A novel assay of bacterial peptidoglycan synthesis for natural product screening.

Although a large number of microbial metabolites have been discovered as inhibitors of bacterial peptidoglycan biosynthesis, only a few inhibitors were reported for the pathway of UDP-MurNAc-pentapeptide formation, partly because of the lack of assays appropriate for natural product screening. Among the pathway enzymes, D-Ala racemase (Alr), D-Ala:D-Ala ligase (Ddl) and UDP-MurNAc-tripeptide:D-Ala-D-Ala transferase (MurF) are particularly attractive as antibacterial targets, because these enzymes are essential for growth and utilize low-molecular-weight substrates. Using dansylated UDP-MurNAc-tripeptide and L-Ala as the substrates, we established a cell-free assay to measure the sequential reactions of Alr, Ddl and MurF coupled with translocase I. This assay is sensitive and robust enough to screen mixtures of microbial metabolites, and enables us to distinguish the inhibitors for D-Ala-D-Ala formation, MurF and translocase I. D-cycloserine, the D-Ala-D-Ala pathway inhibitor, was successfully detected by this assay (IC(50)=1.7 microg ml(-1)). In a large-scale screening of natural products, we have identified inhibitors for D-Ala-D-Ala synthesis pathway, MurF and translocase I.

A-94964, a novel inhibitor of bacterial translocase I, produced by Streptomyces sp. SANK 60404. I. Taxonomy, isolation and biological activity.

Bacterial phospho-N-acetylmuramyl-pentapeptide translocase (translocase I: EC 2.7.8.13) is a key enzyme in peptidoglycan biosynthesis, and a known target of antibiotics. Here we report a novel nucleoside inhibitor against translocase I, A-94964, isolated from the culture broth of the strain Streptomyces sp. SANK 60404. A-94964 inhibited bacterial translocase I with IC50 value of 1.1 microg/ml, and showed antimicrobial activities against Staphylococcus aureus and Enterococcus faecalis with MIC of 100 and 50 microg/ml, respectively. A-94964 did not show cytotoxicity against mammalian cell lines.

A-102395, a new inhibitor of bacterial translocase I, produced by Amycolatopsis sp. SANK 60206.

Bacterial phospho-N-acetylmuramyl-pentapeptide translocase (translocase I: EC 2.7.8.13) is a key enzyme in peptidoglycan biosynthesis, and a known target of antibiotics. Here we report a new nucleoside inhibitor for translocase I, A-102395, isolated from the culture broth of the strain Amycolatopsis sp. SANK 60206. A-102395 is a new derivative of capuramycin that has the benzene with a uniquely substituted chain instead of an aminocaprolactam. A-102395 is a potent inhibitor of bacterial translocase I with IC50 value of 11 nM, but possesses no antimicrobial activity against various strains tested.

Pleofungins, novel inositol phosphorylceramide synthase inhibitors, from Phoma sp. SANK 13899. I. Taxonomy, fermentation, isolation, and biological activities.

In the course of a screening for inositol phosphorylceramide (IPC) synthase inhibitors, the novel inhibitors pleofungins A, B, C, and D were found in a mycelial extract of a fungus, Phoma sp. SANK13899. Purification was performed by 50% methanol and ethyl acetate extraction, reversed phase open-column chromatography, and HPLC separations. Pleofungin A inhibited the IPC synthase of Saccharomyces cerevisiae and Aspergillus fumigatus at IC(50) values of 16 and 1.0 ng/ml, respectively. The inhibitor also suppressed the growth of Candida albicans, Cryptococcus neoformans, and A. fumigatus at MIC values of 2.0, 0.3, and 0.5 mug/ml, respectively. These biological properties indicate that pleofungins belong to a novel class of IPC synthase inhibitors efficacious against A. fumigatus.

Studies on novel bacterial translocase I inhibitors, A-500359s. V. Enhanced production of capuramycin and A-500359 A in Streptomyces griseus SANK 60196.

Streptomyces griseus SANK 60196 produces the novel nucleoside antibiotics A-500359 A, C, D and capuramycin. Enhanced production of capuramycin and A-500359 A was achieved through a number of medium modifications and a series of single colony isolations. The addition of maltose instead of glucose as the carbon source in a primary medium resulted in a 20-fold increase in the productivity of capuramycin. Furthermore, the addition of cobalt chloride (CoCl2) and yeast extract to the medium containing maltose drastically altered the production ratio of A-500359 A to capuramycin. Thus, the yield of A-500359 A increased up to 600 microg/ml in an optimal medium, while the yield in the primary medium was 1 microg/ml.

Deletion of OSH3 gene confers resistance against ISP-1 in Saccharomyces cerevisiae.

Sphingolipids have been reported to regulate the growth and death of mammalian and yeast cells, but their precise mechanisms are unknown. In this paper, it was shown that the deletion of the oxysterol binding protein homologue 3 (OSH3) gene confers hyper resistance against ISP-1, an inhibitor of sphingolipid biosynthesis, in the yeast Saccharomyces cerevisiae. Furthermore, the overexpression of the ROK1 gene, which directly binds to Osh3p, conferred resistance against ISP-1, and the deletion of the KEM1 gene, which regulates microtubule functions, exhibited ISP-1 hypersensitivity. And yet, an ISP-1 treatment caused an abnormal mitotic spindle formation, and the ISP-1-induced cell cycle arrest was rescued by the deletion of the OSH3 gene. Taken together, it is suggested that the expression levels of the OSH3 gene influence the ISP-1 sensitivity of S. cerevisiae, and the sphingolipids are necessary for normal mitotic spindle formation in which the Osh3p may play a pivotal role.

Structure-activity relationships of globomycin analogues as antibiotics.

Globomycin (1a), a signal peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. The hydroxyl group in the L-Ser residue was essential for the antimicrobial activity and the length of the alkyl side chain greatly influenced the activity. In addition, derivatives that had a modified cyclic core exhibited weak activity. One of the analogues showed a wider antimicrobial spectrum, effective against not only Gram-negative but also Gram-positive bacteria.

A-503083 A, B, E and F, novel inhibitors of bacterial translocase I, produced by Streptomyces sp. SANK 62799.

Novel nucleoside antibiotics were isolated from the cultured broth of the strain classified as Streptomyces sp. SANK 62799. The strain produced four novel capuramycin derivatives designated as A-503083 A, B, E and F. Their structures were elucidated as 2'-O-carbamoyl derivatives of A-500359 A, B (capuramycin), E and F, respectively. A-503083 A, B, E and F inhibited bacterial phospho-N-acetylmuramyl-pentapeptide-translocase (translocase I: EC 2.7.8.13) with IC50 values of 0.024, 0.038, 0.135 and 17.9 microM, respectively.

Synthesis and antimycobacterial activity of capuramycin analogues. Part 1: substitution of the azepan-2-one moiety of capuramycin.

Capuramycin analogues with a variety of substituents in place of the azepan-2-one moiety were synthesized from A-500359E and were tested for their translocase I inhibitory activity and in vitro antimycobacterial activity. Phenyl-type moieties were found to be effective substituents for capuramycin analogues.

Synthesis and antimycobacterial activity of capuramycin analogues. Part 2: acylated derivatives of capuramycin-related compounds.

Acylated derivatives of capuramycin and A-500359A were synthesized and tested for antimycobacterial activity. Compound 20 having a decanoyl group showed very potent activity.

Synthesis and antimicrobial activity of novel globomycin analogues.

Globomycin, a signal peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. One of the analogues showed a more potent activity against Gram-negative bacteria than globomycin and also exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Studies on novel bacterial translocase I inhibitors, A-500359s. I. Taxonomy, fermentation, isolation, physico-chemical properties and structure elucidation of A-500359 A, C, D and G.

In the course of our screening for bacterial phospho-N-acetylmuramyl-pentapeptide-translocase (translocase I: EC 2.7.8.13) inhibitors, we found inhibitory activity in the cultured broth of the strain identified as Streptomyces griseus SANK 60196. The strain produced capuramycin and four novel capuramycin derivatives designated as A-500359 A, C, D and G. Purification and structural analysis were performed, and the structures of A-500359 A, C, D and G were elucidated as 6'''-methylcapuramycin, 3'-demethyl-6'''-methylcapuramycin, 2''-deoxy-6'''-methylcapuramycin and 3'-demethylcapuramycin, respectively.

Studies on novel bacterial translocase I inhibitors, A-500359s. III. Deaminocaprolactam derivatives of capuramycin: A-500359 E, F, H; M-1 and M-2.

Novel derivatives of capuramycin were obtained when 10 mM of 2-aminoethyl-L-cysteine (AEC), an inhibitor of aspartokinase, was added to the culture of Streptomyces griseus SANK 60196, the producer of A-500359. They were purified from the culture filtrate and their chemical structures were elucidated as a deaminocaprolactam derivative of capuramycin designated as A-500359 F, A-500359 E, a methyl ester of A-500359 F, and A-500359 H, a 3'-demethyl derivative of A-500359 F. Two other compounds, A-500359 M-1 and A-500359 M-2, were purified from the same medium and their structures were elucidated. A-500359 E, F, H, M-1 and M-2 inhibited bacterial translocase I with an IC50 of 0.027 microM, 1.1 microM, 0.008 microM, 0.058 microM and 0.010 microM, respectively. A-500359 E, M-1 and M-2 inhibited the growth of mycobacteria as well.

Studies on novel bacterial translocase I inhibitors, A-500359s. II. Biological activities of A-500359 A, C, D and G.

A-500359 A, C, D, G and capuramycin inhibited bacterial phospho-N-acetylmuramyl-pentapeptide-translocase (translocase I: EC 2.7.8.13) with IC50 values of 0.017, 0.12, 0.53, 0.14 and 0.018 microM, respectively. Consistently, A-500359 A, C and capuramycin inhibited in vitro peptidoglycan biosynthesis. A-500359 A exhibited reversible inhibition, which was mixed type and noncompetitive with respect to UDP-MurNAc-(N(epsilon)-Dns)pentapeptide (Ki=0.0079 microM) and undecaprenyl-phosphate, respectively. A-500359 A, C, D and G showed antimicrobial activity against Mycobacterium smegmatis. As a single intravenous injection of A-500359 A at a dose of 500 mg/kg showed no toxicity in mice, it was suggested that the capuramycin derivatives might become candidates as novel therapeutic agents for various diseases caused by Mycobacteria including tuberculosis.

Studies on novel bacterial translocase I inhibitors, A-500359s. IV. Biosynthesis of A-500359s.

This report describes the isolation of novel A-500359 analogues from the culture broth of Streptomyces griseus SANK 60196 and 13C-incorporation studies of A-500359 A to reveal the biosynthetic pathway of A-500359 derivatives. As a result, A-500359 M-3 and J were isolated as novel analogues. The former, isolated from a culture broth fed with unnatural amino acids, was a novel amino acid adduct of A-500359, and the latter was found to be a putative precursor of all A-500359 derivatives, on the basis of the structure. Moreover, 13C-incorporation studies revealed the origin of every carbon atom of A-500359 A. From these results, it was revealed that the core skeleton of A-500359 was biosynthesized from uridine and phosphoenolpyruvate in the same manner as for polyoxin, a nucleoside antibiotic. Moreover, the uronic acid and aminocaprolactam moiety was derived from hexose and lysine, respectively, and two methyl groups of A-500359 A were derived from methionine.

Arborcandins A, B, C, D, E and F, novel 1,3-beta-glucan synthase inhibitors: physico-chemical properties and structure elucidation.

Arborcandins A, B, C, D, E and F, which possess potent 1,3-beta-glucan synthase inhibitory activity, were isolated from the cultured broth of a filamentous fungus, strain SANK 17397. The structures of arborcandins A, B, C, D, E and F were elucidated by a combination of NMR and mass spectrometry, and established to be novel cyclic peptides containing uncommon amino acid residues.