RESUMO
The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2-depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2-podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2-expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 µM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.
Assuntos
Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Metaloporfirinas/farmacologia , Animais , Sítios de Ligação , Neoplasias Pulmonares/complicações , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Metaloporfirinas/uso terapêutico , Camundongos , Simulação de Acoplamento Molecular , Metástase Neoplásica/tratamento farmacológico , Agregação Plaquetária/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Trombose/tratamento farmacológicoRESUMO
GPBA is a G protein-coupled receptor that is activated by bile acids. Because activation of GPBA leads to increased cAMP levels and secretion of incretins and insulin, GPBA has been proposed as a promising drug target for the treatment of metabolic syndrome. Previously, we have developed a ligand-screening system to identify novel agonists of GPBA by means of a fusion protein of GPBA with G protein stimulatory α subunit (Gsα) and by a [35S]GTPγS-binding assay. To express the GPBA-Gsα fusion protein, transgenic silkworms were employed in this study, and cell membrane fractions were prepared from their fat body or pupae. We applied them to the screening of a chemical library that contains 10,625 compounds from the RIKEN Natural Products Depository (NPDepo). Eventually, a unique partial agonist, GUM2, was successfully identified. Our results indicated that the GPCR-Gα fusion proteins were beneficial for ligand identification and that the transgenic silkworms were useful for large-scale production of GPCRs. In HEK293 cells transiently expressing GPBA, GUM2 showed 50% effective concentration (EC50) of 3.5 ± 2.4µM and induced GPBA internalization as effectively as did an endogenous agonist, TLC. We also confirmed that GUM2 stimulates insulin secretion in MIN6 cells. Moreover, a single 2mg/kg dose of GUM2 significantly reduced blood glucose levels in mice during an intraperitoneal glucose tolerance test even though GUM2 is only a partial agonist with a low intrinsic activity. We concluded that GUM2 is a good candidate for research on GPBA signaling under physiological conditions and for the development of GPBA-targeting therapeutic compounds.
Assuntos
Produtos Biológicos/farmacologia , Glicemia/metabolismo , Teste de Tolerância a Glucose , Receptores Acoplados a Proteínas G/agonistas , Animais , Células HEK293 , Humanos , Insulina/metabolismo , Secreção de Insulina , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Larva/metabolismo , Camundongos , Pupa/metabolismoRESUMO
We established a facile access to an unexplored mirror-image library of chiral natural product derivatives using d-protein technology. In this process, two chemical syntheses of mirror-image substances including a target protein and hit compound(s) allow the lead discovery from a virtual mirror-image library without the synthesis of numerous mirror-image compounds.
Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Modelos Moleculares , Conformação Proteica , Estereoisomerismo , Interface Usuário-ComputadorRESUMO
The identification of novel synthetic ligands for G protein-coupled receptors (GPCRs) is important not only for understanding human physiology, but also for the development of novel drugs, especially for orphan GPCRs for which endogenous ligands are unknown. One of the orphan GPCR subfamilies, Super conserved Receptor Expressed in Brain (SREB), consists of GPR27, GPR85 and GPR173 and is expressed in the central nervous system. We report herein the identification of inverse agonists for the SREB family without their agonists. We carried out an in vitro screening of 5472 chemical compounds from the RIKEN NPDepo chemical library. The binding of [(35) S]GTPγS to the GPR173-Gsα fusion protein expressed in Sf9 cells was measured and resulted in the identification of 8 novel GPR173 inverse agonists. The most potent compound showed an IC50 of approximately 8 µm. The identified compounds were also antagonists for other SREB members, GPR27 and GPR85. These results indicated that the SREB family could couple Gs-type G proteins, and SREB-Gsα fusion proteins showed significant constitutive activities. Moreover, a molecular model of GPR173 was constructed using the screening results. The combination of computational and biological methods will provide a unique approach to ligand identification for orphan GPCRs and brain research.
Assuntos
Simulação de Acoplamento Molecular/métodos , Proteínas do Tecido Nervoso/química , Receptores Acoplados a Proteínas G/química , Bibliotecas de Moléculas Pequenas/química , Encéfalo/metabolismo , Sistema Nervoso Central/efeitos dos fármacos , Expressão Gênica , Humanos , Ligantes , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Interface Usuário-ComputadorRESUMO
Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.
Assuntos
Comunicação Celular/fisiologia , HIV-1/metabolismo , HIV-1/patogenicidade , Macrófagos/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Western Blotting , Linhagem Celular , Citocinas/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
CONTEXT: Euphorbia hirta L. (Euphorbiaceae) has been used as a folk remedy in Southeast Asia for the treatment of various ailments. OBJECTIVE: The current study evaluates the cytotoxicity, cell-cycle arrest, and apoptotic induction by E. hirta in MCF-7 breast cancer cells. MATERIALS AND METHODS: Cytotoxic activity of methanol extract of whole part of E. hirta was determined by the MTT assay at various concentrations ranging from 1.96 to 250.00 µg/mL in MCF-7 cells. Cell morphology was assessed by light and fluorescence microscopy. Apoptosis and cell-cycle distribution were determined by annexin V staining and flow cytometry. DNA fragmentation, caspase activity, and reactive oxygen species (ROS) assays were performed using the commercially available kits. To identify the cytotoxic fraction, E. hirta extract was subjected to bioassay-guided fractionation. RESULTS: Euphorbia hirta exhibited significant inhibition of the survival of MCF-7 cells and the half inhibitory concentration (IC50) values was 25.26 µg/mL at 24 h. Microscopic studies showed that E. hirta-treated cells exhibited marked morphological features characteristic of apoptosis. Euphorbia hirta extract also had an ignorable influence on the LDH leakage and generating intracellular ROS. The flow cytometry study confirmed that E. hirta extract induced apoptosis in MCF-7 cells. Euphorbia hirta also resulted in DNA fragmentation in MCF-7 cells. Moreover, E. hirta treatment resulted in the accumulation of cells at the S and G2/M phases as well as apoptosis. The caspase activity study revealed that E. hirta extract induced apoptosis through the caspase-3-independent pathway by the activation of caspase-2, 6, 8, and 9. Euphorbia hirta hexane fraction, namely HFsub4 fraction, demonstrated highest activity among all the fractions tested with an IC50 value of 10.01 µg/mL at 24 h. DISCUSSION AND CONCLUSION: This study revealed that E. hirta induced apoptotic cell death and suggests that E. hirta could be used as an apoptosis-inducing anticancer agent for breast cancer treatment with further detailed studies.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Euphorbia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Dano ao DNA , Relação Dose-Resposta a Droga , Euphorbia/química , Feminino , Células HT29 , Células HeLa , Humanos , Concentração Inibidora 50 , Células MCF-7 , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Espécies Reativas de Oxigênio/metabolismo , Fatores de Tempo , Células VeroRESUMO
The radioprotective effect of Polyalthia longifolia was studied in mice. P. longifolia treatment showed improvement in mice survival compared to 100% mortality in the irradiated mice. Significant increases in hemoglobin concentration, and red blood cell, white blood cell and platelet counts were observed in the animals pretreated with leaf extract. Pre-irradiation administration of P. longifolia leaf extract also increased the CFU counts of the spleen colony and increased the relative spleen size. A dose-dependent decrease in lipid peroxidation levels was observed in the animals pretreated with P. longifolia. However, although the animals pretreated with P. longifolia exhibited a significant increase in superoxide dismutase and catalase activity, the values remained below normal in both liver and the intestine. Pre-irradiation administration of P. longifolia also resulted in the regeneration of the mucosal crypts and villi of the intestine. Moreover, pretreatment with P. longifolia leaf extract also showed restoration of the normal liver cell structure and a significant reduction in the elevated levels of ALT, AST and bilirubin. These results suggested the radioprotective ability of P. longifolia leaf extract, which is significant for future investigation for human applications in developing efficient, economically viable, non-toxic natural and clinically acceptable novel radioprotectors.
Assuntos
Fígado/efeitos da radiação , Extratos Vegetais/química , Polyalthia/química , Lesões por Radiação/terapia , Protetores contra Radiação/química , Baço/efeitos da radiação , Animais , Antioxidantes/química , Plaquetas/efeitos da radiação , Catalase/metabolismo , Cromatografia Líquida , Eritrócitos/efeitos da radiação , Leucócitos/efeitos da radiação , Peroxidação de Lipídeos , Masculino , Camundongos , Folhas de Planta/química , Rutina/química , Células-Tronco , Superóxido Dismutase/metabolismo , Espectrometria de Massas em Tandem , Temperatura , Irradiação Corporal Total , Raios XRESUMO
Opioids are the most effective and widely used drugs for pain treatment. Morphine is an archetypal opioid and is an opioid receptor agonist. Unfortunately, the clinical usefulness of morphine is limited by adverse effects such as analgesic tolerance and addiction. Therefore, it is important to study the development of novel opioid agonists as part of pain control. The analgesic effects of opioids are mediated by three opioid receptors, namely opioid µ-, δ-, and κ-receptors. They belong to the G protein-coupled receptor superfamily and are coupled to Gi proteins. In the present study, we developed a ligand screening system to identify novel opioid µ-receptor agonists that measures [(35)S]GTPγS binding to cell membrane fractions prepared from the fat body of transgenic silkworms expressing µ-receptor-Gi1α fusion protein. We screened the RIKEN Natural Products Depository (NPDepo) chemical library, which contains 5848 compounds, and analogs of hit compounds. We successfully identified a novel, structurally unique compound, that we named GUM1, with agonist activity for the opioid µ-receptor (EC50 of 1.2 µM). The Plantar Test (Hargreaves' Method) demonstrated that subcutaneous injection of 3mg/kg of GUM1 into wild-type rats significantly extended latency time. This extension was also observed in a rat model of morphine tolerance and was inhibited by pre-treatment of naloxone. The unique molecular skeleton of GUM1 makes it an attractive molecule for further ligand-opioid receptor binding studies.
Assuntos
Benzilaminas/agonistas , Benzilaminas/farmacologia , Produtos Biológicos/farmacologia , Piranos/agonistas , Piranos/farmacologia , Receptores Opioides mu/agonistas , Analgésicos Opioides/agonistas , Analgésicos Opioides/farmacologia , Animais , Animais Geneticamente Modificados , Bombyx , Tolerância a Medicamentos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Masculino , Medição da Dor/efeitos dos fármacos , Ensaio Radioligante , Ratos , Receptores Opioides mu/genética , Radioisótopos de Enxofre/metabolismoRESUMO
Prior to viral DNA replication, baculovirus IE1 exhibits a focal distribution within the cell nucleus. During DNA replication, the IE1 foci apparently expand and develop into a virus replication center called the virogenic stroma (VS). In our search for chemical compounds capable of modulating Bombyx mori nucleopolyhedrovirus (BmNPV: a prototype of baculovirus) replication, we found an inhibitor (dBIQdO) of IE1 focus formation. VS formation, however, was not affected, suggesting that IE1 foci are not essential for VS formation and that IE1 possesses two independent mechanisms for sub-nuclear localization. In addition to inhibition of IE1 focus formation, dBIQdO also reduced viral titers following infection at a low MOI. Comparison of the effects of three chemicals, dBIQdO, aphidicolin and caffeine, on IE1 localization allowed us to detect a shift from focal distribution to VS localization, suggesting that IE1 foci are disassembled prior to VS formation.
Assuntos
Núcleo Celular/virologia , Proteínas Imediatamente Precoces/metabolismo , Nucleopoliedrovírus/metabolismo , Animais , Bombyx , Linhagem Celular , Replicação do DNA , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Nucleopoliedrovírus/genética , Transporte ProteicoRESUMO
Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate; that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers.
Assuntos
Regulação Neoplásica da Expressão Gênica , Processamento de Proteína Pós-Traducional , Espectinomicina/farmacologia , Enzimas de Conjugação de Ubiquitina/metabolismo , Linhagem Celular Tumoral , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Ligação Proteica , Salicilatos/química , Salicilatos/isolamento & purificação , Salicilatos/farmacologia , Transdução de Sinais , Espectinomicina/química , Sumoilação , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.
Assuntos
Acetiltransferases/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Fatores de Tempo , Valeratos/química , Valeratos/farmacologiaRESUMO
MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein-protein interactions (PPIs) of MDM2-p53 and MDMX-p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2-p53 and MDMX-p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2-p53 and MDMX-p53 interactions.
Assuntos
Ensaios de Triagem em Larga Escala , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/metabolismoRESUMO
Plant activators are agrochemicals that protect crops from diseases by activating the plant immune system. To isolate lead compounds for use as practical plant activators, we screened two different chemical libraries composed of various bioactive substances by using an established screening procedure that can selectively identify immune-priming compounds. We identified and characterized a group of sulfonamide compounds - sulfameter, sulfamethoxypyridazine, sulfabenzamide, and sulfachloropyridazine - among the various isolated candidate molecules. These sulfonamide compounds enhanced the avirulent Pseudomonas-induced cell death of Arabidopsis suspension cell cultures and increased disease resistance in Arabidopsis plants against both avirulent and virulent strains of the bacterium. These compounds did not prevent the growth of pathogenic bacteria in minimal liquid media at 200 µM. They also did not induce the expression of defense-related genes in Arabidopsis seedlings, at least not at 24 and 48 h after treatment, suggesting that they do not act as salicylic acid analogs. In addition, although sulfonamides are known to be folate biosynthesis inhibitors, the application of folate did not restore the potentiation effects of the sulfonamides on pathogen-induced cell death. Our data suggest that sulfonamides potentiate Arabidopsis disease resistance by their novel chemical properties.
RESUMO
We found while screening a chemical library that indomethacin, an inhibitor of prostaglandin biosynthesis, induced strobilation (metamorphosis from the asexual to sexual stage) in the moon jellyfish, Aurelia aurita. Indomethacin initiated strobilation in a dose-dependent manner, but was not involved in the progression of strobilation. Pharmacological experiments suggested that indomethacin could induce strobilation independently of prostaglandin biosynthesis.
Assuntos
Indometacina/farmacologia , Larva/efeitos dos fármacos , Metamorfose Biológica/efeitos dos fármacos , Cifozoários/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Ensaios de Triagem em Larga Escala , Larva/fisiologia , Metamorfose Biológica/fisiologia , Prostaglandinas/metabolismo , Cifozoários/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Glycosylation is now recognized as one of the most important modifications of eukaryotic proteins. In cancer biology, alterations in cell surface glycosylation have been exploited as valuable biomarkers, and the relationship of this modification to the metastatic characteristics of cancer cells has also been well-documented. Chemicals that can alter cell surface glycosylation patterns will therefore become attractive lead compounds for controlling the metastatic characteristics of cancer cells, one of the critical factors in their malignancy and prognosis of the disease. In this study, we established a system for screening compounds that have the potential to alter cell surface glycosylation by taking advantage of the susceptibility of cells toward various lectins. Through our screening of a chemical library, we were able to identify two compounds that augment the sensitivity of Chinese Hamster Ovary (CHO-K1) cells against the L4-PHA lectin. Surprisingly, these compounds did not result in alterations in cell surface glycan structures. Instead, they appeared to render the cells to be more sensitive to various lectins with distinct carbohydrate specificities. These compounds promise to be valuable, not only as tools for providing insights into the intracellular signaling of lectin-mediated growth arrest, but also as potential lead compounds for use as therapeutic, anti-cancer drugs.
Assuntos
Lectinas/farmacologia , Fito-Hemaglutininas/farmacologia , Bibliotecas de Moléculas Pequenas , Animais , Células CHO , Cricetinae , Ensaios de Seleção de Medicamentos Antitumorais , Glicosilação , Lectinas/químicaRESUMO
To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8ß-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8ß-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8ß-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.
Assuntos
Ceramidas/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Limoninas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Esfingomielinas/biossíntese , Animais , Células CHO , Varredura Diferencial de Calorimetria , Cricetinae , Células HeLa , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Microscopia Confocal , Esfingolipídeos/metabolismoRESUMO
The RIKEN Natural Products Depository (NPDepo) is a public depository of small molecules. Currently, the NPDepo chemical library contains 39,200 pure compounds, half of which are natural products and their derivatives. In order to reinforce the uniqueness of our chemical library, we have improved our strategies for the collection of microbial natural products. Firstly, a microbial metabolite fraction library coupled with an MP (microbial products) plot database provides a powerful resource for the efficient isolation of microbial metabolites. Secondly, biosynthetic studies of microbial metabolites have enabled us to not only access ingenious biosynthetic machineries, but also obtain a variety of biosynthetic intermediates. Our chemical library contributes to the discovery of molecular probes for increasing our understanding of complex biological processes and for eventually developing new drug leads.
Assuntos
Bactérias/metabolismo , Produtos Biológicos/química , Bases de Dados Factuais , Animais , Bactérias/química , Produtos Biológicos/metabolismo , Humanos , Internet , Engenharia de ProteínasRESUMO
Two new 6,6-spiroacetal polyketides, spirotoamides A (1) and B (2), were isolated from a microbial metabolite fraction library of Streptomyces griseochromogenes JC82-1223 by screening of structurally unique compounds based on a search of spectral database. The fraction library was constructed using a systematic separation method to efficiently discover new metabolites from microbial sources such as actinomycetes and fungi. The structures of 1 and 2 were elucidated by 2D-NMR and mass spectrometric measurements. They belong to a class of polyketides, and contain a 6,6-spiroacetal core structure and a carboxamide group. The biosynthetic pathway of 1 and 2 is discussed in the text.
RESUMO
An automated fluorescence microscopy assay using a nontoxic cholesterol binding protein, toxin domain 4, (D4), was developed in order to identify chemical compounds modifying intracellular cholesterol metabolism and distribution. Using this method, we screened a library of 1,056 compounds and identified 35 compounds that decreased D4 binding to the cell surface. Among them, 8 compounds were already reported to alter the biosynthesis or the intracellular distribution of cholesterol. The remaining 27 hit compounds were further analyzed biochemically and histochemically. Cell staining with another fluorescent cholesterol probe, filipin, revealed that 17 compounds accumulated cholesterol in the late endosomes. Five compounds decreased cholesterol biosynthesis, and two compounds inhibited the binding of D4 to the membrane. This visual screening method, based on the cholesterol-specific probe D4 in combination with biochemical analyses, is a cell-based, sensitive technique for identifying new chemical compounds and modifying cholesterol distribution and metabolism. Furthermore, it is suitable for high-throughput analysis for drug discovery.
Assuntos
Colesterol/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Microscopia de Fluorescência/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/biossíntese , Cricetinae , Cricetulus , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Filipina/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Elastase PancreáticaRESUMO
An acinar morphogenesis inhibitor named fusarisetin A (1) that possesses both an unprecedented carbon skeleton and a new pentacyclic ring system has been identified from an in-house fractionated fungal library using a three-dimensional matrigel-induced acinar morphogenesis assay system. The structure of 1 was determined in detail by NMR and circular dichroism spectroscopy, X-ray analysis, and chemical reaction experiments.