RESUMO
We screened for bacterial phospho-N-acetylmuramyl-pentapeptide-translocase (MraY: EC 2.7.8.13) inhibitors with the aim of discovering novel antibiotics and observed inhibitory activity in the culture broth of an actinomycete, SANK 60501. The active compounds, muraminomicins A, B, C, D, E1, E2, F, G, H, and I exhibited strong inhibitory activity against MraY with IC50 values of 0.0105, 0.0068, 0.0104, 0.0099, 0.0115, 0.0109, 0.0089, 0.0134, 0.0186, and 0.0094 µg ml-1, respectively. Although muraminomicin F exhibited favorable antibacterial activity against drug-resistant Gram-positive bacteria, this activity was reduced with the addition of serum. To efficiently supply the core component for chemical modification studies, production was carried out in a controlled trial by adding myristic acid to the medium, and a purification method suitable for large-scale production was successfully developed.
Assuntos
Actinomycetales/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Actinomycetales/genética , Antibacterianos/biossíntese , Proteínas de Bactérias/antagonistas & inibidores , Ácidos Graxos/química , Fermentação , Bactérias Gram-Positivas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Transferases/antagonistas & inibidores , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
Cinnamaldehyde is stereospecifically converted to (2S,3R) 5-phenylpent-4-ene-2,3-diol, an important starting material for the synthesis of biologically active compounds, by the budding yeast Saccharomyces cerevisiae. Immobilization of the yeast in calcium alginate capsules suppressed the formation of by-products and increased accumulation of the diol compounds. The mechanism of cinnamaldehyde conversion was investigated by using recombinant strains of Escherichia coli and S. cerevisiae carrying the pyruvate decarboxylase gene PDC1. As a result, condensation of the substrate with acetaldehyde was enhanced by PDC and flow to the diol product was altered.
Assuntos
Acroleína/análogos & derivados , Álcoois/metabolismo , Piruvato Descarboxilase/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Acetaldeído/metabolismo , Acroleína/química , Acroleína/metabolismo , Álcoois/química , Alginatos , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Glucurônico , Ácidos Hexurônicos , Piruvato Descarboxilase/genética , Saccharomyces cerevisiae/genéticaRESUMO
We report here that flavonol (3-hydroxyflavone) was O-glucosylated efficiently by filamentous fungus Cunninghamella echinulata. Kaemferol and some other flavonols were also glucosylated. The novel conversion is expected to be applicable to prepare glycosylated flavonoids which are commonly found in plants and mammalian metabolites of related compounds.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Flavonóis/biossíntese , Cunninghamella , Macrolídeos/metabolismoRESUMO
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.
Assuntos
Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Produtos Biológicos/farmacologia , Peptidoglicano/biossíntese , Bactérias/genética , Produtos Biológicos/química , Sequência de Carboidratos , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Ciclosserina/farmacologia , Escherichia coli/metabolismo , Fermentação , Fluorescência , Conformação Molecular , Dados de Sequência Molecular , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/metabolismo , Peptidoglicano/química , Plasmídeos/genética , Espectrometria de Fluorescência , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
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.
Assuntos
Antibacterianos/isolamento & purificação , Bactérias/enzimologia , Dissacarídeos/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Nucleotídeos de Pirimidina/isolamento & purificação , Streptomyces/classificação , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Antibacterianos/farmacologia , Dissacarídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Nucleotídeos de Pirimidina/farmacologia , Streptomyces/metabolismo , Tunicamicina/farmacologiaRESUMO
In our screening for translocase I inhibitors, we found the novel nucleoside antibiotic, A-94964 in the culture broth of Streptomyces sp. SANK 60404. The structure of A-94964 was elucidated primarily by various NMR studies, including a 1H-31P HMBC experiment. A-94964 has a unique structure which possesses a nucleoside moiety and an N-acylglucosamine moiety connected via a phosphate.
Assuntos
Antibacterianos/química , Bactérias/enzimologia , Dissacarídeos/química , Nucleotídeos de Pirimidina/química , Streptomyces/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Espectroscopia de Ressonância MagnéticaRESUMO
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.
Assuntos
Aminoglicosídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Positivas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Aminoglicosídeos/química , Fenômenos Químicos , Físico-Química , Meios de Cultura/química , Fermentação , Bactérias Gram-Positivas/classificação , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Espectrofotometria UltravioletaRESUMO
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.
Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Inibidores Enzimáticos/metabolismo , Nucleosídeos/biossíntese , Streptomyces griseus/metabolismo , Cobalto/metabolismo , Meios de Cultura/química , Glucose/metabolismo , Maltose/metabolismoRESUMO
Candida albicans ERG3 encodes a sterol C5,6-desaturase which is essential for synthesis of ergosterol. Defective sterol C5,6 desaturation has been considered to be one of the azole resistance mechanisms in this species. However, the clinical relevance of this resistance mechanism is still unclear. In this study, we created a C. albicans erg3/erg3 mutant by the "Ura-blaster" method and confirmed the expected azole resistance using standard in vitro testing and the presence of ergosta-7,22-dien-3beta-ol instead of ergosterol. For in vivo studies, a wild-type URA3 was placed back into its native locus in the erg3 homozygote to avoid positional effects on URA3 expression. Defective hyphal formation of the erg3 homozygote was observed not only in vitro but in kidney tissues. A marked attenuation of virulence was shown by the longer survival and the lower kidney burdens of mice inoculated with the reconstituted Ura+ erg3 homozygote relative to the control. To assess fluconazole efficacy in a murine model of disseminated candidiasis, inoculum sizes of the control and the erg3 homozygote were chosen which provided a similar organ burden. Under these conditions, fluconazole was highly effective in reducing the organ burden in both groups. This study demonstrates that an ERG3 mutation causing inactivation of sterol C5,6-desaturase cannot confer fluconazole resistance in vivo by itself regardless of resistance measured by standard in vitro testing. The finding questions the clinical significance of this resistance mechanism.
Assuntos
Antifúngicos/uso terapêutico , Candidíase/tratamento farmacológico , Fluconazol/uso terapêutico , Oxirredutases/fisiologia , Animais , Candida albicans/química , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/patogenicidade , Candidíase/patologia , Farmacorresistência Fúngica , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Mutação , Oxirredutases/genética , Esteróis/análise , VirulênciaRESUMO
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.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos/química , Bioquímica/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
Arborcandin C is a novel antibiotic with potent antifungal activity that inhibits 1,3-beta-glucan synthase in fungi. We examined spontaneous Saccharomyces cerevisiae mutants which are selectively resistant to arborcandin C and revealed that a single amino acid replacement in Fks1p of Asn(470) with Lys or of Leu(642) with Ser confers selective resistance on Fks1p mutants.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/genética , Proteínas de Membrana/genética , Peptídeos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Schizosaccharomyces pombe , Sequência de Aminoácidos , DNA Fúngico/genética , Farmacorresistência Fúngica , Equinocandinas , Glicopeptídeos , Dados de Sequência Molecular , Mutação/genética , Plasmídeos/genética , Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Antibacterianos/isolamento & purificação , Azepinas/isolamento & purificação , Bactérias/enzimologia , Inibidores Enzimáticos/isolamento & purificação , Streptomyces/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Uridina/análogos & derivados , Uridina/isolamento & purificação , Antibacterianos/química , Antibacterianos/farmacologia , Azepinas/química , Azepinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Streptomyces/classificação , Uridina/química , Uridina/farmacologiaRESUMO
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.
Assuntos
Aminoglicosídeos/síntese química , Aminoglicosídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Azepinas/química , Azepinas/farmacologia , Aminoglicosídeos/química , Antibacterianos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Relação Estrutura-Atividade , Transferases/antagonistas & inibidoresRESUMO
Acylated derivatives of capuramycin and A-500359A were synthesized and tested for antimycobacterial activity. Compound 20 having a decanoyl group showed very potent activity.
Assuntos
Aminoglicosídeos/síntese química , Aminoglicosídeos/farmacologia , Antibacterianos/síntese química , Antibacterianos/farmacologia , Acilação , Aminoglicosídeos/química , Antibacterianos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Isomerismo , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Transferases/antagonistas & inibidoresRESUMO
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).
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Indicadores e Reagentes , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Aminoglicosídeos , Antibacterianos/química , Antibacterianos/farmacologia , Azepinas/farmacologia , Bactérias/enzimologia , Inibidores Enzimáticos/farmacologia , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Uridina/análogos & derivados , Azepinas/química , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/química , Estrutura Molecular , Espectrofotometria Ultravioleta , Uridina/química , Uridina/farmacologiaRESUMO
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.
Assuntos
Aminoglicosídeos , Antibacterianos/biossíntese , Azepinas/metabolismo , Inibidores Enzimáticos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores , Uridina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacologia , Azepinas/química , Azepinas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fermentação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Uridina/biossíntese , Uridina/química , Uridina/farmacologiaRESUMO
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.