ASP2397: a novel antifungal agent produced by Acremonium persicinum MF-347833.

The novel antifungal agent ASP2397 (Vical's compound ID VL-2397) is produced by the fungal strain MF-347833 that was isolated from Malaysian leaf litter and is identified here as an Acremonium species based on its morphology, physiological properties and 28S ribosomal DNA sequence. Because of its potential importance for producing novel antifungal agents, we determined the taxonomic and biologic properties of MF-347833. We show here that ASP2397 is a cyclic hexapeptide that chelates aluminum ion and is therefore similar to ferrichrome, a hydroxamate siderophore. However, ASP2397 differs structurally from licensed antifungal agents such as amphotericin B, triazoles and echinocandins. To understand the relationship between chemical structure and biological function, we isolated certain ASP2397 derivatives from the culture broth, and we further chemically converted the metal-free form to other derivatives.

Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887.

FR901459, a product of the fungus Stachybotrys chartarum No. 19392, is a derivative of cyclosporin A (CsA) and a powerful immunosuppressant that binds cyclophilin. Recently, it was reported that CsA was effective against hepatitis C virus (HCV). However, FR901459 lacks active moieties, which are essential for synthesizing more potent and safer derivatives of this anti-HCV agent. Here we identified an actinomycete strain (designated 7887) that was capable of efficient bioconversion of FR901459. Structural elucidation of the isolated bioconversion products (1-7) revealed that compounds 1-4 were mono-hydroxylated at the position of 1-MeBmt or 9-MeLeu, whereas compounds 5-7 were bis-hydroxylated at both positions. The results of morphological and chemical characterization, as well as phylogenetic analysis of 16S ribosomal DNA (rDNA), suggested that strain 7887 belonged to the genus Lentzea. Comparison of the FR901459 conversion activity of strain 7887 with several other Lentzea strains revealed that although all examined strains metabolized FR901459, strain 7887 had a characteristic profile with respect to bioconversion products. Taken together, these findings suggest that strain 7887 can be used to derivative FR901459 to produce a chemical template for further chemical modifications that may provide more effective and safer anti-HCV drugs.

Identification of ten KB425796-A congeners from Paenibacillus sp. 530603 using an antifungal assay against Aspergillus fumigatus in combination with micafungin.

The discovery and characterization of natural congeners is one approach for understanding the relationship between chemical structure and biological function. We recently isolated the novel antifungal metabolite KB425796-A produced by the recently isolated bacterium Paenibacillus sp. 530603. On the basis of morphological changes of Aspergillus fumigatus induced by KB425796-A in combination with micafungin, we developed a highly sensitive screening method for the specific detection of KB425796-A congeners. Using this method, we isolated ten congeners of KB425796-A, named KB425796-B, -C, -D, -E, -F, -G, -H, -I, -J and -K, which exhibited diverse antifungal potencies against A. fumigatus. One of the most potent congeners, KB425796-C, had antifungal activities against several micafungin-resistant infectious fungi. KB425796-C can be a potential drug candidate for treating micafungin-resistant fungal infections.

KB425796-A, a novel antifungal antibiotic produced by Paenibacillus sp. 530603.

The novel antifungal macrocyclic lipopeptidolactone, KB425796-A (1), was isolated from the fermentation broth of bacterial strain 530603, which was identified as a new Paenibacillus species based on morphological and physiological characteristics, and 16S rRNA sequences. KB425796-A (1) was isolated as white powder by solvent extraction, HP-20 and ODS-B column chromatography, and lyophilization, and was determined to have the molecular formula C79H115N19O18. KB425796-A (1) showed antifungal activities against Aspergillus fumigatus and the micafungin-resistant infectious fungi Trichosporon asahii, Rhizopus oryzae, Pseudallescheria boydii and Cryptococcus neoformans.

Bioconversion of AS1387392: bioconversion studies involving Amycolatopsis azurea JCM 3275.

We screened actinomycetes capable of converting AS1387392 to AS1429716 and identified those strains capable of hydroxylation. Amycolatopsis azurea JCM 3275 was found to be a particularly efficient strain, capable of converting AS1387392 to AS1429716, with a yield of 44% after 9 h. This strain can metabolize not only the hydroxylation of phenylalanine at the meta and para positions but also the reduction of hydroxyketones, as shown by the isolation of bioconversion products. Examination of more suitable conversion conditions showed that pH 7.8 and 25 °C were the optimum pH and temperature for bioconversion, respectively. We also demonstrated the effect of carbon and nitrogen sources in the culture media on hydroxylation. Using this strain, we were able to efficiently produce AS1429716 as a chemical template. Further derivatization studies may provide more effective, safer immunosuppressants than those that are currently on-market.

Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392.

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.

Bioconversion of AS1387392: screening and characterization of actinomycetes that convert AS1387392 to AS1429716.

AS1387392 was a novel and powerful histone deacetylase inhibitor with an excellent oral absorption profile, but this compound was lacking in active moieties, which are essential to synthesize more derivatives. In our screening program to identify actinomycetes capable of converting AS1387392 to AS1429716, which has an active moiety to synthesize more derivatives, we identified 12 strains capable of efficient hydroxylation. Results of phylogenetic analysis of 16S rDNA sequences suggested that these strains belonged to the genera Lentzea, Saccharopolyspora, Sphaerisporangium and Amycolatopsis. Morphological and chemical characteristics as well as results of phylogenetic analysis suggested that strain No. 7980 was a new species belonging to the genus Amycolatopsis, according to the FASTA search result of 16S rDNA gene sequence. Using these strains, we can easily produce AS1429716 as a chemical template for further chemical modifications, which may provide more effective and safer immunosuppressant.

AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase.

The novel immunosuppressant AS1387392 has been isolated from Acremonium sp. No. 27082. This compound showed a strong inhibitory effect against mammalian histone deacetylase and T-cell proliferation. Further, AS1387392 showed a good oral absorption, and its plasma concentration was higher than that of FR235222, an analog of AS1387392 that inhibited histone deacetylase previously reported. Given these findings, AS1387392 may represent an important new lead in developing immunosuppressant.

Naphthalecin, a novel antibiotic produced by the anaerobic bacterium, Sporotalea colonica sp. nov.

A novel antibiotic naphthalecin was purified and isolated from the cells of an anaerobic bacterium isolated from a soil sample. This antibiotic contained a naphthalene moiety, so named as naphthalecin, and showed antibacterial activity against gram positive species. The producing strain, an obligate anaerobe, was identified as a new species of the genus Sporotalea. Identification of the bacterium, cultivation, purification, structure determination, and antibacterial activity are shown.

FR209602 and related compounds, novel antifungal lipopeptides from Coleophoma crateriformis no.738. I. Taxonomy, fermentation, isolation and physico-chemical properties.

Novel antifungal lipopeptides, FR209602, FR209603 and FR209604, were isolated from the fermentation broth of a fungal strain No. 738 which was identified as Coleophoma crateriformis from morphological and physiological characteristics. The antibiotics were purified by solvent extraction, HP-20, YMC-ODS and silica gel column chromatography and lyophilization. These compounds were structurally similar to FR901379 previously reported by ourselves which had a sulfate residue in the cyclic peptide portion.

FR227673 and FR190293, novel antifungal lipopeptides from Chalara sp. No. 22210 and tolypocladium parasiticum No. 16616.

Novel antifungal lipopeptides, FR227673 and FR190293, were isolated from the fermentation broths of fungal strains Chalara sp. No. 22210 and Tolypocladium parasiticum No. 16616, respectively. These compounds have the same cyclic peptide nuclear structure as FR901379, with different side chains, and showed antifungal activity against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-beta-glucan synthesis.

FR220897 and FR220899, novel antifungal lipopeptides from Coleophoma empetri no. 14573.

Novel antifungal lipopeptides, FR220897 and FR220899, were isolated from the fermentation broth of a fungal strain No. 14573. This strain was identified as Coleophoma empetri No. 14573 from morphological and physiological characteristics. FR220897 and FR220899 showed antifungal activities against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-beta-glucan synthesis. Furthermore, FR220897 was effective in a murine model of systemic candidiasis.

Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24.

Cytochrome P450 enzymes are a major class of biocatalysts related to the oxidative metabolism of many drugs, assisted by electron transfer partners. The functional expression of the P450 gene in a heterologous host will lead to efficient biotransformation and biodegradation, which are useful in pharmaceutical improvement or environmental cleanup. The soluble cytochrome P450 monooxygenase systems CYP105D1 and CYP107B1 involved in the biotransformation of some xenobiotics, such as secondary metabolites or environmental pollutants, were expressed in Streptomyces lividans TK24 with the Streptomyces expression vector pIJ6021. In whole-cell biotransformation assay using these recombinant strains, the oxidative dealkylation of 7-ethoxycoumarin was detected without any foreign redox partners in the case of CYP107B1, while the activity of CYP105D1 was not monitored until this gene was coexpressed with the ferredoxin gene located downstream of the CYP105D1 gene, and the ferredoxin reductase gene SCF 15.02 from Streptomyces coelicolor A3(II). This result suggests that CYP107B1 is capable of utilizing an endogenous electron transfer partner from the host but not CYP105D1, and that CYP105D1 is complemented by some redox partner imported from closely related strains.

FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354. I. Taxonomy, fermentation, isolation and biological activities.

FR258900 is a novel glycogen synthesis activator produced by Fungus No. 138354. This compound was isolated from the culture broth by solvent extraction and reverse-phase column chromatography. FR258900 stimulated glycogen synthesis and glycogen synthase activity in primary rat hepatocytes. FR258900 exhibited a potent inhibitory effect on the activity of liver glycogen phosphorylase, suggesting that this compound may activate hepatic glycogen synthesis via glycogen phosphorylase inhibition. Thus, this glycogen phosphorylase inhibitor may be useful in the treatment of postprandial hyperglycemia in type 2 diabetes.

FR207944, an antifungal antibiotic from Chaetomium sp. No. 217 II. Isolation and structure elucidation.

We discovered FR207944 produced by Chaetomium sp. No. 217 in the course of screening for antifungal antibiotics from natural products. FR207944 is identical with fuscoatroside, described in the preceding paper as an anti-Aspergillus flavus agent. Determination of the relative stereochemistry of fuscoatroside was made formally by comparison with WF11605 (16-Oxo-FR207944). We confirmed the stereochemistry on the basis of single crystal X-ray analysis.

FR207944, an antifungal antibiotic from Chaetomium sp. no. 217 I. Taxonomy, fermentation, and biological properties.

An antifungal antibiotic, FR207944, was isolated from the culture broth of a fungal strain Chaetomium sp. no. 217. FR207944 is a triterpene glucoside with antifungal activity against Aspergillus fumigatus and Candida albicans. Specifically, FR207944 exhibits in vitro and in vivo antifungal activity against A. fumigatus. The effects of FR207944 on the morphology of A. fumigatus were shown to be similar to those of FR901379, a known 1,3-beta-glucan synthase inhibitor. The MECs of FR207944 against A. fumigatus FP1305 and C. albicans FP633 in micro-broth dilution test were 0.039 and 1.6 mug/ml respectively. FR207944 showed good potency by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 5.7 and 17 mg/kg respectively.

FR227244, a novel antifungal antibiotic from Myrothecium cinctum No. 002. II. Biological properties and mode of action.

FR227244 is a novel triterpene glycoside that exhibits in vitro antifungal activity against filamentous fungi such as Aspergillus sp. and Trichophyton sp. and yeast such as Candida utilis and Candida parapsilosis but shows low activity against Candida albicans, Candida krusei and Candida tropicalis. Specifically, FR227244 exhibits in vitro and in vivo antifungal activity against Aspergillus fumigatus. The minimum effective concentration (MEC) of FR227244 against A. fumigatus FP1305 in a micro-broth dilution test was 0.031 microg/ml. FR227244 showed good efficacy by subcutaneous injection and oral administration against A. fumigatus in a murine systemic infection model, with ED(50)s of 1.9 and 18 mg/kg, respectively. FR227244 inhibited glucan synthesis in a 1,3-beta-glucan synthase assay weakly and in whole cells strongly, but did not effect other macromolecule synthesis, including protein, nucleic acids, mannan and chitin. These results and the effect on hyphal morphology of A. fumigatus suggested that FR227244 showed antifungal activity based on inhibition of glucan synthesis.

The novel gluconeogenesis inhibitors FR225659 and related compounds that originate from Helicomyces sp. No. 19353. I. Taxonomy, fermentation, isolation and physico-chemical properties.

FR225659 and four related compounds are novel gluconeogenesis inhibitors that consist of a novel acyl-group and three abnormal amino acids. They were isolated from the culture broth of Helicomyces sp. No. 19353 and can be purified by absorptive resin and reverse-phase column chromatography. They are potent inhibitors of gluconeogenesis in primary cultured rat hepatocytes and thus may be useful as anti-diabetic agents.

FR252921, a novel immunosuppressive agent isolated from Pseudomonas fluorescens no. 408813. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities of FR252921, FR252922 and FR256523.

Novel immunosuppressive agents, FR252921, FR252922 and FR256523 were isolated from the cultured broth of a bacterial strain No. 408813. The strain was identified Pseudomonas fluorescens from morphological and physiological characteristics. FR252921, FR252922 and FR256523, novel compounds containing macrolactone ring, showed immunosuppressive activity against murine splenocyte proliferation stimulated with lipopolysaccharide (LPS) or anti-CD3 mAb in vitro.

FR235222, a fungal metabolite, is a novel immunosuppressant that inhibits mammalian histone deacetylase (HDAC). I. Taxonomy, fermentation, isolation and biological activities.

A cyclic tetrapeptide FR235222, a novel immunosuppressant, has been isolated from the fermentation broth of a fungus, Acremonium sp. No. 27082. FR235222 showed potent and selective inhibitory effects on both T cell proliferation and lymphokine production. Further study has revealed this compound exhibits potent inhibitory effects on the activity of mammalian histone deacetylases (HDACs).