Light-Induced Transformation of the Aromatic Heptaene Antifungal Antibiotic Candicidin D into Its All-Trans Isomer.

Illumination of the aromatic heptaene macrolide antifungal antibiotic candicicin D with UV light results in an isomerization of the molecule. The product formed after irradiation of the candicidin complex with UV light (λ = 365 nm), namely, iso-candicidin D, was isolated and subjected to 2D NMR studies, consisting of DQF-COSY, ROESY, TOCSY, HSQC, and HMBC experiments. The obtained spectral data unambiguously evidenced that iso-candicidin D was the all-trans isomer of the native antibiotic, and straightening of the heptaenic chromophore was the only light-induced structural change that occurred. Hence, iso-candicidin D was proclaimed to be a prototype of a novel class of polyene macrolide antifungal antibiotics: the all-trans aromatic heptaenes, containing a macrolide ring similar to that of amphotericin B.

Monosaccharides as Potential Chiral Probes for the Determination of the Absolute Configuration of Secondary Alcohols.

Herein, a new method for the elucidation of the absolute configuration of chiral secondary alcohols is proposed. This method is an alternative for a widely used approach reported by Mosher and Dale and similar methods that are based on the 1H NMR shift (δ) changes of protons that are attached to the substituents of the oxymethine carbon atom. The presented method is not based on tracking the chemical shift changes and utilizes stereochemically defined monosaccharides as chiral probes. A secondary alcohol is glycosylated, and the resulting glycoside is subjected to NMR studies. The observation of dipolar couplings between the protons of the monosaccharide moiety and the protons of the secondary alcohol moiety via the NOESY/ROESY spectra enables the determination of the absolute configuration of the oxymethine carbon atom.

Intramolecular transformation of an antifungal antibiotic nystatin A1 into its isomer, iso-nystatin A1 - structural and molecular modeling studies.

Nystatin A1 , a polyene macrolide antifungal antibiotic, in a slightly basic or acidic solution undergoes an intramolecular transformation, yielding a structural isomer, the translactonization product, iso-nystatin A1 with lactone ring diminished by two carbon atoms. Structural evidence is provided by advanced NMR and Mass Spectrometry (MS) studies. Molecular dynamics simulations and quantum mechanics calculations gave the insight into the course and mechanism of the transformation and its effect on the conformation of the subject molecule. Copyright © 2016 John Wiley & Sons, Ltd.

Novel Nystatin A1 derivatives exhibiting low host cell toxicity and antifungal activity in an in vitro model of oral candidosis.

Opportunistic oral infections caused by Candida albicans are frequent problems in immunocompromised patients. Management of such infections is limited due to the low number of antifungal drugs available, their relatively high toxicity and the emergence of antifungal resistance. Given these issues, our investigations have focused on novel derivatives of the antifungal antibiotic Nystatin A1, generated by modifications at the amino group of this molecule. The aims of this study were to evaluate the antifungal effectiveness and host cell toxicity of these new compounds using an in vitro model of oral candidosis based on a reconstituted human oral epithelium (RHOE). Initial studies employing broth microdilution, revealed that against planktonic C. albicans, Nystatin A1 had lower minimal inhibitory concentration than novel derivatives. However, Nystatin A1 was also markedly more toxic against human keratinocyte cells. Interestingly, using live/dead staining to assess C. albicans and tissue cell viability after RHOE infection, Nystatin A1 derivatives were more active against Candida with lower toxicity to epithelial cells than the parent drug. Lactate dehydrogenase activity released by the RHOE indicated a fourfold reduction in tissue damage when certain Nystatin derivatives were used compared with Nystatin A1. Furthermore, compared with Nystatin A1, colonisation of the oral epithelium by C. albicans was notably reduced by the new polyenes. In the absence of antifungal agents, confocal laser scanning microscopy showed that C. albicans extensively invaded the RHOE. However, the presence of the novel derivatives greatly reduced or totally prevented this fungal invasion.

The Substantial Improvement of Amphotericin B Selective Toxicity Upon Modification of Mycosamine with Bulky Substituents.

BACKGROUND: It is assumed that the unfavorable selective toxicity of an antifungal drug Amphotericin B (AmB) can be improved upon chemical modification of the antibiotic molecule. OBJECTIVE: The aim of this study was verification of the hypothesis that introduction of bulky substituents at the amino sugar moiety of the antibiotic may result in diminishment of mammalian in vitro toxicity of thus prepared AmB derivatives. METHODS: Twenty-eight derivatives of AmB were obtained upon chemical modification of the amino group of mycosamine residue. This set comprised 10 N-succinimidyl-, 4 N-benzyl-, 5 Nthioureidyl- and 9 N-aminoacyl derivatives. Parameters characterizing biological in vitro activity of novel compounds were determined. RESULTS: All the novel compounds demonstrated lower than AmB antifungal in vitro activity but most of them exhibited negligible cytotoxicity against human erythrocytes and three mammalian cell lines. In consequence, the selective toxicity of majority of novel antifungals, reflected by the selective toxicity index (STI = EH50/IC50) was improved in comparison with that of AmB, especially in the case of 5 compounds. The novel AmB derivatives with the highest STI, induced substantial potassium efflux from Candida albicans cells at concentrations slightly lower than IC50s but did not trigger potassium release from human erythrocytes at concentrations lower than 100 µg/mL. CONCLUSION: Some of the novel AmB derivatives can be considered promising antifungal drug candidates.

Interactions of amphotericin B derivatives with lipid membranes--a molecular dynamics study.

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.

Cell Density-Dependent Cytological Stage Profile and Its Application for a Screen of Cytostatic Agents Active Toward Leukemic Stem Cells.

Proliferation and expansion of leukemia is driven by leukemic stem cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in acute myeloid leukemia (AML) treatment. In this study, we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells, which include LSCs determining the overall stage profile. This change manifested in morphology, expression of both cell surface markers and redox-state proteins, as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Among the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of reactive oxygen species (ROS). Furthermore, it had low toxicity in vivo and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C-123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogeneic transplantation and/or ex vivo treatment preceding autologous transplantation.

Modulation of polyene antibiotics self-association by ions from the Hofmeister series.

The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers-sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers-perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.

The mechanism of overcoming multidrug resistance (MDR) of fungi by amphotericin B and its derivatives.

Comparative studies were performed to determine the activity and cytotoxicity of amphotericin B (AmB) and its derivatives on standard strain of Saccharomyces cerevisiae and its transformants with cloned genes from Candida albicans encoding multidrug resistance (MDR) pumps of ATP-binding cassette and major facilitator superfamilies. The AmB derivatives: amphotericin B 3-dimethylaminopropyl amide and N-methyl-N-D-fructopyranosylamphotericin B methyl ester were shown to be fungistatic and fungicidal towards MDR strains, by membrane permeabilization mechanism. Antibiotic-cell interaction monitored by energy transfer method indicates similar membrane affinity in parent strain and its MDR transformants. Experiments with fungal cells loaded with rhodamine 6G point to lack of competition between this dye and AmB and its derivatives for efflux driven by CDR2p. It can be thus assumed that AmB and its derivatives overcome fungal MDR by not being substrates of the multidrug exporting pumps, presumably due to their large molecular volumes.

Analytical studies on ascosin, candicidin and levorin multicomponent antifungal antibiotic complexes. The stereostructure of ascosin A2.

In the class of polyene macrolides, there is a subgroup of aromatic heptaenes, which exhibit the highest antifungal activity within this type of antibiotics. Yet, due to their complex nature, aromatic heptaenes were not extensively studied and their potential as drugs is currently underexploited. Moreover, there are many inconsistencies in the literature regarding the composition and the structures of the individual components of the aromatic heptaene complexes. Inspired by one of such cases, herein we conducted the analytical studies on ascosin, candicidin and levorin using HPLC-DAD-(ESI)Q-TOF techniques. The resulting chromatograms and the molecular masses of the individual components of these three complexes strongly indicated that the major components of ascosin, candicidin and levorin are structurally identical. In order to validate these results, the main component of previously structurally uncharacterized ascosin was derivatized, isolated and subjected to 2D NMR studies. The resulting structure of the ascosin's main component, herein named ascosin A2, was shown to be identical with the earlier reported structures of the main components of candicidin and levorin complexes: candicidin D and levorin A2. In the end, all the structural knowledge regarding these three antibiotic complexes was gathered, systematized and completed, and the new nomenclature was proposed.

Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7.

Anthrapyridazones, imino analogues of anthraquinone, constitute a family of compounds with remarkable anti-cancer activity. To date, over 20 derivatives were studied, of which most displayed nanomolar cytotoxicity towards broad spectrum of cancer cells, including breast, prostate and leukemic ones. BS-154, the most potent derivative, had IC50 values close to 1 nM, however, it was toxic in animal studies. Here, we characterize another anthrapyridazone, PDZ-7, which retains high cytotoxicity while being well tolerated in mice. PDZ-7 is also active in vivo against anthracycline-resistant tumor in a mouse xenograft model and induces DNA damage in proliferating cells, preferentially targeting cells in S and G2 phases of the cell cycle. Activation of Mre11-Rad50-Nbs1 (MRN) complex and phosphorylation of H2AX suggest double-stranded DNA breaks as a major consequence of PDZ-7 treatment. Consistent with this, PDZ-7 treatment blocked DNA synthesis and resulted in cell cycle arrest in late S and G2 phases. Analysis of topoisomerase IIα activity and isolation of the stabilized covalent topoisomerase IIα - DNA complex in the presence of PDZ-7 suggests that this compound is a topoisomerase IIα poison. Moreover, PDZ-7 interfered with actin polymerization, thereby implying its action as a dual inhibitor of processes critical for dividing cells. Using nuclear magnetic resonance (NMR) spectroscopy we show that PDZ-7 interacts with DNA double helix and quadruplex DNA structure. Taken together, our results suggest that PDZ-7 is a unique compound targeting actin cytoskeleton and DNA.

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels.

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.

Strategies for overcoming ABC-transporters-mediated multidrug resistance (MDR) of tumor cells.

The development of multidrug resistance (MDR) of tumors is a major cause of failure in antitumor chemotherapy. This type of cross-resistance is due to the expression of ABC transporter glycoproteins actively effluxing the drug from the cells against the concentration gradient at the expense of metabolic energy, thus preventing the accumulation in cells of therapeutic concentration of active agents. In this review strategies for overcoming this adverse phenomenon are discussed. They comprise the control of expression of MDR glycoprotein transporters and control of the functioning of the expressed transporter proteins. The latter approach is discussed in more detail, comprising the following general strategies: (i) development of compounds that are not substrates of efflux pump(s), (ii) use of agents that inactivate (inhibit) MDR proteins, (iii) design of cytostatics characterized by fast cellular uptake, surpassing their mediated efflux, (iv) use of compounds competing with the drug for the MDR protein-mediated efflux. Positive and negative aspects of these strategies are analysed, with special attention put on strategy based on the use of MDR modulators in combination therapy, allowing the restoration of cytotoxic activity of clinical cytostatics towards resistant tumor cells.

Molecular modelling of membrane activity of amphotericin B, a polyene macrolide antifungal antibiotic.

Amphotericin B (AmB) is a well known polyene macrolide antibiotic used to treat systemic fungal infections. Despite its toxicity AmB is still regarded as a life-saving drug. The lack of adequate knowledge of the AmB mechanism of action is a serious obstacle to efficient development of new less toxic derivatives. Complementary to various experimental approaches, computational chemistry methods were used to study AmB mechanism of action. A programme lasting for a decade, that was run by our group covered studies of: i) molecular properties of AmB and its membrane targets, ii) structure and properties of AmB membrane channels, and iii) interaction of AmB with the membrane.

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design.

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.

Molecular basis of the low activity of antitumor anthracenediones, mitoxantrone and ametantrone, in oxygen radical generation catalyzed by NADH dehydrogenase. Enzymatic and molecular modelling studies.

Synthetic antitumor anthracenedione drugs, in contrast to anthracycline antibiotics, are ineffective in free radical formation in NADH dehydrogenase system. Our results have indicated that neither the reduction potential nor the side chain conformation and the energies of border orbitals (HOMO and LUMO) determine the ability of anthracenediones to stimulate reactive oxygen species formation in NADH dehydrogenase system. It was shown that the distribution of the molecular electrostatic potential (MEP), around the quinone system was crucial for this ability. We have found for non-stimulating anthracenediones that the clouds of positive MEP cover the quinone carbon atoms while for agents effective in stimulating reactive oxygen species formation the clouds of negative MEP cover continuously the aromatic core together with the quinone system.

The stereostructure of candicidin D.

The candicidin D stereostructure was established based on NMR studies including DQF-COSY, ROESY, HSQC and HMBC experiments. The relative configurations of the candicidin D stereogenic centers were assigned as the following: 9R*, 11S*, 13S*, 15R*, 17S*, 18R*, 19S*, 21R*, 36S*, 37R*, 38S*, 40S* and 41S*. The geometry of the heptaene chromophore was defined as 22E, 24E, 26Z, 28Z, 30E, 32E and 34E.

The role of structural factors in the kinetics of cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines: implications for overcoming multidrug resistance towards leukaemia K562/DOX cells.

The appearance of multidrug resistance (MDR) of tumour cells to a wide array of antitumour drugs, structurally diverse and having different mechanisms of action, constitutes the major obstacle to the successful treatment of cancer. Our approach to search for non-cross resistant antitumour agents is based on the rational design of derivatives, which have a high kinetics of passive cellular uptake rendering their active efflux by MDR exporting pumps inefficient. Recently, two families of acridine cytotoxic agents were obtained, pyrazoloacridines (PACs) and pyrazolopyrimidoacridines (PPACs). The aim of this study was to examine molecular basis of the reported differences in retaining cytotoxic activity of these derivatives at cellular level against resistant erythroleukaemia K562/DOX (overexpressing P-glycoprotein) cell line. The study was performed using a spectrofluorometric method, which allows continuous monitoring of the uptake and efflux of fluorescent molecules by living cells. It was demonstrated that the presence of two additional rings, pyrazole and pyrimidine, fused to the acridine chromophore structure (PPAC) favoured more rapid cellular diffusion than the presence of only one additional pyrazole ring (PAC). The presence of hydrophobic substituent OCH3 markedly favoured the cellular uptake of pyrazoloacridines and pyrazolopyrimidoacridines while compounds having hydrophilic substituent OH exhibited very low kinetics of cellular uptake. In contrast, it was found that neither structure of the ring system nor the hydrophobic/hydrophilic character of examined substituents determined the rate of active efflux of these compounds by P-glycoprotein. Our data showed that a nearly linear relation exists between the resistance factor (RF) and lnV+ reflecting the impact of the cellular uptake rate (V+) on the ability of these compounds to overcome MDR.

Comparative in vitro studies on liposomal formulations of amphotericin B and its derivative, N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME).

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MFAME) is a semisynthetic derivative of the antifungal antibiotic amphotericin B (AMB). In contrast to the parent antibiotic, the derivative is characterised by low toxicity to mammalian cells and good solubility in water of its salts. Comparative studies on biological properties of free MFAME, AMB and their liposomal formulations were performed. To obtain liposomal forms, the antibiotics were incorporated into small unilamellar vesicles composed of dimyristoyl phosphatidylcholine (DMPC) and DMPC:cholesterol or ergosterol, 8:2 molar ratio. The effectivity of the liposomal and free forms of AMB and MFAME were compared by determination of fungistatic and fungicidal activity against Candida albicans ATCC 10261, potassium release from erythrocytes, and haemolysis. The results obtained indicate that in contrast to AMB, incorporation of MFAME into liposomes did not further improve its selective toxicity. Studies on the antagonistic effect of ergosterol and cholesterol on the antifungal activity of the antibiotics indicated that sterol interference was definitely less pronounced in the case of MFAME than in the case of AMB.