essential for the AmB biological activity, that is, for the formation of transmembrane ion channels. The selective toxicity of AmB for fungal cells is attributed to the fact that it is more potent against fungal cell membranes containing ergosterol than against the mammalian membranes with cholesterol. According to the "primary complex" hypothesis, AmB associates with sterols in a membrane to form binary complexes, which may subsequently assemble into a barrel-stave channel. To elucidate the molecular nature of the AmB selectivity for ergosterol-containing membranes, in the present work, we used computational methods to study the formation of the putative AmB/sterol complexes in a lipid bilayer. The free energy profiles for the AmB-sterol association in phospholipid bilayers containing 30 mol % of sterols were calculated and thoroughly analyzed. The results obtained confirm the formation of specific AmB/ergosterol complexes and are used to determine the energetic and structural origin of the enhanced affinity of AmB for ergosterol than for cholesterol. The significance of this affinity difference for the mechanism of action of AmB is discussed. The data obtained allowed us also to suggest a possible origin of the increased selectivity of a novel class of less toxic AmB derivatives.     

Large molecular assembly of amphotericin B formed in ergosterol-containing membrane evidenced by solid-state NMR of intramolecular bridged derivative

Amphotericin B (AmB 1) is known to assemble and form an ion channel across biomembranes. We have recently reported that conformation-restricted derivatives of AmB 2-4 show different ergosterol preferences in ion-channel assays, which suggested that the orientation of the mycosamine strongly affects the sterol selectivity of AmB. The data allowed us to assume that compound 3 showing the highest selectivity would reflect the active conformation of AmB in the channel assembly. In this study, to gain further insight into the active conformation of AmB, we prepared a new intramolecular-bridged derivative 5, where the linker encompassed a hydrophilic glycine moiety. The derivative has almost equivalent ion-channel activity to those of AmB and 3. The antifungal activity of 5 compared with 3 improves significantly, possibly because the increasing hydrophilicity in the linker enhances the penetrability through the fungal cell wall. Conformation of 5 was well converged and very similar to that of 3, thus further supporting the notion that the conformations of these derivatives reproduce the active structure of AmB in the channel complex. Then we used the derivative to probe the mobility of AmB in the membrane by solid-state NMR. To measure dipolar couplings and chemical shift anisotropies, we incorporated [1-(13)C,(15)N]glycine into the linker. The results indicate that 5 is mostly immobilized in ergosterol-containing DMPC bilayers, implying formation of large aggregates of 5. Meanwhile some fraction of 5 remains mobile in sterol-free DMPC bilayers, suggesting promotion of AmB aggregation by ergosterol.     

Mycosamine orientation of amphotericin B controlling interaction with ergosterol: sterol-dependent activity of conformation-restricted derivatives with an amino-carbonyl bridge

Amphotericin B (AmB 1) is known to assemble together and form an ion channel across biomembranes. The antibiotic consists of mycosamine and macrolactone moieties, whose relative geometry is speculated to be determinant for the drug's channel activity and sterol selectivity. To better understand the relationship between the amino-sugar orientation and drug's activity, we prepared conformation-restricted derivatives 2-4, in which the amino and carboxyl groups were bridged together with various lengths of alkyl chains. K+ influx assays across the lipid-bilayer membrane revealed that ergosterol selectivity was markedly different among derivatives; short-bridged derivative 2 almost lost the selectivity, while 3 showed higher ergosterol preference than AmB itself. Monte Carlo conformational analysis of 2-4 based on NOE-derived distances indicated that the amino-sugar moiety of 2 comes close to C41 because of the short bridge, whereas those of 3 and 4 are pointing outward. The mutual orientation of the amino-sugar moiety and macrolide ring is so rigid in derivatives 2 and 3 that these conformations should be unchanged upon complex formation in lipid membranes. These results strongly suggest that the large difference in sterol preference between derivatives 2 and 3 is ascribed to the different orientation of amino-sugar moieties. These findings allowed us to propose a simple model accounting for AmB-sterol interactions, in which hydrogen bonding between 2'-OH of AmB and 3beta-OH of ergosterol plays an important role.     

Amphotericin B covalent dimers forming sterol-dependent ion-permeable membrane channels

Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for a long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K+ influx actions monitored by measuring the pH of the liposome lumen by 31P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in 31P NMR spectra, the dimers appeared to induce "all-or-none"-type ion flux across the liposome membrane in the presence of ergosterol, which suggested that the ion channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements.     

Modulation of amphotericin B membrane interaction by cholesterol and ergosterol--a molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. According to a well-documented hypothesis, molecules of AmB form ionic membrane channels that are responsible for chemotherapeutic action. These channels disturb the barrier function of the cell membrane which, in consequence, leads to cell death. The presence of sterols in the cell membrane is necessary for full manifestation of the antibiotic's ionophoric activity, at least in vivo. Ergosterol-containing fungal membranes are targeted more efficiently by AmB than mammalian membranes containing cholesterol. However, a similar level of disturbance of fungal and mammalian membranes is responsible for serious toxicity of the antibiotic. Due to the importance of AmB and lack of better antifungal alternatives, the search for new less toxic derivatives of this antibiotic still continues. Therefore, studies of the AmB-membrane interaction are very important. The present work constitutes a continuation of a broad program of study on AmB mode of action in our group. In particular, molecular dynamics simulations of AmB monomers inside the bilayers of three different compositions (pure dimiristoylphosphatidylcholine (DMPC) and DMPC bilayer containing approximately 25 mol % of cholesterol or ergosterol) were carried out. In general, analysis of generated trajectories resulted in identifying many significant differences in the behavior of AmB monomers depending on the membrane environment. In particular, it was established that the antibiotic increases the internal order of DMPC bilayer containing 25 mol % of cholesterol, while it has no effect on the order of the bilayer with the same amount of ergosterol. Performed calculations also revealed that relatively rigid and elongated AmB molecules exhibit higher affinity toward the sterol-containing lo phases and, therefore, may be cumulated in ordered membrane domains (e.g., lipid rafts). Since the partition coefficient between the ld and lo phase appears to be greater in the case of the ergosterol- compared to cholesterol-containing membrane, this effect can be also discussed as the possible origin of AmB-selective toxicity and indirect sterol involvement in expression of AmB activity.     

Synthesis of 6-F-ergosterol and its influence on membrane-permeabilization of amphotericin B and amphidinol 3

Two well-known antifungals, amphotericin B (AmB) and amphodinol 3 (AM3), are thought to exert antifungal activity by forming ion-permeable channels or pores together with sterol molecules. However, detailed molecular recognitions for AmB-sterol and AM3-sterol in lipid bilayers have yet to be determined. Toward (19)F NMR-based investigation of the molecular recognition underlying their potent antifungal activity, we synthesized 6-fluoro-ergosterol in five steps via ring opening of (5α,6α)-epoxide of ergosterol acetate with using novel combination of TiF(4) and n-Bu(4)N(+)Ph(3)SiF(2)(-). Then we evaluated its activity of promoting pore formation of AmB and AM3, and found that pore formation of AmB was barely promoted by 6-F-ergosterol in clear contrast to the dramatic promotion effect of unmodified ergosterol, whereas AM3 activity was markedly enhanced in the presence of 6-F-ergosterol, which was comparable to that of unmodified ergosterol. These results indicate that the introduction of an F atom at C6 position of ergosterol plays an inhibitory role in interacting with AmB, but it is not the case with AM3.     

Ion selectivity, transport properties and dynamics of amphotericin B channels studied over a wide range of acidity changes

Amphotericin B (AmB) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. Herein we studied the influence of pH upon the selectivity and the transport properties of AmB channels inserted in reconstituted, ergosterol-containing zwitterionic lipid membranes. Our electrophysiology experiments carried out on single and multiple AmB channels prove that at pH 2.8 these channels are anion selective, whereas at neutral and alkaline pH's (pH 7 and pH 11) they become cation selective. We attribute this to the pH-dependent ionization state of the carboxyl and amino groups present at the mouth of AmB molecules. Surprisingly, our data reveal that the single-molecule ionic conductance of AmB channels varies in a non-monotonic fashion with pH changes, which we attribute to the pH-dependent variation of the surface and dipole membrane potential. We demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low pH values, AmB channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition.     

Study of penetration of amphotericin B into cholesterol or ergosterol containing dipalmitoyl phosphatidylcholine Langmuir monolayers

The interactions of amphotericin B (AmB) with sterols and phospholipids have been studied by adsorption of AmB from aqueous solutions into Langmuir monolayers from dipalmitoyl phosphatidylcholine (DPPC), ergosterol, cholesterol and their mixtures. The results show that AmB exhibits stronger interaction with cholesterol than ergosterol in one-component monolayers. However, for DPPC–sterol monolayers, the effectiveness of AmB penetration depends on the proportion of both film components in the mixed film as well as on the strength of interaction between DPPC and particular sterol.     

An amphotericin B-ergosterol covalent conjugate with powerful membrane permeabilizing activity

Amphotericin B-sterol conjugates were synthesized and examined for their membrane permeabilizing activity. Ergosterol and cholesterol, each connected with amphotericin B via an ethylenecarbamate or hexamethylenecarbamate linker, were examined by K(+) flux assays using liposomes and by single-channel recording across phospholipid membrane. Among four conjugates tested, AmB-ergosterol bearing an ethylenecarbamate linker exhibited the most powerful activity, which substantially exceeded that of the cholesterol homolog. Single-channel recording clearly exhibited that the ergosterol conjugate elicited channel current with the conductance of 28 pS, which was comparable with those by AmB, and revealed a higher channel open probability than the cholesterol conjugate. These results imply that direct interaction between amphotericin B and ergosterol is reproduced by their conjugate, which may serve as a model compound for understanding the drug's selective toxicity.     

Tetraazachlorin-fullerene conjugates: on-off control of electronic communication enabled by push-pull substituents

Novel tetraazachlorin (TAC)-fullerene (C60) conjugates (TAC-C60) and their analogues (TAiBC-C60 and TABC-C60 where TAiBC = tetraazaisobacteriochlorin and TABC = tetraazabacteriochlorin) have been synthesized by condensing 1,2-dicyanofullerene (1) and phthalonitrile derivatives (2) in the presence of nickel chloride in quinoline, and fully characterized using mass spectrometry and 1H and 13C NMR. By arranging the TAC and C60 units at the minimum distance, and taking also into account the molecular symmetry, the resultant conjugates show on-off electronic communication behavior, depending on the push-pull properties of the peripheral substituents in the TAC moiety. Consequently, the UV-vis absorption spectrum of the electron-releasing butyloxy-substituted TAC-C60 (3a) contains an unusual group of three absorption bands in the Q-band region (500-900 nm) as a result of a strong electronic communication between the two moieties. On the other hand, the absorption spectrum of the electron-withdrawing butylsulfonyl-substituted TAC-C60 (3b) comprises a typically normal TAC spectrum with markedly split two-peak Q-bands. A similar phenomenon is observed between alkoxy-substituted TAiBC-C60 (4a) and butylsulfonyl-substituted TAiBC-C60 (4b). This study reveals that the electron-donating or -withdrawing nature of the peripheral substituents on an azachlorin moiety has an important effect on the electronic structures of our novel azachlorin-C60 conjugates, although the linking carbon atoms are aliphatic sp3 carbon atoms which generally do not contribute to aromaticity. The electronic structures of the conjugates have been investigated in detail using spectroscopic and electrochemical techniques with the aid of DFT calculations.     

Conformation and position of membrane-bound amphotericin B deduced from NMR in SDS micelles

Amphotericin B (AmB) is known to self-assemble to form an ion channel across lipid bilayer membranes. To gain insight into the conformation of AmB in lipidic environments, AmB in SDS micelles was subjected to high-resolution NMR and CD measurements, and the NMR-derived conformation thus obtained was refined by molecular mechanics calculations. These results indicate that AmB in SDS micelles is conformationally fixed particularly for the macrolide moiety. Paramagnetic relaxation experiments with the use of Mn2+ reveal that AmB is shallowly embedded in the micelle with the polyhydroxyl chain being close to the water interface and the side of polyene portion facing to the micelle interior. CD measurements demonstrate that AmB is in a monomeric form in SDS micelles. The structure of AmB in the micelles obtained in the present study may reproduce the initial stage of membrane interaction of AmB prior to the assembly formation in biomembranes.     

Specific adenine alkylation by pyrrole-imidazole CBI conjugates

We examined DNA alkylation by pyrrole (Py)-imidazole (Im) hairpin polyamides, which possess 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) or cyclopropapyrroloindole (CPI) as DNA alkylating moieties. High-resolution denaturing gel electrophoresis revealed that alkylation by CBI conjugates 2 and 4 occurred specifically at adenines (A) in matched sequences, whereas CPI conjugates 1 and 3 alkylated both A and guanines (G) in matched sequences. The origin of the different reactivity of CBI and CPI conjugates is discussed in relation to the electrophilicity of the cyclopropane moiety. The high selectivity of the CBI conjugate gives additional sequence specificity relative to CPI conjugates that would be useful for the biological applications.     

Synthesis and properties of fluorene‐based fluorinated polymers

Fluorene‐based polymers containing various fluorinated benzene (fluorobenzene, p‐difluorobenzene, and tetrafluorobenzene) moieties were synthesized. In addition, perfluorooctylation of poly‐[(9,9‐dioctylfluorene‐2,7‐diyl)‐co‐(fluorene‐2,7‐diyl)] was carried out to afford fluorene‐based polymers with perfluorooctyl moiety at the 9‐position on the fluorene ring. To evaluate the effect of fluorine moiety, polymers containing nonfluorinated benzene moieties and nonfluorinated octyl groups were synthesized. The photoluminescence measurements indicated that all these polymers exhibited blue emission in solution, but a polymer containing a perfluorooctyl group did not emit in the film state. Polymers containing various fluorinated benzene moieties showed higher fluorescence quantum yields and thermal stability than those containing nonfluorinated benzene. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 3143–3150, 2001     

Reaction of the 1,8-bis(diphenylmethylium)naphthalenediyl dication with fluoride: formation of a cation containing a C-F-->C bridge

Treatment of 1,8-bis(diphenylhydroxymethyl)naphthalene with a mixture of [HBF(4)](aq) and (CF(3)CO)(2)O affords the corresponding dication, 1,8-bis(diphenylmethylium)naphthalenediyl (1(2+)), which was isolated as the [BF4]- salt. This dication has been fully characterized, and its structure has been studied computationally. The (13)C NMR resonance of the methylium centers appears at 207.7 ppm. As indicated by an X-ray single-crystal analysis, the vicinal methylium centers are separated by 3.112(4) A. Dication (1(2+)) reacts with fluoride to afford [1-F]+ which has been isolated as the [BF4]- salt. The fluorine atom of [1-F](+) is connected to one of the former methylium centers through a typical C-F bond of 1.424(2) A and forms a long interaction of 2.444(2) A with the other methylium center. While the structure of [1-F]+ can be largely accounted for by considering a simple methylium formulation, density functional calculations followed by an Atom In Molecules analysis as well as a calculation of the Boys localized orbitals indicate that the long C-F interaction of 2.444(2) A corresponds to a dative bond. Hence, formulation of [1-F]+ as an unsymmetrical fluoronium must also be considered. As indicated by 1H NMR spectroscopy, the structure of this ion is fluxional; the fluorine atom oscillates between the former methylium centers with apparent activation parameters of DeltaH++ = 52(+/-3) kJ mol(-1) and DeltaS++ = -18(+/-9) J K(-1) mol(-1) as derived from line shape analysis. This dynamic process, which has also been studied theoretically by B3LYP density functional theory and M?ller-Plesset second-order perturbation theory methods, involves symmetrical fluoronium ions as low-energy transition states.     

Dinicotinamidium squarate

The crystal structure determination of the dinicotinamidium squarate salt, 2C₆H₇N₂O⁺·C₄O₄²⁻, is reported, with the squarate dianion residing on an inversion centre and the unique cation in a general position. Salt formation occurs by donation of two H atoms from squaric acid to the nicotin­amide base. The crystal packing is derived from three types of hydrogen bonding. The primary hydrogen bond involves a squarate anion O atom and an H atom of the protonated pyridine group of the nicotin­amide, with an N⋯O distance of 2.5760 (13) Å. The second hydrogen bond involves a second anion O atom and an amide H atom, with an N⋯O distance of 2.8374 (14) Å. Thirdly, an intermolecular interaction between two coplanar nicotin­amide moieties occurs between an amide O atom and a symmetry‐related amide H atom, with an N1⋯O3 distance of 2.8911 (15) Å. These hydrogen bonds are also responsible for the planarity of the nicotin­amide moiety in the salt.     

Electron-rich tetrathiafulvalene-triarylamine conjugates: synthesis and redox properties

By combining tetrathiafulvalenes (TTFs) and triarylamines, four TTF-triarylamine conjugates bridged by an annulated pyrrole ring were designed and synthesized by an N-arylation reaction. Electrochemical and photophysical investigations suggest that these novel conjugates possess very strong electron-donating ability with very high HOMO energy levels of around -4.70 eV; the HOMOs are mainly located on the TTF moiety. We observed significant electronic coupling between the TTF moieties and the triarylamine groups. However, no evidence for such electronic communication between end-capping TTF units (conjugates 5 and 7) or between two terminal triarylamine groups (conjugate 9) could be found. Differential scanning calorimetry (DSC) measurements together with PM3-optimized geometries suggest that conjugates 5 and 7, which adopt three-dimensional propeller-shaped structures, may easily pack and crystallize in the solid state because of the large rigid planar blades consisting of TTF and one of the phenyl rings of the triarylamine moiety. However, conjugate 9, with two bulky end-capping triarylamine groups, forms an amorphous material with a glass transition at 74.5 degrees C.     

Identification of aromatic moieties and mycosamine in antifungal heptaenes with high-performance liquid chromatography, high-performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.

A high-performance liquid chromatographic (HPLC) method for the determination of the aromaticity of heptaene polyene antibiotics has been developed. The released aromatic moiety of the heptaene polyenes aureofungin, candicidin, candimycin, hamycin and trichomycin was assayed after alkaline hydrolysis. The presence of p-aminoacetophenone (PAAP) and N-methyl-p-aminoacetophenone (N-methyl-PAAP) in the hydrolysates was determined by HPLC, HPLC-mass spectrometry (HPLC-MS) and gas chromatography-MS (GC-MS). Candicidin and hamycin contained only the PAAP residue; aureofungin contained both PAAP and N-methyl-PAAP. Trichomycin contained PAAP and also some unknown component of molecular weight 179. The aromatic nature of the individual components of the heptaene complex was demonstrated using radioactivity flow detection for the determination of the incorporation of [14C]-p-aminobenzoic acid to individual candicidin components. Ammonia chemical ionization MS was successfully used for the GC-MS identification of the acetylated mycosamine moiety of heptaenes.     

1hJFH coupling in 2‐fluorophenol revisited: Is intramolecular hydrogen bond responsible for this long‐range coupling?

The present study shows that a hydrogen bond between the OH group and the fluorine atom is not involved in the (1h)J(FH) spin-spin coupling transmission either for 4-bromo-2-fluorophenol or 2-fluorophenol. In fact, according to a quantum theory of atoms in molecules analysis, no bond critical point is found between O-H and F moieties. The nature of the transmission mechanism of the Fermi contact term of the (1h)J(FH) spin-spin coupling is studied by analyzing canonical molecular orbitals (see J. Phys. Chem. A 2010, 114, 1044), and it is observed that virtual orbitals play only a quite minor role in its transmission. This is typical of a Fermi contact term transmitted mainly through exchange interactions owing to the overlap of proximate electronic clouds; therefore, it is suggested to identify them as (nTS)J(FH) coupling where n stands for the number of formal bonds separating the coupling nuclei. In the cases studied in this work is n = 4. Results presented in this work could provide an interesting rationalization for different experimental signs known in the current literature for proximate J(FH) couplings.     

Ligandenaustauschreaktionen von Fluoratomen mit Chloratomen bei Phosphorverbindungen der Koordinationszahl 3 und 4

Ligand-exchange reactions of fluorine versus chlorine atoms in phosphorus compounds with coordination number 3 or 4 Fluorine versus chlorine ligand-exchange reactions were carried out on the following phosphorus-based central moieties: CH₃P <, CH₃P(O)<, CH₃P(S)< and CH₃P(Se)<. The measured equilibrium distributions were shown to favour the species which result from an accumulation of like substituents on the central moieties, whereas the equilibrium distribution of chlorine vs bromine on CH₃P(O) < was found to be almost random. These fluorine exchanging systems thus represent the unusual case of large positive deviation from random distribution. It is also noteworthy that the equilibrium distributions depend only very little on the nature of the atom which is bonded to phosphorus in the “isolated” position. System CH₃PF₂/CH₃PCl₂ could not be studied quantitatively owing to the competitive dismutation of CH₃PF₂; however, evidence is given for fast initial redistribution of the halogens on the methylphosphorus moiety and for the transient formation of CH₃PFCl.     

Multivalent Siderophore–DOTAM Conjugates as Theranostics for Imaging and Treatment of Bacterial Infections

There is a strong need to better diagnose infections at deep body sites through noninvasive molecular imaging methods. Herein, we describe the synthesis and characterization of probes based on siderophore conjugates with catechol moieties and a central DOTAM scaffold. The probes can accommodate a metal ion as well as an antibiotic moiety and are therefore suited for theranostic purposes. The translocation of the conjugates across the outer and inner cell membranes of E. coli was confirmed by growth recovery experiments with enterobactin-deficient strains, by the antibacterial activity of ampicillin conjugates, and by confocal imaging using a fluorogen-activating protein–malachite green system adapted to E. coli. The suitability of the probes for in vivo imaging was demonstrated with a Cy5.5 conjugate in mice infected with P. aeruginosa.