RESUMO
We previously studied 2-aryl-2-(3-indolyl)acetohydroxamates as potential agents against melanoma. These compounds were ineffective in a mouse melanoma xenograft model, most likely due to unfavorable metabolic properties, specifically due to glucuronidation of the N-hydroxyl of the hydoxamic moiety. In the present work, we prepared a series of analogues, 2-aryl-2-(3-indolyl)acetamides and their oxazoline derivatives, which do not contain the N-hydroxyl group. We investigated the structure-activity relationship in both series of compounds and found that the 2-naphthyl is a preferred group at C-2 of the indole in the amide series, whereas the tetralin moiety is favorable in the same location in the oxazoline series. Overall, three compounds in the amide series have GI50 values as low as 0.2-0.3 µM and the results clearly indicate that the N-hydroxyl group is not necessary for high potency in vitro.
Assuntos
Antineoplásicos , Melanoma , Humanos , Animais , Camundongos , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Caffeic acid phenethyl ester (CAPE) is a phenolic natural product with a wide range of biological activities, including anticancer activity; however, the ester group of CAPE is metabolically labile. The corresponding amide, CAPA, has improved metabolic stability but limited anticancer activity relative to CAPE. We report the synthesis using flow and on-water Wittig reaction approaches of five previously reported and five novel CAPA analogues. All of these analogues lack the reactive catechol functionality of CAPA and CAPE. Cytotoxicity studies of CAPE, CAPA, and these CAPA analogues in HeLa and BE(2)-C cells were carried out. Surprisingly, we found that CAPA is cytotoxic against the neuroblastoma BE(2)-C cell line (IC50 = 12 µM), in contrast to the weak activity of CAPA against HeLa cells (IC50 = 112 µM), and the literature reports of the absence of activity for CAPA against a variety of other cancer cell lines. One novel CAPA analogue, 3f, was identified as having cytotoxic activity similar to CAPE in HeLa cells (IC50 = 63 µM for 3f vs. 32 µM for CAPE), albeit with lower activity against BE(2)-C cells (IC50 = 91 µM) than CAPA. A different CAPA analogue, 3g, was found to have similar effects against BE(2)-C cells (IC50 = 92 µM). These results show that CAPA is uniquely active against neuroblastoma cells and that specific CAPA analogues that are predicted to be more metabolically stable than CAPE can reproduce CAPA's activity against neuroblastoma cells and CAPE's activity against HeLa cells.
Assuntos
Antineoplásicos , Ácidos Cafeicos , Álcool Feniletílico , Humanos , Ácidos Cafeicos/farmacologia , Ácidos Cafeicos/química , Ácidos Cafeicos/síntese química , Células HeLa , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/farmacologia , Álcool Feniletílico/química , Álcool Feniletílico/síntese química , Água/química , Linhagem Celular Tumoral , Amidas/farmacologia , Amidas/química , Sobrevivência Celular/efeitos dos fármacosRESUMO
BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.
Assuntos
Indústria Farmacêutica , Medicamentos de Ervas Chinesas , Flavonoides , Microbiologia Industrial , Catálise , Medicamentos de Ervas Chinesas/síntese química , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Escherichia coli/genética , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Sphingomonadaceae/enzimologia , Sphingomonadaceae/genética , Paenibacillus/enzimologia , Paenibacillus/genética , Microbiologia Industrial/métodos , Indústria Farmacêutica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Flavonoides/biossíntese , HidróliseRESUMO
Previously, we developed an innovative high-content screening (HCS) approach to quantify neuroblastoma cell differentiation based on neurite outgrowth, a morphological differentiation marker of neuroblastoma cells. Here, we report the utilization of this platform to identify 1-methyl-5-(ethylsulfonyl)-1H-tetrazole (3a) as a new neuroblastoma differentiation agent using the ChemBridge DiversetTM commercial synthetic small molecule compound library. We show that this activity can be extended to a group of analogues, which can be accessed via a short two-step synthetic sequence. A new analogue, 5-(allylsulfonyl)-1-methyl-1H-tetrazole (3c) was identified in this synthetic effort as a compound that has even more pronounced differentiation and cytotoxic activities than the original hit compound 3a.
Assuntos
Antineoplásicos , Neuroblastoma , Humanos , Neuroblastoma/tratamento farmacológico , Antineoplásicos/farmacologia , Diferenciação Celular , Biblioteca Gênica , Crescimento NeuronalRESUMO
microRNA mimics are synthetic RNA molecules that imitate the mature miRNA duplexes and their functions. These mimics have shown promise in treating cancers. Nucleotide chemical modifications of microRNA mimics have been investigated and have improved the stability of miRNA mimics. However, the potential therapeutic benefit of mimic analogs based on sequence modifications has not been explored. miR-506-3p was identified as a differentiation-inducing microRNA in neuroblastoma cells, suggesting the potential of applying the miR-506-3p mimic in neuroblastoma differentiation therapy. In this study, we explored the possibility of developing shortened miR-506-3p analogs that can maintain differentiation-inducing activities comparable to the wild-type miR-506-3p mimic. We found that deleting up to two nucleotides at either the 3' end or within the middle region of the miR-506-3p sequence fully maintained the differentiation-inducing activity when compared to the wild-type mimic. Deleting up to four nucleotides from the 3' end or deleting three nucleotides in the middle positions diminished the differentiation-inducing activity, but the analogs still maintained differentiation-inducing activities that were significantly higher than the negative control oligo. The shortened analog designs potentially benefit patients from two perspectives: (1) the reduced cost of manufacturing shortened analogs, and (2) the reduced non-specific toxicity due to their smaller molecular sizes.
Assuntos
MicroRNAs , Células-Tronco Neurais , Neuroblastoma , Humanos , MicroRNAs/genética , Diferenciação Celular , Neuroblastoma/genética , NucleotídeosRESUMO
A 15-step chemoenzymatic total synthesis of C-1 methoxycarbonyl narciclasine (10) was accomplished. The synthesis began with the toluene dioxygenase-mediated dihydroxylation of ortho-dibromobenzene to provide the corresponding cis-dihydrodiol (12) as a single enantiomer. Further key steps included a nitroso Diels-Alder reaction and an intramolecular Heck cyclization. The C-1 homolog 10 was tested and evaluated for antiproliferative activity against natural narciclasine (1) as the positive control. Experimental and spectral data are reported for all novel compounds.
Assuntos
Alcaloides de Amaryllidaceae , Alcaloides de Amaryllidaceae/farmacologia , Ciclização , Estrutura Molecular , Fenantridinas/farmacologia , EstereoisomerismoRESUMO
Resveratrol, an ingredient of traditional Chinese medicine, has beneficial effects on human health and huge potential for application in modern medicine. Polydatin is extracted from plants and then deglycosylated into resveratrol; enzymatic methods are preferred for this reaction. In this study, a ß-D-glucosidase from Sphingomonas showed high efficiency in transforming polydatin into resveratrol and was tolerant toward organic solvents. Applying this enzyme in a biphasic transformation system resulted in 95.3% conversion of 20% concentration crude polydatin to resveratrol in 4 h. We thus report a new method for high-efficiency, clean production of resveratrol.
Assuntos
Glucosídeos , EstilbenosRESUMO
During the search for a general, efficient route toward the synthesis of C-1 analogues of narciclasine, natural narciclasine was protected and converted to its C-1 enol derivative using a novel semi-synthetic route. Attempted conversion of this material to its triflate in order to conduct cross-coupling at C-1 resulted in a triflate at C-6 that was successfully coupled with several functionalities. Four novel compounds were fully deprotected after seven steps and subjected to evaluation for cytotoxic activity against three cancer cell lines. Only one derivative showed moderate activity compared to that of narciclasine. Spectral and physical data are provided for all new compounds.
Assuntos
Alcaloides de Amaryllidaceae , Antineoplásicos , Neoplasias , Alcaloides de Amaryllidaceae/química , Antineoplásicos/química , Humanos , Fenantridinas/químicaRESUMO
Plant polyphenols, such as the African potato (Hypoxis hemerocallidea)-derived bis-catechol rooperol, can display promising anticancer activity yet suffer from rapid metabolism. Embarking upon a program to systematically examine potentially more metabolically stable replacements for the catechol rings in rooperol, we report here a general, scalable synthesis of rooperol and analogues that builds on our previous synthetic approach incorporating a key Pd-catalyzed decarboxylative coupling strategy. Using this approach, we have prepared and evaluated the cancer cell cytotoxicity of rooperol and a series of analogues. While none of the analogues examined here were superior to rooperol in preventing the growth of cancer cells, analogues containing phenol or methylenedioxyphenyl replacements for one or both catechol rings were nearly as effective as rooperol.
Assuntos
Catecóis , Neoplasias , Catecóis/farmacologia , Neoplasias/tratamento farmacológico , Fenol , Fenóis , PolifenóisRESUMO
Indolizines and pyrazolo[1,5-a]pyridines were prepared via [3 + 2]-cycloaddition of pyridinium ylides to 1-chloro-2-nitrostyrenes. The synthesized molecules were evaluated for antiproliferative activities against a BE(2)-C neuroblastoma cell line with several compounds decreasing the viability of cancer cells. Indolizine 9db showed higher potency than that of all-trans-retinoic acid, an approved cancer drug. Mechanistically, it was found to inhibit tubulin polymerization and it is thus proposed that the discovered chemistry can be exploited for the development of novel microtubule-targeting anticancer agents.
Assuntos
Moduladores de TubulinaRESUMO
A chemoenzymatic convergent synthesis of 10-benzyloxy narciclasine from bromobenzene was accomplished in 16 steps. The key transformations included toluene dioxygenase-mediated hydroxylation, nitroso Diels-Alder reaction and intramolecular Heck cyclization. The unnatural derivative of narciclasine was subjected to biological evaluation and its activity was compared to other C-10 and C-7 compounds prepared previously.
RESUMO
We discovered a reaction of nitroalkanes with 2-hydrazinylquinolines, 2-hydrazinylpyridines and bis-2,4-dihydrazinylpyrimidines in polyphosphoric acid (PPA) affording 1,2,4-triazolo[4,3-a]quinolines, 1,2,4-triazolo[4,3-a]pyridines and bis[1,2,4]triazolo[4,3-a:4',3'-c]pyrimidines, respectively. The reaction expands the scope of heterocyclic annulations involving phosphorylated nitronates, believed to be the electrophilic intermediates formed from nitroalkanes in PPA. Several of the synthesized triazoles showed promising anticancer activity by inducing differentiation in neuroblastoma cancer cells. Due to the urgent need for novel differentiation agents for neuroblastoma therapy, this finding warrants further evaluation of this class of compounds against neuroblastoma.
RESUMO
An acid-assisted [4 + 1]-cycloaddition of indoles with nitrostyrenes affords 4' H-spiro[indole-3,5'-isoxazoles] in a diastereomerically pure form. Several of these spirocyclic molecules exhibit promising anticancer activity by reducing viability and inducing differentiation of neuroblastoma cells.
Assuntos
Alcenos/química , Antineoplásicos/farmacologia , Indóis/farmacologia , Isoxazóis/farmacologia , Nitrocompostos/química , Compostos de Espiro/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclização , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/síntese química , Indóis/química , Isoxazóis/síntese química , Isoxazóis/química , Estrutura Molecular , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
A convenient and effective sucrose transport assay for Clostridium strains is needed. Traditional methods, such as 14C-sucrose isotope labelling, use radioactive materials and are not convenient for many laboratories. Here, a sucrose transporter from potato was introduced into Clostridium, and a fluorescence assay based on esculin was used for the analysis of sucrose transport in Clostridium strains. This showed that the heterologously expressed potato sucrose transporter is functional in Clostridium. Recombinant engineering of high-level sucrose transport would aid sucrose fermentation in Clostridium strains. The assay described herein provides an important technological platform for studying sucrose transporter function following heterologous expression in Clostridium.
Assuntos
Bioensaio , Clostridium/genética , Clostridium/metabolismo , Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Plantas/genética , Solanum tuberosum/metabolismo , Sacarose/metabolismo , Transporte Biológico , Fluorescência , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Metastatic disease is a primary cause of cancer-related death, and factors governing tumor cell metastasis have not been fully elucidated. Here, we address this question by using tumor cell lines derived from mice that develop metastatic lung adenocarcinoma owing to expression of mutant K-ras and p53. Despite having widespread somatic genetic alterations, the metastasis-prone tumor cells retained a marked plasticity. They transited reversibly between epithelial and mesenchymal states, forming highly polarized epithelial spheres in three-dimensional culture that underwent epithelial-to-mesenchymal transition (EMT) following treatment with transforming growth factor-beta or injection into syngeneic mice. This transition was entirely dependent on the microRNA (miR)-200 family, which decreased during EMT. Forced expression of miR-200 abrogated the capacity of these tumor cells to undergo EMT, invade, and metastasize, and conferred transcriptional features of metastasis-incompetent tumor cells. We conclude that tumor cell metastasis is regulated by miR-200 expression, which changes in response to contextual extracellular cues.
Assuntos
Espaço Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Metástase Neoplásica/fisiopatologia , Adenocarcinoma/fisiopatologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Neoplasias Pulmonares/fisiopatologia , Camundongos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Objective: The aim of this study was to characterize ß-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification. Mehods: A bacterial strain with ß-glucosidase activity was screened from the soil around Wuming sugar mill in Guangxi by esculin-ferric ammonium citrate selecting plate. The 16S rDNA of the strain was obtained and analyzed. By searching GenBank database, the genes encoding ß-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding ß-glucosidase was amplified by PCR. The recombinant plasmid pQE-cbgl was constructed. The recombinant protein was purified with Ni-NTA. The enzyme properties of the recombinant protein CBGL were studied in detail. At last, the wild enzyme CBGL was reformed by error-prone PCR and site-directed random mutagenesis. Results: C. koser GXW-1 with ß-glucosidase activity was isolated from the soil. A gene encoding ß-glucosidase was cloned from the wild strain GXW-1. The properties of CBGL were identified. Its optimal pH and temperature were 6.0 and 45â. Its Km and Vmax value were (11.280±1.073) mmol/L and (0.1704±0.0073) µmol/(mg·min), respectively. Its Ki values was (66.84±3.40) mmol/L. CBGL can hydrolyze α-pNPG, stevioside, daidzin and genistin. CBGL was modified by error-prone PCR and site directed random mutagenesis. A positive mutant W147F was obtained successfully. Its Vmax was 2.54 times that of the wild enzyme CBGL. Conclusion: CBGL not only can hydrolyze ß-glycosidic bond, but also can hydrolyze the α-glycosidic bond in α-pNPG. Furthermore, CBGL can hydrolyze stevioside, daidzin and genistin. These characteristics indicate that the ß-glucosidase CBGL has important applications in theoretical research and in industry.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Citrobacter/enzimologia , beta-Glucosidase/química , beta-Glucosidase/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Citrobacter/genética , Citrobacter/isolamento & purificação , Citrobacter/metabolismo , Diterpenos do Tipo Caurano/metabolismo , Estabilidade Enzimática , Glucosídeos/metabolismo , Concentração de Íons de Hidrogênio , Isoflavonas/metabolismo , Cinética , Filogenia , Microbiologia do Solo , Especificidade por Substrato , Temperatura , beta-Glucosidase/isolamento & purificação , beta-Glucosidase/metabolismoRESUMO
microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms--inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Diferenciação Celular/genética , Genes Supressores de Tumor , MicroRNAs/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Regiões 3' não Traduzidas/genética , Apoptose/genética , Sequência de Bases , Proliferação de Células , Sobrevivência Celular/genética , Quinase 6 Dependente de Ciclina/metabolismo , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , MicroRNAs/genética , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Análise de SobrevidaRESUMO
In order to produce rubusoside, enzymes with preferential specificity for the saccharide sophorose were tested for ability to produce rubusoside from stevioside. We identified BGL1, a ß-glucosidase from Streptomyces sp. GXT6, as an enzyme for rubusoside production. Out of several saccharide substrates, BGL1 showed the most affinity to sophorose. This enzyme only hydrolyzes the glucose moiety of the sophoroside at C-13 in stevioside. Production of rubusoside was determined by (1)H and (13)C nuclear magnetic resonance (NMR). Thus, rubusoside was produced from stevioside and the stevioside conversion rate was 98.2 %. The production yield of rubusoside was 78.8 % in 6 h.
Assuntos
Diterpenos do Tipo Caurano/biossíntese , Diterpenos do Tipo Caurano/metabolismo , Glucanos/metabolismo , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Streptomyces/enzimologia , beta-Glucosidase/metabolismo , Espectroscopia de Ressonância MagnéticaRESUMO
OBJECTIVE: To characterize a neutral invertase from Enterobacter cloacae GX-3. METHODS: By searching GenBank database, we found the genes encoding invertase from the same genus Enterobacter. These sequences were aligned and analyzed. Then, a gene encoding neutral invertase was amplified by PCR. The recombinant plasmid pQE-Einv was constructed. We purified the expressed protein Einv with nickel-nitrilotriacetic acid chromatography. At last, the characterics of the recombinant protein Einv were studied in detail. RESULTS: A gene encoding neutral invertase was discovered and cloned from E. cloacae GX-3. The recombinant enzyme Einv was characterized. Einv had an optimum pH of 6.5 and an optimum temperature of 40 degrees C. The results of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and gel permeation chromatography ( GPC) showed that Einv was a homo-dimer protein. Einv retained 80% activity at sucrose concentrations up to 1170 mmol/L. But, Einv had no transglycosylation activity at high sucrose concentration. It could hydrolyze raffinose, 1-kestose, nystose, fructofuranosylnystose and stachyose. CONCLUSION: It is first reported that an invertase from Enterobacter cloacae is a beta-fructofuranosidase at neutral pH range. It only has hydrolysis activity without tranglycosylation activity. These characteristics indicate that the neutral invertase Einv has important applications in food industry.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Enterobacter cloacae/enzimologia , beta-Frutofuranosidase/química , beta-Frutofuranosidase/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Enterobacter cloacae/química , Enterobacter cloacae/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Especificidade por Substrato , Temperatura , beta-Frutofuranosidase/isolamento & purificação , beta-Frutofuranosidase/metabolismoRESUMO
In view of the important role of isoflavonoids and their related glycoconjugates in human health, there is considerable interest in their enzymatic conversion. SBGL, a novel ß-glucosidase isolated from Novosphingobium sp. GX9, was expressed in Escherichia coli and found to have high activity for hydrolysis of daidzin and genistin. SBGL showed very low K m values for daidzin and genistin, and the k cat/K m values for these substrates were 33,300 and 19,200 s(-1) mM(-1), respectively. The SBGL glucosidase could also transglycosylate the flavanol (+)-catechin at saturating acceptor concentrations, which has not previously been reported for a ß-glucosidase and is difficult to achieve synthetically.