RESUMEN
The limitations of commonly used sodium ascorbate-based catalyst system for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction include excess production of reactive oxygen species and rapid catalyst deactivation. In this study instead of using a highly active reducing agent, such as, sodium ascorbate, we chose reducing sugar as a mild reducing agent to build up the catalyst system for CuAAC reaction. Interestingly, the bicinchoninic acid (BCA) assay system containing reducing sugar satisfies the essential elements of the catalyst system for CuAAC reaction. We found that CuSO4/BCA/Reducing sugar system can catalyze the CuAAC reaction but with low yield. Rational analyses of various parameters in CuSO4/BCA/Glucose catalyst system suggested storage at room temperature might enhance the catalytic activity, which was proven to be the case. Importantly, the system remains stable at room temperature and minimal H2O2 was detected. Notably, our study showed that the coordination between the slow reduction of Cu(I) by reducing sugar and the selective chelation of Cu(I) by BCA is key to developing this system. The CuSO4/BCA/Reducing sugar catalyst system was successfully applied to various CuAAC reaction based bioanalyses, and it is suitable for the CuAAC reaction based bioanalyses that are sensitive to ROS or request long reaction time.
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Alquinos , Azidas , Sulfato de Cobre , Cobre , Reacción de Cicloadición , Catálisis , Cobre/química , Azidas/química , Alquinos/química , Sulfato de Cobre/química , Estructura Molecular , Especies Reactivas de Oxígeno/química , QuinolinasRESUMEN
Taking advantage of key interactions between sulfoxide and heme cofactor, we used the sulfoxide as the anchor functional group to develop two series of indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors: 2-benzylsulfinylbenzoxazoles (series 1) and 2-phenylsulfinylbenzoxazoles (series 2). In vitro enzymatic screening shows that both series can inhibit the activity of IDO1 in low micromolar (series 1) or nanomolar (series 2) levels. They also show inhibitory selectivity between IDO1 and tryptophan 2, 3-dioxygenase 2. Interestingly, although series 1 is less potent IDO1 inhibitors of these two series, it exhibited stronger inhibitory activity toward kynurenine production in interferon-γ stimulated BxPC-3 cells. Enzyme kinetics and binding studies demonstrated that 2-sulfinylbenzoxazoles are non-competitive inhibitors of tryptophan, and they interact with the ferrous form of heme. These results demonstrated 2-sulfinylbenzoxazoles as type II IDO1 inhibitors. Furthermore, molecular docking studies supports the sulfoxide being of the key functional group that interacts with the heme cofactor. Compound 22 (series 1) can inhibit NO production in a concentration dependent manner in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and can relieve pulmonary edema and lung injury in LPS induced mouse acute lung injury models.
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Inhibidores Enzimáticos , Hemo , Indolamina-Pirrol 2,3,-Dioxigenasa , Animales , Humanos , Ratones , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Hemo/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad , Imidazoles/síntesis química , Imidazoles/química , Imidazoles/farmacología , Benzoxazoles/síntesis química , Benzoxazoles/química , Benzoxazoles/farmacologíaRESUMEN
Herein we report our investigation concerning the development of Human neutrophil elastase (hNE) inhibitors for the treatment of Acute Respiratory Distress Syndrome (ARDS). Various benzenesulfonic acid derived compounds were synthesized and evaluated as competitive inhibitors of hNE. Biological screening revealed that compound 4f shows moderate inhibitory activity (IC50 = 35.2 µM) against hNE. Compound 4f was also superimposed onto the active center of hNE to understand the binding mode.
RESUMEN
Indoleamine 2,3-dioxygenase (IDO) 1 is the key enzyme for regulating tryptophan metabolism and is an important target for interrupting tumor immune escape. In this study, we designed four series of compounds as potential IDO1 inhibitors by attaching various fragments or ligands to indole or phenylimidazole scaffolds to improve binding to IDO1. The compounds were synthesized and their inhibitory activities against IDO1 and tryptophan 2,3-dioxygenase were evaluated. The cytotoxicities of the compounds against two tumor cell lines were also determined. Two compounds with a phenylimidazole scaffold (DX-03-12 and DX-03-13) showed potent IDO1 inhibition with IC50 values of 0.3-0.5 µM. These two IDO1 inhibitors showed low cell cytotoxicity, which indicated that they may exert their anti-tumor effect via immune modulation. Compound DX-03-12 was investigated further by determining the in vivo pharmacokinetic profile and anti-tumor efficacy. The pharmacokinetic study revealed that DX-03-12 had satisfactory properties in mice, with rapid absorption, moderate plasma clearance (â¼36% of hepatic blood flow), acceptable half-life (â¼4.6 h), and high oral bioavailability (â¼96%). Daily oral administration of 60 mg/kg of compound DX-03-12 decreased tumor growth by 72.2% after 19 days in a mouse melanoma cell B16-F10 xenograft model compared with the untreated control. Moreover, there was no obvious weight loss in DX-03-12-treated mice. In conclusion, compound DX-03-12 is a potent lead compound for developing IDO1 inhibitors and anti-tumor agents.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Inyecciones Intravenosas , Masculino , Ratones Endogámicos ICR , Modelos Moleculares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer's disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.
Asunto(s)
Precursor de Proteína beta-Amiloide/efectos de los fármacos , Iridoides/farmacología , Neuronas/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/fisiología , Neuronas/metabolismo , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
The 5-bromo-2'-deoxyuridine (BrdU) incorporation cell proliferation assay is the most commonly used method for assessing DNA replication. The current detection of BrdU in cells relies on antibody immunostaining, but has various limitations including low antibody specificity and poor tissue penetration. In this study, we utilised a Suzuki-Miyaura reaction to develop a chemical method to label cellular BrdU with fluorescent boronic acid probes. The coupling conditions were optimised for complex cellular environments, and the key observation was the need to use oxygen scavengers and zerovalent palladium to prevent side reactions and increase the rate of coupling. The reliability and specificity of the BrdU Suzuki-Miyaura labelling method were verified under various biological conditions. The applicability of the BrdU Suzuki-Miyaura labelling methodology was also investigated, and we show that labelling cellular BrdU is highly sensitive and reliable, which is comparable to the ideal performance of BrdU immunostaining. Moreover, the Suzuki-Miyaura reaction protocol provides high BrdU recognition specificity. Taken together, the BrdU Suzuki-Miyaura labelling protocol provides an attractive alternative to the more traditional cell proliferation assay.
Asunto(s)
Bromodesoxiuridina/química , Proliferación Celular , Replicación del ADN , Coloración y Etiquetado/métodos , Anticuerpos , Línea Celular Tumoral , Fluorescencia , Colorantes Fluorescentes , Humanos , Paladio , Reproducibilidad de los ResultadosRESUMEN
C5-modified uridines are a valuable class of nucleoside analogues, both as potent chemotherapy agents and through their use as the conjunction site in DNA labeling strategies. As an important C5-modified uridine, BrdU has been used in cell proliferation assays since the 1980s. Currently, the detection of BrdU relies on traditional immunostaining; however, this approach has its limitations. Thus, it is desirable, albeit difficult, to develop chemistry methods to fluorescently label BrdU in a cellular context. In the present study, we report our efforts toward developing a robust chemistry methodology for BrdU fluorescent labeling. The Sonogashira reaction was chosen as the key reaction, and various alkynyl groups (aliphatic or aryl) containing fluorescent dyes were synthesized to cross-couple with BrdU. Various bases and catalyst systems were screened to evaluate the optimum conditions. A mild aqueous Sonogashira reaction (K2PdCl4, S-Phos, n-Bu4NâºOH-, Sodium d-isoascorbate, EtOH/H2O = 1:1, 37 °C, Ar) was obtained to enable high-yielding BrdU fluorescent labeling.
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Alquinos/síntesis química , Bromodesoxiuridina/química , Colorantes Fluorescentes/síntesis química , Catálisis , Cumarinas/síntesis química , Colorantes Fluorescentes/química , Humanos , Indoles/síntesis química , Estructura Molecular , Espectrometría de Fluorescencia/métodosRESUMEN
Decaprenylphosphoryl-ß-d-ribose oxidase (DprE1) is the flavoprotein subunit of decaprenylphosphoryl-d-ribose epimerase involved in cell wall synthesis in Mycobacterium tuberculosis and catalyzes the conversion of decaprenylphosphoryl ribose to decaprenylphosphoryl arabinose. DprE1 is a potential target against tuberculosis, including multidrug-resistant tuberculosis. We identified potential DprE1 inhibitors from the ChemDiv dataset through virtual screening based on pharmacophore and molecular docking. Thirty selected compounds were subjected to absorption, distribution, metabolism, excretion, and toxicity prediction with the Discovery Studio software package. Two compounds were obtained as hits for inhibiting DprE1 activity in M. tuberculosis and are suitable for further in vitro and in vivo evaluation.
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Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/química , Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Simulación por Computador , Descubrimiento de Drogas , Descubrimiento de Drogas/métodos , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Bibliotecas de Moléculas Pequeñas , Relación Estructura-ActividadRESUMEN
Chromenone-derived natural products include chromones (flavone, isoflavone) and coumarins. Chromenone compounds not only exhibit impressive biological activities, but also are an important resource of experimentally used fluorophores, such as, 7-amino-4-methylcoumarin (AMC). Various chromenone compounds have reported to have weak fluorescence, and this has the potential to interfere with the measurements during AMC fluorogenic assays and result in non-robust assay readouts. Several flavones and isoflavones were found as SIRT1 activators, while fluorogenic sirtuin assays utilized AMC labelled peptides as the substrates. In this study we investigated whether the fluorescent properties of chromenone-derived natural products interrupt the measurement of SIRT1/2 modulated activities. We found that the reported SIRT1 activators: flavones were detected with the SIRT1 activation activity, but isoflavones were not detected with SIRT1 activation activity, and instead that they were found to be fluorogenic compounds. Another chromenone compound, osthole, exhibited a moderate SIRT2 inhibitory activity with an IC50 of 10 µM. In conclusion, the fluorescent properties of these chromenone compounds do affect the measurement of the sirtuin activities of both inhibitors and activators. However, if the possible fluorescence properties are mitigated in the assay readout, these fluorogenic assays enable the screening of activity modulators.
Asunto(s)
Cumarinas/química , Flavonas/química , Fluorescencia , Isoflavonas/química , Sirtuina 1/química , HumanosRESUMEN
Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds.
Asunto(s)
Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citotoxinas/administración & dosificación , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Pruebas de Toxicidad/métodos , Apoptosis/fisiología , Bioensayo/métodos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/fisiología , Citoplasma/químicaRESUMEN
A series of benzofuran derivatives were designed and synthesized, and their inhibitory activites were measured against the SIRT1-3. The enzymatic assay showed that all the compounds showed certain anti-SIRT2 activity and selective over SIRT1 and SIRT3 with IC50 (half maximal inhibitory concentration) values at the micromolar level. The preliminary structure-activity relationships were analyzed and the binding features of compound 7e (IC50 3.81 µM) was predicted using the CDOCKER program. The results of this research could provide informative guidance for further optimizing benzofuran derivatives as potent SIRT2 inhibitors.
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Benzofuranos/síntesis química , Benzofuranos/farmacología , Técnicas de Química Sintética/métodos , Sirtuina 2/antagonistas & inhibidores , Sitios de Unión , Diseño de Fármacos , Escherichia coli , Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Simulación del Acoplamiento Molecular/métodos , Estructura Molecular , Unión Proteica , Sirtuina 2/genética , Relación Estructura-ActividadRESUMEN
Cell multinucleation is closely related to chromosomal instability. We report a simple, convenient method to assess cell multinucleation with cytoplasm-localized fluorescent probes (CLFP) which is superior to conventional nuclear staining methods. The CLFP method provides high-resolution images that enable the accurate calculation of the number of nuclear fragments.
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Núcleo Celular , Citoplasma/química , Colorantes Fluorescentes , Compuestos de Boro , Línea Celular Tumoral , Fragmentación del ADN , Compuestos de Dansilo , Humanos , Coloración y EtiquetadoRESUMEN
In this study, we evaluated the applicability of various superoxide anion sensors which were designed based on either redox or non-redox mechanisms. Firstly, both redox- and non-redox-based superoxide anion probes were designed and synthesized using either coumarin or chromone as the fluorophores, and the photophysical properties of these probes were measured. Subsequently, the sensing preference of both types of probes toward various reactive oxygen species (ROS) was evaluated. We found that non-redox-based O2 â¢- probes exhibited broad sensing ability toward various ROS. By contrast, redox based O2 â¢- probes showed a clear reactivity hierarchy which was well correlated to the oxidizing strength of the ROS. Lastly, the detection selectivity of redox-based O2 â¢- recognizing probes was also observed when balancing various factors, such as reactant ROS concentrations, temperature, and changing reaction transformation rates. Herein, we concluded the selectivity advantage of redox-based O2 â¢- probes.
RESUMEN
Herein, we report our work exploring the essential requirements for fluorophore selection during the development of various fluorescence applications. We assembled a library of chromone-derived fluorophores with diverse structure-fluorescence properties, which allowed us to choose the fluorophore pairs with similar structures but differing fluorescence properties and compared the performance of the selected fluorophore pairs in three types of commonly used fluorescence applications. We found that the selection standard of a suitable fluorophore is variable depending on the application. (1) In fluorescence imaging, fluorophores with strong and constant fluorescence under various conditions, such as a large pH range, are preferred. Notably, (2) in the detection of bioactive species, fluorophores with relatively lower fluorescence quantum yield favor the detection sensitivity. Furthermore, (3) in enzymatic assays employing fluorescence, the key parameter is the binding affinity between the fluorophore and the enzyme.
Asunto(s)
Cromonas/química , Colorantes Fluorescentes/química , Línea Celular Tumoral , Supervivencia Celular , Enzimas/química , Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Simulación del Acoplamiento Molecular , Imagen Óptica/métodos , Sensibilidad y Especificidad , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Tripsina/químicaRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the world's leading causes of death among cancer patients. It is important to find a new biomarker that diagnoses HCC and monitors its treatment. In our previous work, we screened a single-chain antibody (scFv) N14, which could specifically recognize human HepG2 HCC cells but not human non-cancerous liver LO2 cells. However, the antigen it recognized in the cells remained unknown. METHODS: Recombinant scFv N14 antibody was expressed as an active antibody. Using this antibody with a combination of immunological and proteomic approaches, we identified the antigen of scFv N14 antibody as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). The expression of hnRNP A2/B1 in HCC cells was then investigated by semi-quantitative RT-PCR and immunohistochemistry. RESULTS: We found that the up-regulation of hnRNP A2/B1 was measured at both transcriptional and translational levels in rat HCC cells but not in rat hepatic cells. We also found that in various human hepatic tissues, hnRNP A2/B1 was highly expressed in both human hepatitis virus positive liver tissues and human HCC tissues but not in normal liver tissues. Interestingly, we observed that the localization of hnRNP A2/B1 in HCC cells was altered during the development of HCC. In human hepatitis virus infected tissues hnRNP A2/B1 resides exclusively in the nuclei of hepatocytes. However, when the HCC progressed from a well differentiated to a poorly differentiated stage, hnRNP A2/B1 was increasingly localized in the cytoplasm. In contrast, the HCC tissues with hnRNP A2/B1 highly expressed in the nucleus decreased. CONCLUSIONS: This work is the first to show that hnRNP A2/B1 is the antigen specifically recognized by the scFv N14 antibody in HCC cells. The over-expression of hnRNP A2/B1 was confirmed in cultured human and rat HCC cell lines, human virus related hepatitis liver tissues and human HCC tissues. The increased localization of hnRNP A2/B1 in the cytoplasm of HCC cells was revealed during the dedifferentiation of hepatocellular carcinoma. Therefore, we suggest that the increased expression and cytoplasmic localization of hnRNP A2/B1 can be used as a diagnostic biomarker to assess the risk of human liver cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Citoplasma/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Neoplasias Hepáticas/metabolismo , Secuencia de Aminoácidos , Animales , Biomarcadores de Tumor/genética , Western Blotting , Carcinoma Hepatocelular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/patología , Femenino , Técnica del Anticuerpo Fluorescente , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hepatocitos/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/inmunología , Humanos , Técnicas para Inmunoenzimas , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/aislamiento & purificación , Regulación hacia ArribaRESUMEN
Plasmodium falciparum thymidylate kinase (PfTMPK) shows a broad range of substrate tolerance when compared to the corresponding human enzyme. Besides 2'-deoxythymidine monophosphate (dTMP), PfTMPK can phosphorylate 3'-azido-2',3'-dideoxythymidine monophosphate (AZTMP) very efficiently. In contrast, human thymidylate kinase (hTMPK) is 200 times less active towards AZTMP. We were interested to see if we could use PfTMPK to activate 3'-azido-2',3'-deoxythymidine (AZT) derivatives as a strategy to treat malaria. P. falciparum lacks a pyrimidine nucleoside kinase which usually activates nucleoside and nucleoside analogues to the corresponding monophosphates. Therefore, several prodrug analogues of AZT and related nucleoside monophosphates were prepared and analysed for antiparasitic activity. The prodrugs showed an increase in activity over the parent nucleoside analogues, which showed no inhibition of parasite growth at the concentration tested. The evidence here reported provides a strategy that could be exploited for further anti-malarial design.
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Antimaláricos/química , Nucleósido-Fosfato Quinasa/antagonistas & inhibidores , Nucleósidos/química , Plasmodium falciparum/enzimología , Antimaláricos/síntesis química , Antimaláricos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Nucleósido-Fosfato Quinasa/metabolismo , Nucleósidos/síntesis química , Nucleósidos/farmacología , Plasmodium falciparum/efectos de los fármacos , Profármacos/síntesis química , Profármacos/química , Profármacos/farmacología , Zidovudina/síntesis química , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
Tetramethylpyrazine (TMP) has neuroprotective effects in the pathogenesis of some human diseases, such as Parkinson's disease. The present study aimed to investigate the role of TMP in isoflurane-induced cognitive dysfunction in rats, and further identify the mechanisms involved in the protective effects of TMP. The Morris water maze test was used to evaluate the cognitive function of rats exposed to isoflurane or treated with TMP. ELISA was conducted to evaluate the effects of isoflurane or TMP on neuroinflammation. The expression of microRNA-150 (miR-150) was measured using reverse transcription-quantitative PCR, and the potential target genes of miR-150 were predicted and verified. The impaired cognitive function induced by isoflurane in the rats was significantly ameliorated by treatment with TMP. In addition, TMP treatment in rats attenuated neuroinflammation caused by isoflurane. The expression of miR-150 was inhibited by isoflurane exposure, but was enhanced by TMP treatment in rats. Furthermore, the overexpression of miR-150 alleviated the isoflurane-induced cognitive dysfunction and neuroinflammation, while the neuroprotective effects of TMP were significantly abrogated by the knockdown of miR-150. AKT3 was a direct target of miR-150, and its mRNA expression was significantly decreased by the overexpression of miR-150 in isoflurane- and TMP-treated rats. These results demonstrated the protective effects of TMP against isoflurane-induced cognitive dysfunction, which were achieved by attenuating neuroinflammation via the regulation of the miR-150/AKT3 pathway. In addition, miR-150 may serve as a novel therapeutic target for the alleviation of cognitive dysfunction induced by anesthetics.
RESUMEN
The aromatisation of 7-diethylamino-3,4-dihydrocoumarin provides an alternative fluorescent probing technique to selectively detect the concentration of superoxide anion in solution. In addition, we reported the advantage of evaluating O2Ë- sensing probes in anhydrous DMSO instead of in aqueous buffers when using KO2 as the surrogate of O2Ë-.
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Cumarinas/química , Espectrometría de Fluorescencia/métodos , Superóxidos/análisis , Aniones/química , Colorantes Fluorescentes/química , Óxidos/química , Compuestos de Potasio/química , Superóxidos/química , Agua/químicaRESUMEN
Optochin, a cinchona alkaloid derivative discovered over 100 years ago, possesses highly selective antibacterial activity toward Streptococcus pneumoniae. Pneumococcal disease remains the leading source of bacterial pneumonia and meningitis worldwide. The structure-activity relationships of optochin were examined through modification to both the quinoline and quinuclidine subunits, which led to the identification of analogue 48 with substantially improved activity. Resistance and molecular modeling studies indicate that 48 likely binds to the c-ring of ATP synthase near the conserved glutamate 52 ion-binding site, while mechanistic studies demonstrated that 48 causes cytoplasmic acidification. Initial pharmacokinetic and drug metabolism analyses of optochin and 48 revealed limitations of these quinine analogues, which were rapidly cleared, resulting in poor in vivo exposure through hydroxylation pendants to the quinuclidine and O-dealkylation of the quinoline. Collectively, the results provide a foundation to advance 48 and highlight ATP synthase as a promising target for antibiotic development.
Asunto(s)
Antibacterianos/farmacología , Alcaloides de Cinchona/farmacología , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Streptococcus pneumoniae/enzimología , Antibacterianos/química , Antibacterianos/metabolismo , Sitios de Unión , Alcaloides de Cinchona/química , Alcaloides de Cinchona/metabolismo , Farmacorresistencia Microbiana , Pruebas de Sensibilidad Microbiana , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Relación Estructura-ActividadRESUMEN
Current methods used to evaluate in vivo target efficacy of selected compound include western blot to semi-quantitatively analyze protein expression. However, problems arise as it is difficult to compare in vivo target efficacy of anti-tumor agents with the same mode of action. It is therefore desirable to develop a protocol that can quantitatively display in vivo target efficacy while also providing other useful information. In this study EdU labeling was used to mark out the proliferating area. The tumor tissue was accordingly divided into proliferating and non-proliferating areas. Fifteen tumor related proteins were stained by immunofluorescence and were found to express in either the proliferating or non-proliferating areas. This allows the quantitative analysis of protein expressions within the precise area. With simple image analysis, our method gave precise percent changes of protein expression and cell proliferation between the drugs treated group and the control group. Additional information, such as, the status of protein expression can also be obtained. This method exhibits high sensitivity, and provides a quantitative approach for in vivo evaluation of target efficacy.