RESUMO
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.
Assuntos
Actinomycetales/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Imunossupressores/metabolismo , Peptídeos Cíclicos/metabolismo , Actinomycetales/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Hidroxilação , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Streptomyces/genéticaRESUMO
Excessive hepatic glucose production through the gluconeogenesis pathway is partially responsible for the elevated glucose levels observed in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box O1 (Foxo1) plays a crucial role in mediating the effect of insulin on hepatic gluconeogenesis. Here, using a db/db mouse model, we demonstrate the effectiveness of Foxo1 inhibitor, an orally active small-molecule compound, as a therapeutic drug for treating T2DM. Using mass spectrometric affinity screening, we discovered a series of compounds that bind to Foxo1, identifying among them the compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), which potently inhibits human Foxo1 transactivation and reduces glucose production through the inhibition of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase mRNA levels in a rat hepatic cell line. Oral administration of AS1842856 to diabetic db/db mice led to a drastic decrease in fasting plasma glucose level via the inhibition of hepatic gluconeogenic genes, whereas administration to normal mice had no effect on the fasting plasma glucose level. Treatment with AS1842856 also suppressed an increase in plasma glucose level caused by pyruvate injection in both normal and db/db mice. Taken together, these findings indicate that the Foxo1 inhibitor represents a new class of drugs for use in treating T2DM.
Assuntos
Fatores de Transcrição Forkhead/antagonistas & inibidores , Hiperglicemia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Quinolonas/farmacologia , Animais , Linhagem Celular Tumoral , Jejum , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Glucose/biossíntese , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/genética , Humanos , Hiperglicemia/metabolismo , Hipoglicemiantes/uso terapêutico , Masculino , Espectrometria de Massas , Camundongos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (GTP)/antagonistas & inibidores , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Ácido Pirúvico/farmacologia , Quinolonas/uso terapêutico , RNA Mensageiro/antagonistas & inibidores , Ratos , Relação Estrutura-Atividade , Ativação TranscricionalRESUMO
FR225659 was originally isolated as a novel gluconeogenesis inhibitor produced by fungal strain Helicomyces sp. No. 19353. To identify the target protein of FR225659, we synthesized high-performance affinity latex beads that immobilized FR225659 derivative FR253761 or FR259383. Using these beads, we identified FR225659 binding proteins as serine/threonine protein phosphatase type1 (PP1) and type2A (PP2A) from rat hepatocyte crude extract. FR225659 and its synthetic derivatives were strongly inhibited the enzyme activities of purified catalytic subunits of PP1 and PP2A in vitro.
Assuntos
Proteínas de Transporte/isolamento & purificação , Oligopeptídeos/metabolismo , Fosfoproteínas Fosfatases/isolamento & purificação , Animais , Proteínas de Transporte/metabolismo , Gluconeogênese , Masculino , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Ratos , Ratos WistarRESUMO
A new HIV-inhibitory cyclic depsipeptide, neamphamide A (2), was isolated from a Papua New Guinea collection of the marine sponge Neamphius huxleyi. Its structure was established through interpretation of spectroscopic data and by acid hydrolysis, derivatization of the free amino acids, and LC-MS analysis of the derivatives. Neamphamide A (2) contains 11 amino acid residues and an amide-linked 3-hydroxy-2,4,6-trimethylheptanoic acid moiety. The amino acid constituents were identified as L-Leu, L-NMeGln, D-Arg, D- and L-Asn, two residues of D-allo-Thr, L-homoproline, (3S,4R)-3,4-dimethyl-L-glutamine, beta-methoxytyrosine, and 4-amino-7-guanidino-2,3-dihydroxyheptanoic acid. In a cell-based XTT assay, 2 exhibited potent cytoprotective activity against HIV-1 infection with an EC50 of approximately 28 nM.
Assuntos
Fármacos Anti-HIV/isolamento & purificação , Depsipeptídeos , HIV-1/efeitos dos fármacos , Peptídeos Cíclicos/isolamento & purificação , Poríferos/química , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Estrutura Molecular , Papua Nova Guiné , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , EstereoisomerismoRESUMO
During the course of screening for novel gluconeogenesis inhibitors, FR225659 and its related compounds were isolated from a fermentation broth of Helicomyces sp. No. 19353. Spectroscopic analysis concluded that FR225659 is an N-acyl tripeptide consisting of a novel acyl, a 3-chloro-4-hydroxyarginine, a 3-hydroxy-3-methylproline and a dehydrovaline. Degradation study allowed assignment of the absolute configuration of the 3-hydroxy-3-methylproline to be (2S,3R). FR225656 was shown to possess a dehydroisoleucine instead of the dehydrovaline of FR225659.