Polyene Macrolide Research Articles

Studies on the Total Synthesis of Antibiotic Macrolactin S: A Conventional Approach for the Synthesis of the C1–C9 and C10–C24 Fragments

The C1–C9 and C10–C24 segments of the 24-membered polyene macrolide macrolactin S were synthesized by routes involving an epoxide-ring-opening reaction, an Ohira–Bestmann alkyne formation, a chelation-controlled nucleophilic addition reaction, and a Still–Gennari olefination as key steps. A chiron approach , starting from readily available glucose diacetonide, was used to synthesize a key intermediate, and a convergent approach was adopted for the synthesis of the key C10–C24 fragment.

Does the use of antifungal agents in agriculture and food foster polyene resistance development? A reason for concern.

Environmental fungicides are used in agriculture to reduce fungal spoilage of crops to a minimum, and the polyene macrolide natamycin is used as a food preservative. The use of natamycin in yoghurt has recently been authorised in the USA and some other countries. However, resistance development is a serious risk associated with the use of antimicrobials as food additives and environmental fungicides. Cross-resistance between agricultural and medical azoles and between azoles and amphotericin B (AMB) not being used in agriculture has been demonstrated in clinical and environmental isolates. Polyene resistance can be elicited in vitro by the use of subinhibitory polyene concentrations and a large number of transfers. This condition may mirror the exposure of faecal Candida spp. to natamycin following consumption of natamycin-containing food. A large number of environmental and clinical isolates are resistant to AMB, and strong evidence linking farm antibiotic use and multidrug resistance, including AMB resistance, in human infections has been provided. In contrast to the acquisition of resistant environmental strains, consumption of natamycin-containing food may expose the gastrointestinal fungal flora directly to resistance selective pressure. So far, whether natamycin itself may cause the emergence of polyene resistance in gastrointestinal fungal flora and/or may act as an AMB resistance selector is probable but speculative. Use of any anti-infective agent as a food preservative should be limited to an absolute minimum as the clinical efficacy of anti-infectives used to treat serious life-threatening infections has to be preserved.

Structural modifications and yield improvements of polyene macrolide antibiotics

Polyene macrolide antibiotics, which selectively bind ergosterols in fungal membranes, are important antifungal agents for the treatment of both superficial and invasive fungal infections. But severe side effects, such as nephrotoxicity, are caused by their binding to cholesterol in mammalian membranes and undermine their therapeutic values. Nevertheless, because of their excellent fungicidal efficacy, broad spectrum of activities and minimal resistance development, polyene macrolide antibiotics continue to command interest. Therefore, the pharmacological and yield improvement of polyene macrolide antibiotics becomes urgent. In past few decades, much attention was paid on the genetic and chemical modifications of polyene macrolides, and a variety of genetic engineering strategies were applied to improve the yield of polyene macrolides. Based on the literature, we summarized the structural modifications of polyene macrolides in polyene region, polyol region, exocyclic carboxyl group and sugar moiety. The heptaene nystatin analog S44HP shows considerably higher antifungal activity than that of nystatin and equal to that of amphotericin B. While the exocyclic carboxyl modifications of polyene macrolides usually led to comparable or slightly improved antifungal activity and significantly reduced hemolytic toxicity, most of alterations in polyol region resulted in decreased antifungal activity and hemolytic toxicity. Furthermore, the polyene macrolides harboring a di–sugar moiety exhibited reduced hemolytic toxicity and improved water solubility, and the modifications in C3′ amino group or C2′ hydroxyl group considerably improved the pharmacological properties. Moreover, the action mechanisms and structure–activity relationship of polyene macrolide antibiotics were elucidated, according to the data of structural modifications. It indicated that the modifications in exocyclic carboxyl group, C3′ amino group or C2′ hydroxyl group disrupt the intramolecular polar interactions and thus favor a shift to an alternate conformer that selectively binds ergosterol rather than cholesterol. In addition, the strategies for yield improvement of polyene macrolide antibiotics were summarized, including the engineering of pathway-specific and pleiotropic regulatory genes, engineering of quorum–sensing communication, and optimization of precursor supply. Although transcriptional regulation has been investigated for amphotericin, nystatin and candicidin/FR–008 biosynthesis, most detailed genetic engineering strategies for yield improvement were only carried out on pimaricin. The yield of pimaricin was improved via the overexpression of pathway- specific or pleiotropic positive regulatory genes and GBL biosynthetic genes, the disruption of pathway-specific or pleiotropic negative regulatory genes and GBL receptor encoding genes, and the addition of inducing compounds or small molecular precursors. Finally, we pointed out the challenges and problems in the structural modifications and yield improvement of polyene macrolide antibiotics, such as the possibility to obtain more modified polene analogs with exocyclic carboxyl or C3′ amino group via genetic engineering, combinational strategies for further yield improvement of polyene macrolides, and so on.

Biosynthesis regulation of natamycin production from Streptomyces natalensis HDMNTE-01 enhanced by response surface methodology

ABSTRACTNatamycin has been widely applied in medical treatments and food protection widely due to its effective inhibition to the growth of yeast and mold. As polyene macrolide antibiotic, the biosynthesis pathway of natamycin is relatively clear. To regulate the biosynthesis of natamycin, additions of precursors affecting cell growth and natamycin production were investigated. The results showed that 0.003% (w/v) potassium ferrocyanide and sodium propionate: n-butanol at a ratio of 4:1 was added into the broth at 0 and 24 hr, respectively, and they contributed to yield natamycin, reaching 1.62 g L−1 (174.6% higher than control). Furthermore, response surface methodology was undertaken to enhance natamycin production by Streptomyces natalensis HDMNTE-01 (a wild strain). The optimum conditions determined were: glucose 3.97%; soya peptone 2%; yeast extract 0.5%; original pH 7.0; inoculum volume 6%; growth in a 250-mL flask containing 24.68 mL of medium; shaken (220 rpm) at 28°C for 4 days. Under the optimized conditions, the yield was 2.81 g L−1 natamycin in 5-L fermentor when the fermentation was processed.

Improved recovery and biological activities of an engineered polyene NPP analogue in Pseudonocardia autotrophica.

NPP A1 produced by Pseudonocardia autotrophica is a unique disaccharide-containing polyene macrolide. NPP A1 was reported to have higher water solubility and lower hemolytic toxicity than nystatin A1 while retaining its antifungal activity. An engineered NPP A1 analogue, NPP A2, was generated by inactivation of the nppL gene, encoding a P450 monooxygenase in P. autotrophica. The resulting compound exhibited the corresponding chemical structure of NPP A1 but lacked a C10 hydroxyl group. In this study, newly developed crystallization recovery methods for NPP A2 purification, followed by an evaluation of in vitro antifungal activity and hemolytic activity, were performed. The crystallization methods were designed to eliminate the undesired viscous impurities encountered during the NPP A2 purification process, resulting in improved purity from 5.3 to 83.5% w/w. NPP A2 isolated from the improved purification process also exhibited two times higher antifungal activity and 1.8 times higher hemolytic toxicity than those of NPP A1. These results suggest that the minor structural modification of disaccharide-containing polyene macrolides, such as removing a C10 hydroxyl group, might require an alternative recovery process, such as crystallization, to confirm its improved biological activity.

A multi-purpose tool for food inspection: Simultaneous determination of various classes of preservatives and biogenic amines in meat and fish products by LC-MS

This paper describes an innovative fast and multipurpose method for the chemical inspection of meat and fish products by liquid chromatography-tandem mass spectrometry. Solid-liquid extraction and low temperature partitioning were applied to 17 analytes, which included large bacteriocins (3.5kDa) and small molecules (organic acids, heterocyclic compounds, polyene macrolides, alkyl esters of the p-hydroxybenzoic acid, aromatic, and aliphatic biogenic amines and polyamines). Chromatographic separation was achieved in 10min, using stationary phase of di-isopropyl-3-aminopropyl silane bound to hydroxylated silica. Method validation was in accordance to Commission Decision 657/2002/CE. Linear ranges were among 1.25–10.0mgkg−1 (natamycin and parabens), 2.50–10.0mgkg−1 (sorbate and nisin), 25.0–200mgkg−1 (biogenic amines, hexamethylenetetramine, benzoic and lactic acids), and 50.0–400mgkg−1 (citric acid). Expanded measurement uncertainty (U) was estimated by single laboratory validation combined to modeling in two calculation approaches: internal (U = 5%) and external standardization (U = 24%). Method applicability was checked on 89 real samples among raw, cooked, dry fermented and cured products, yielding acceptable recoveries. Many regulatory issues were revealed, corroborating the need for enhancement of the current analytical methods. This simple execution method dispenses the use of additional procedures of extraction and, therefore, reduces costs over time. It is suitable for routine analysis as a screening or confirmatory tool for both qualitative and quantitative results, replacing many time consuming analytical procedures.

Redesign of antifungal polyene glycosylation: engineered biosynthesis of disaccharide-modified NPP.

Polyene macrolides such as nystatin A1 and amphotericin B have been known to be potent antifungal antibiotics for several decades. Because the therapeutic application of polyenes is restricted by severe side effects such as nephrotoxicity, various chemical and biological studies to modify the polyene structure have been conducted to develop less-toxic polyene antifungals. A newly discovered nystatin-like polyene compound NPP was shown to contain an aglycone that was identical to nystatin but harbored a unique di-sugar moiety, mycosaminyl-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic toxicity. Additionally, a NPP-specific second sugar extending gene, nppY, was recently identified to be responsible for the transfer of a second sugar, N-acetyl-glucosamine, in NPP biosynthesis. In this study, we investigated biosynthesis of the glycoengineered NPP analog through genetic manipulation of the NPP A1 producer, Pseudonocardia autotrophica KCTC9441. NypY is another second sugar glycosyltransferase produced by Pseudonocardia sp. P1 that is responsible for the transfer of a mannose to the mycosaminyl sugar residue of nystatin. We blocked the transfer of a second sugar through nppY disruption, then expressed nypY in P. autotrophica △nppY mutant strain. When compared with nystain A1 and NPP A1, the newly engineered mannosylated NPP analog showed reduced in vitro antifungal activity, while exhibiting higher nephrotoxical activities against human hepatocytes. These results suggest for the first time that not only the number of sugar residues but also the type of extended second sugar moiety could affect biological activities of polyene macrolides.

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.

For Debate: May the Use of the Polyene Macrolide Natamycin as a Food Additive Foster Emergence of Polyene-Resistance in Candida Species?

Natamycin is approved almost worldwide as a food additive for surface treatment of cheese and sausages. Its use is considered to be safe as Natamycin is extremely sensitive to ultraviolet light and an acidic pH, so that products exposed to light in the retail industry and food stores are likely free from Natamycin. However, the use of an acid-, heat- and light stable Natamycin formulation in yoghurt has recently been authorized in the USA as well as in Australia and New Zealand. Furthermore, yoghurt is stored in sealed cups in refrigerated shelves, so it will not be exposed to light and thus not be inactivated during storage. Consequently, the resident flora will be exposed to Natamycin and it may exert a resistance selective pressure on faecal Candida spp. hypothetically selecting strains being resistant to Amphothericin B. In this review literature has been evaluated addressing the questions if Natamycin may foster emergence of polyene-resistance. This concern is supported by the facts that first, polyene-resistance could be elicited in vitro and in vivo . Second, as Azoles being used in agriculture and hospitals as well as polyenes share some common resistance mechanisms a polyene-resistance reservoir does exist in environmental and clinical fungal isolates. Third, Natamycin may amplify Amphothericin B resistance as fourth, resistance can in principal be spread amongst fungi by horizontal gene transfer. To preserve clinical efficacy of Amphothericin B for treatment of serious, life threatening infections, the use of Natamycin as a food-preservative should be limited to an absolute minimum.

Engineered biosynthesis and characterisation of disaccharide-modified 8-deoxyamphoteronolides.

Several polyene macrolides are potent antifungal agents that have severe side effects. Increased glycosylation of these compounds can improve water solubility and reduce toxicity. Three extending glycosyltransferases are known to add hexoses to the mycosaminyl sugar residues of polyenes. The Actinoplanes caeruleus PegA enzyme catalyses attachment of a D-mannosyl residue in a β-1,4 linkage to the mycosamine of the aromatic heptaene 67-121A to form 67-121C. NppY from Pseudonocardia autotrophica adds an N-acetyl-D-glucosamine to the mycosamine of 10-deoxynystatin. NypY from Pseudonocardia sp. P1 adds an extra hexose to a nystatin, but the identity of the sugar is unknown. Here, we express the nypY gene in Streptomyces nodosus amphL and show that NypY modifies 8-deoxyamphotericins more efficiently than C-8 hydroxylated forms. The modified heptaene was purified and shown to be mannosyl-8-deoxyamphotericin B. This had the same antifungal activity as amphotericin B but was slightly less haemolytic. Chemical modification of this new disaccharide polyene could give better antifungal antibiotics.

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin's shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin's biosynthesis including a subcluster of genes consistent with selvamicin's 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.

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.

Multigram Scale Synthesis of A21, A New Antibiotic Equally Effective and Less Toxic than Amphotericin B

A21 (2) is a new polyene macrolide Amphotericin B amide antibiotic derived from amphotericin B AmB (1), which has been tested extensively on preclinical trials showing the same antimycotic effectiv...

Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A (13)C MAS NMR Study.

The molecular action of polyene macrolides with antifungal activity, amphotericin B and natamycin, involves recognition of sterols in membranes. Physicochemical and functional studies have contributed details to understanding the interactions between amphotericin B and ergosterol and, to a lesser extent, with cholesterol. Fewer molecular details are available on interactions between natamycin with sterols. We use solid state 13C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing membranes, amphotericin B addition resulted in marked increase in both DOPC and cholesterol 13C MAS NMR linewidth, reflecting membrane insertion and cooperative perturbation of the bilayer. By contrast, natamycin affects little either DOPC or cholesterol linewidth but attenuates cholesterol resonance intensity preferentially for sterol core with lesser impact on the chain. Ergosterol resonances, attenuated by amphotericin B, reveal specific interactions in the sterol core and chain base. Natamycin addition selectively augmented ergosterol resonances from sterol core ring one and, at the same time, from the end of the chain. This puts forward an interaction model similar to the head-to-tail model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces. Low toxicity of natamycin is attributed to selective, non-cooperative sterol engagement compared to cooperative membrane perturbation by amphotericin B.

Structural and Functional Relationship of Macrolide Polyene Antiobiotics in Plasmatic and Bilayer Lipid Membranes

Structural and Functional Relationship of Macrolide Polyene Antiobiotics in Plasmatic and Bilayer Lipid Membranes

The Structure of the Bimolecular Complex between Amphotericin B and Ergosterol in Membranes Is Stabilized by Face-to-Face van der Waals Interaction with Their Rigid Cyclic Cores.

Amphotericin B (AmB) is a polyene macrolide antibiotic isolated from Streptomyces nodosus. The antifungal activity of AmB can be attributed to the formation of an ion-channel assembly in the presence of ergosterol (Erg), in which there are two different AmB-Erg orientations, parallel and antiparallel, as reported previously. In this study, to elucidate the structures of those AmB-Erg complexes based on solid-state nuclear magnetic resonance, a (19)F-labeled AmB derivative was newly prepared by a hybrid synthesis that utilized degradation products from the drug. Using the 2-(trimethylsilyl)ethoxymethyl (SEM) group as the protecting group for the carboxylic acid moiety of AmB, the fully deprotected labeled AmB compounds were obtained successfully. Then, these labeled AmBs were subjected to (13)C{(19)F} rotational-echo double-resonance (REDOR) experiments in hydrated lipid bilayers. The results indicated the coexistence of parallel and antiparallel orientations for AmB and Erg pairing, at a ratio of 7:3. A total of six distances between AmB and Erg were successfully obtained. Geometry analysis using the distance constraints derived from the REDOR experiments provided the plausible AmB-Erg complex structure for both the parallel and antiparallel interactions. The flat macrolide of AmB and the tetracyclic core of Erg closely contacted in a face-to-face manner, thus maximizing the van der Waals interaction between the two molecules. This interaction can be attributed to the coexistence of both the parallel and antiparallel orientations.

Onychomycosis and its Chemotherapy.

Onychomycosis (fungal nail infections) is very common worldwide but is fortunately not often lethal. Several powerful drugs have been introduced into clinical practice in recent years, but these infections remain difficult to cure primarily due to the difficulty of penetration of drug to the site of the infection in therapeutic concentrations. The nature of the disease, the causative fungi, and the characteristics of the drugs employed to treat this condition are discussed in this review.

Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies.

The polyene macrolide group includes important antifungal drugs, to which resistance does not arise readily. Chemical and biological methods have been used in attempts to make polyene antibiotics with fewer toxic side effects. Genome sequencing of producer organisms is contributing to this endeavour, by providing access to new compounds and by enabling yield improvement for polyene analogues obtained by engineered biosynthesis. This recent work is also enhancing bioinformatic methods for deducing the structures of cryptic natural products from their biosynthetic enzymes. The stereostructure of candicidin D has recently been determined by NMR spectroscopy. Genes for the corresponding polyketide synthase have been uncovered in several different genomes. Analysis of this new information strengthens the view that protein sequence motifs can be used to predict double bond geometry in many polyketides.Chemical studies have shown that improved polyenes can be obtained by modifying the mycosamine sugar that is common to most of these compounds. Glycoengineered analogues might be produced by biosynthetic methods, but polyene glycosyltransferases show little tolerance for donors other than GDP-α-D-mycosamine. Genome sequencing has revealed extending glycosyltransferases that add a second sugar to the mycosamine of some polyenes. NppY of Pseudonocardia autotrophica uses UDP-N-acetyl-α-D-glucosamine as donor whereas PegA from Actinoplanes caeruleus uses GDP-α-D-mannose. These two enzymes show 51% sequence identity and are also closely related to mycosaminyltransferases. These findings will assist attempts to construct glycosyltransferases that transfer alternative UDP- or (d)TDP-linked sugars to polyene macrolactones.

Genome mining reveals the biosynthetic potential of the marine-derived strain Streptomyces marokkonensis M10

Marine streptomycetes are rich sources of natural products with novel structures and interesting biological activities, and genome mining of marine streptomycetes facilitates rapid discovery of their useful products. In this study, a marine-derived Streptomyces sp. M10 was revealed to share a 99.02% 16S rDNA sequence identity with that of Streptomyces marokkonensis Ap1T, and was thus named S. marokkonensis M10. To further evaluate its biosynthetic potential, the 7,207,169 bps of S. marokkonensis M10 genome was sequenced. Genomic sequence analysis for potential secondary metabolite-associated gene clusters led to the identification of at least three polyketide synthases (PKSs), six non-ribosomal peptide synthases (NRPSs), one hybrid NRPS-PKS, two lantibiotic and five terpene biosynthetic gene clusters. One type I PKS gene cluster was revealed to share high nucleotide similarity with the candicidin/FR008 gene cluster, indicating the capacity of this microorganism to produce polyene macrolides. This assumption was further verified by isolation of two polyene family compounds PF1 and PF2, which have the characteristic UV adsorption at 269, 278, 290 nm (PF1) and 363, 386 and 408 nm (PF2), respectively. S. marokkonensis M10 is therefore a new source of polyene metabolites. Further studies on S. marokkonensis M10 will provide more insights into natural product biosynthesis potential of related streptomycetes. This is also the first report to describe the genome sequence of S. marokkonensis-related strain.

Polyene macrolide antibiotics: Mechanisms of inactivation, ways of stabilization, and methods of disposal of unusable drugs (Review)

Published data concerning various mechanisms of the inactivation of polyene macrolide antibiotics, ways of their stabilization, and search for new stable dosage forms prepared with the use of nanotechnology techniques were summarized. Environmentally acceptable ways of disposal of pharmaceutical waste, and in particular polyene macrolide antibiotics, were discussed.