Polyene Macrolide Antibiotic Research Articles

Deciphering the roles of fatty acids and oils in fungichromin enhancement from Streptomyces padanus

Fungichromin (FC), a polyene macrolide antibiotic, was found to be useful in the treatment of resistance for plant diseases such as Rhizoctonia damping-off of cabbage and tomato late blight. A novel strain, Streptomyces padanus PMS-702, was employed to study the cultivation conditions for FC production. When using linoleic acid as an additive, a negative effect on FC production was observed. On the contrary, when using oleic acid in concentration of 0.01 M (2.82 g/L), a 3.5-fold enhancement of FC production with a slight increase in cell biomass was obtained. To study the effects of plant oils, various plant oils were used as additives. It was found that all the plant oils (namely, corn oil, olive oil and etc.) promote FC production up to about 4.9-fold as compared to that without such an addition. A quantitative analysis on FC yield indicated that, at a lower concentration, fatty acids and plant oils play roles as both a stimulator and a carbon source whereas, at a higher concentration, they serve mainly as the carbon source. This morphological study showed that the addition of fatty acids and oils in the medium vary the cell membrane permeability. The fact that FC could be rapidly secreted out into the medium after its synthesis as well as its stimulatory effect attributed to a dramatic increase in FC production.

A Simple and General Platform for Generating Stereochemically Complex Polyene Frameworks by Iterative Cross‐Coupling

A Simple and General Platform for Generating Stereochemically Complex Polyene Frameworks by Iterative Cross‐Coupling

ChemInform Abstract: Stereoselective Synthesis and the Polyene Macrolide Antibiotics

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Structure of Cytochrome P450 PimD Suggests Epoxidation of the Polyene Macrolide Pimaricin Occurs via a Hydroperoxoferric Intermediate

We present the X-ray structure of PimD, both substrate-free and in complex with 4,5-desepoxypimaricin. PimD is a cytochrome P450 monooxygenase with native epoxidase activity that is critical in the biosynthesis of the polyene macrolide antibiotic pimaricin. Intervention in this secondary metabolic pathway could advance the development of drugs with improved pharmacologic properties. Epoxidation by P450 typically includes formation of a charge-transfer complex between an oxoferryl pi-cation radical species (Compound I) and the olefin pi-bond as the initial intermediate. Catalytic and structural evidence presented here suggest that epoxidation of 4,5-desepoxypimaricin proceeds via a hydroperoxoferric intermediate (Compound 0). The oxygen atom of Compound 0 distal to the heme iron may insert into the double bond of the substrate to make an epoxide ring. Stereoelectronic features of the putative transition state suggest substrate-assisted proton delivery.

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the "filipin complex." CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1', respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B' helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.

Preparation, relative toxicity and therapeutic efficacy in mice and rats of liposomal HA-1-92, a new oxohexaene polyene macrolide antibiotic.

HA-1-92, a new polyene oxohexaene macrolide antibiotic isolated from Streptomyces CDRIL-312, was incorporated into liposomes containing phosphotidyl choline and cholesterol. The liposomal incorporated HA-1-92 considerably decreased toxicity when compared with free HA-1-92 in mice. Liposomal HA-1-92 showed improved pharmacokinetic profiles in rats. When administered to aspergillosis- and cryptococcosis-infected Balb/c mice, liposomal HA-1-92 showed increased antifungal activity, compared with free HA-1-92, with improved survival rate and decreased colony-forming units in lung, liver, spleen and kidney. These results suggest that liposomal HA-1-92 is more effective than free HA-1-92 in controlling experimental aspergillosis and cryptococcosis in Ba1b/c mice.

Redesign of Polyene Macrolide Glycosylation: Engineered Biosynthesis of 19-( O)-Perosaminyl- Amphoteronolide B

Most polyene macrolide antibiotics are glycosylated with mycosamine (3,6-dideoxy-3-aminomannose). In the amphotericin B producer, Streptomyces nodosus, mycosamine biosynthesis begins with AmphDIII-catalyzed conversion of GDP-mannose to GDP-4-keto-6-deoxymannose. This is converted to GDP-3-keto-6-deoxymannose, which is transaminated to GDP-mycosamine by the AmphDII protein. The glycosyltransferase AmphDI transfers mycosamine to amphotericin aglycones (amphoteronolides). The aromatic heptaene perimycin is unusual among polyenes in that the sugar is perosamine (4,6-dideoxy-4-aminomannose), which is synthesized by direct transamination of GDP-4-keto-6-deoxymannose. Here, we use the Streptomyces aminophilus perDII perosamine synthase and perDI perosaminyltransferase genes to engineer biosynthesis of perosaminyl-amphoteronolide B in S. nodosus. Efficient production required a hybrid glycosyltransferase containing an N-terminal region of AmphDI and a C-terminal region of PerDI. This work will assist efforts to generate glycorandomized amphoteronolides for drug discovery.

10.1007/s11176-008-2021-y

Reactions of the polyene macrolide antibiotic pimaricin with aromatic aldehydes and hypophosphorous acid are shown to afford pimaricin hydrophosphoryl derivatives. Physicochemical and biological properties of these compounds are explored. Biological studies indicated that the pimaricin hydrophosphoryl derivatives are low toxic agents possessing a high antifungal activity.

Targeted yeast vacuole disruption by polyene antibiotics with a macrocyclic lactone ring

In this study, the polyene macrolide antibiotics amphotericin B (AmB), nystatin and filipin III were evaluated for their fungicidal activity and their ability to produce vacuole disruption as well as enhancement of these activities by allicin using Saccharomyces cerevisiae. Nystatin has a macrocyclic lactone ring analogous to AmB and their fungicidal activities were both increased by allicin, an allyl-sulfur compound, whereas filipin III, a pentaene macrolide, did not show an increase in fungicidal activity in the presence of allicin. Vacuole staining with the fluorescent probe FM4-64 showed that both AmB and nystatin induced vacuole membrane disintegration at their lethal concentrations; in addition, the vacuole disruptive effect was also enhanced by allicin. In contrast, filipin III did not affect vacuole morphology and addition of allicin had no effect despite filipin III localising to the cell cytoplasm. Isolated S. cerevisiae vacuoles were disrupted following treatment both with nystatin and AmB, though this activity was not potentiated in the presence of allicin. In contrast, filipin III had little effect on the vacuole architecture. This study reveals differential effects of polyene antibiotics on vacuoles in S. cerevisiae, which may be due to differences in the structure of the macrocyclic ring .

Amphotericin B-induced nephrotoxicity: characterization of blood and urinary biochemistry and renal morphology in mice

This study was conducted to characterize blood and urinary biochemistry, and renal morphology, after single or 1-week repeated dosing of mice with the polyene macrolide antibiotic, amphotericin B (AMB). AMB was intravenously administered to mice at 2 or 4 mg/kg for the single-dose experiment or once daily at 1 or 2 mg/kg for 1 week for the repeated-dose experiment. The most prominent histopathological findings included necrosis of the tubular epithelial cells in the thick ascending limb of Henle's loop in the renal outer medulla at a single dose of 2 or 4 mg/kg, and the severity of the lesion was dose-dependent. Blood chemistry and urinalysis revealed several changes suggestive of renal dysfunction such as reduction of plasma filtration ability (increases in plasma creatinine and blood urea nitrogen, a decrease in creatinine clearance) and polyuria accompanied with dehydration (decrease in renal water reabsorption, increases in plasma total protein and albumin) at a dose of 4 mg/kg in the single-dose experiment. Among the parameters analyzed, urinary lactate dehydrogenase was the most sensitive and reliable parameter for the prediction of AMB-induced nephrotoxicity in mice. These data provided comprehensive information on the nephrotoxicity of AMB and indicate useful markers for the sensitive detection of AMB-induced renal injury in mice.

Nanodisks protect amphotericin B from ultraviolet light and oxidation‐induced damage

Macrolide polyene antibiotics possess potent broad-spectrum antifungal properties. Use of these agents in the field or in controlled environments is impeded by their poor water solubility and susceptibility to oxidation- and/or light-induced degradation. While typically used for human disease therapy, there is potential to expand the utility of polyene macrolide antibiotics, such as amphotericin B, for control of fungal disease infestation in agricultural settings. Thus, the susceptibility of this antibiotic to exposure-induced activity loss was evaluated. Incubation of the prototype polyene amphotericin B (AMB) with phospholipid vesicles and apolipoprotein A-I results in the formation of nanoscale complexes, termed nanodisks (NDs), capable of solubilizing significant quantities of AMB. To evaluate whether AMB incorporation into NDs conferred protection against light- or oxidation-induced damage, yeast growth inhibition assays were conducted. Compared with AMB solubilized in detergent micelles, AMB incorporated into NDs was protected from damage caused by exposure to UV light as well as by KMnO(4)-induced oxidation. Furthermore, AMB-NDs inhibited growth of the turfgrass fungus Marasmius oreades Fr. Results suggest that this water-soluble formulation of a natural, biodegradable, antifungal agent represents a potential cost-effective, non-toxic and environmentally friendly substitute for chemical agents currently employed to control a range of fungal infestations.

Molecular modeling of amphotericin B–ergosterol primary complex in water II

The work presented is a part of our continual study on the behavior of the polyene macrolide antibiotic amphotericin B (AmB) complexes with sterols on the molecular level. In contrast to the previously researched AmB–ergosterol binary complex, the AmB–ergosterol–AmB aggregates simulated of 2:1 stoichiometry retain significantly higher stability and relatively rigid, “sandwich” geometry. Van der Waals forces with a considerable share of the electrostatic interactions are responsible for such behavior. System of the intermolecular hydrogen bonds also seems to be of notable importance for the complex's structure preservation. The most energetically favored geometries match fairly close the geometric criteria and the network of interactions postulated in the contemporary hypothetical and computational models of antibiotic–sterol complexes. On the basis of works previously published and the present study novel hypotheses on the AmB selectivity towards sterols varying in chemical structure and on the possible mechanisms of channel structure formation were presented.

Crystal Structures of Cytochrome P450 105P1 from Streptomyces avermitilis : Conformational Flexibility and Histidine Ligation State

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1'-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate.

Chemical Modification and Biological Evaluation of New Semisynthetic Derivatives of 28,29-Didehydronystatin A1 (S44HP), a Genetically Engineered Antifungal Polyene Macrolide Antibiotic

Twenty-three new derivatives of the heptaene nystatin analogue 28,29-didehydronystatin A(1) (1) (S44HP) were obtained by chemical modification of C16 carboxyl and amino groups of mycosamine. These derivatives comprised 15 carboxamides, 4 N-alkyl derivatives, 3 N-derivatives containing additional N-linked monosaccharide or disaccharide moiety (products of Amadori rearrangement), and 1 N-aminoacyl derivative. The derivatives have been tested in vitro against yeasts Candida albicans, Cryptococcus humicolus, and filamentous fungi (molds) Aspergillus niger and Fusarum oxysporum, as well as for hemolytic activity against human erythrocytes. Structure-activity relationships for the compounds obtained are discussed. The most active and least hemolytic derivative 3-(N,N-dimethylamino)propylamide of S44HP (6) was tested for acute toxicity and antifungal activity in animal model. Whereas amphotericin B and S44HP were active in vivo at doses close to the maximal tolerated dose, 6 was considerably less toxic and more active compared to these two antibiotics.

Chemotherapeutic activity of liposomal SJA-95: A new polyene macrolide antibiotic in experimental aspergillosis and cryptococcosis

The incidence of systemic fungal infections that has risen dramatically over the past three decades has propelled a continuous need for more potent antifungal drugs. The purpose of this research was to evaluate the chemotherapeutic activity of a new heptaene polyene macrolide antibiotic (SJA-95) and liposomal incorporated SJA-95 (lip. SJA-95) in a mouse model of aspergillosis and cryptococcosis respectively. Lip. SJA-95 was prepared in our laboratory by the proliposome method involving incorporation of the antifungal into the proliposome mixture and its subsequent conversion into a liposomal dispersion by a simple dilution step. Treatment with free SJA-95 and lip. SJA-95, both in aspergillosis and cryptococcosis, progressively prolonged the survival time and decreased the fungal loads in vital organs respectively. A higher LD 50 value of lip. SJA as compared to that of free SJA-95 was indicative of reduced toxicity of lip. SJA-95. Our findings suggest lip. SJA-95 treatment results in prolonged survival time, effective microbiological clearance and reduced toxicity that might help to establish its usefulness as a chemotherapeutic agent in systemic fungal infections with fewer adverse reactions.

Synthesis and antifungal activity of N-trialkylsilyl derivatives of nystatin

Reactions of the polyene macrolide antibiotic nystatin with trialkylchlorosilanes led to the formation of N-alkylsilyl derivatives. The physicochemical and biological properties of the resulting nystatin derivatives were studied. Organic silicon derivatives of nystatin had high levels of antifungal activity against a wide set of test strains. Biological studies showed that the acute toxicity (LD50) of the resulting nystatin derivatives were 2–3 times lower than that of the initial antibiotic.

Roles of integral protein in membrane permeabilization by amphidinols

Amphidinols (AMs) are a group of dinoflagellate metabolites with potent antifungal activity. As is the case with polyene macrolide antibiotics, the mode of action of AMs is accounted for by direct interaction with lipid bilayers, which leads to formation of pores or lesions in biomembranes. However, it was revealed that AMs induce hemolysis with significantly lower concentrations than those necessary to permeabilize artificial liposomes, suggesting that a certain factor(s) in erythrocyte membrane potentiates AM activity. Glycophorin A (GpA), a major erythrocyte protein, was chosen as a model protein to investigate interaction between peptides and AMs such as AM2, AM3 and AM6 by using SDS-PAGE, surface plasmon resonance, and fluorescent-dye leakages from GpA-reconstituted liposomes. The results unambiguously demonstrated that AMs have an affinity to the transmembrane domain of GpA, and their membrane-permeabilizing activity is significantly potentiated by GpA. Surface plasmon resonance experiments revealed that their interaction has a dissociation constant of the order of 10 μM, which is significantly larger than efficacious concentrations of hemolysis by AMs. These results imply that the potentiation action by GpA or membrane integral peptides may be due to a higher affinity of AMs to protein-containing membranes than that to pure lipid bilayers.

Synthesis of hydrophospohoryl derivatives of the antifungal antibiotic pimaricin by the Kabachnik-Fields reaction

Reactions of the polyene macrolide antibiotic pimaricin with aromatic aldehydes and hypophosphorous acid are shown to afford pimaricin hydrophosphoryl derivatives. Physicochemical and biological properties of these compounds are explored. Biological studies indicated that the pimaricin hydrophosphoryl derivatives are low toxic agents possessing a high antifungal activity.

Optimization of cultivation conditions for fungichromin production from Streptomyces padanus PMS-702

A novel strain, Streptomyces padanus PMS-702, was employed to produce fungichromin (FC, a polyene macrolide antibiotic) in a shake flask cultivation. In comparing the effects of various carbon and nitrogen sources on PMS-702 cultivation, it was found that glucose and soybean meal medium yielded the highest FC within 2 days. Factors such as medium composition, cultivation temperatures, and initial pH were optimized for FC production with response surface methodology (RSM). The optimal cultural condition obtained is as follows: glucose 11.2 g/L, soybean meal 11.2 g/L, CaCO 3 0.46 g/L, temperature 31.7 °C, and an initial pH 5.5. Under these conditions, FC production reached 112 mg/L, about an increase of 2.86 times, as compared to results under basic conditions.

Preparation, Relative Toxicity, Chemotherapeutic Activity, and Pharmacokinetics of Liposomal SJA-95: A New Polyene Macrolide Antibiotic

The research work was designed to compare the relative toxicity, chemotherapeutic activity, and pharmacokinetic parameters of liposomal incorporated SJA-95 with that of free SJA-95, with an objective to reduce toxicity and improve therapeutic activity in vivo. Liposomal-incorporated SJA-95 (Lip SJA-95), prepared using the proliposome method, was found to exhibit a higher LD50 value in mice, and the relative toxicity was about 2.5 times lower than that of the free drug. Lip SJA-95 treatment in experimental mice model of Candidiasis showed increased survival and reduced fungal loads in various organs. The pharmacokinetic profile of the free and liposomal drug was evaluated by administration of free and Lip SJA-95 intravenously to healthy albino rabbits in a crossover fashion. Lip SJA-95 showed an initial fall in plasma levels and longer half-life. The improved microbial clearance following treatment with Lip SJA-95 could be attributed partly to an increased tissue uptake, which was reflected in a marked increase in volume of distribution (Vd) and longer half-life (T1/2). The present results clearly indicated that Lip SJA-95 treatment led to prolonged survival time, effective microbiological clearance, and reduced toxicity in the mice model of Candidiasis.