RESUMEN
Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Colorantes Fluorescentes/química , Rodaminas , IonóforosRESUMEN
MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Inhibidores de Proteasas , Acústica , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , SARS-CoV-2RESUMEN
To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.
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Bibliotecas de Moléculas Pequeñas/química , Difusión , Membranas Artificiales , Estructura Molecular , Permeabilidad , SolubilidadRESUMEN
Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.
Asunto(s)
Catecoles/farmacología , Epigénesis Genética , Redes y Vías Metabólicas , S-Adenosilmetionina/metabolismo , Animales , Catecol O-Metiltransferasa/metabolismo , Células HT29 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Ca2+ is one of the most important second messengers in cells. A far-red to near-infrared (NIR) Ca2+ fluorescent probe is useful for multi-color imaging in GFP or YFP-expressing biosamples. Here we developed a cytosolically localized far-red to NIR rhodamine-based fluorescent probe for Ca2+, CaSiR-2 AM, while rhodamine dyes are basically localized to mitochondria or lysosomes in cells.
Asunto(s)
Calcio , Colorantes Fluorescentes , Iones , Lisosomas , RodaminasRESUMEN
Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs inâ vivo, the near-infrared (NIR) region (650-900â nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30â min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.
Asunto(s)
Colorantes Fluorescentes/química , Receptores de Folato Anclados a GPI/metabolismo , Regulación Neoplásica de la Expresión Génica , Imagen Molecular/métodos , Relación Señal-Ruido , Animales , Línea Celular Tumoral , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Ácido Fólico/metabolismo , Humanos , Ratones , Rodaminas/síntesis química , Rodaminas/química , Rodaminas/metabolismo , Factores de TiempoRESUMEN
Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.
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Ácido Hipocloroso/análisis , Compuestos de Organosilicio/química , Rodaminas/química , Animales , Diseño de Fármacos , Humanos , Ácido Hipocloroso/química , Rayos Infrarrojos , Masculino , Ratones Endogámicos BALB C , Compuestos de Organosilicio/síntesis química , Compuestos de Organosilicio/efectos de la radiación , Técnicas Fotoacústicas/métodos , Rodaminas/síntesis química , Rodaminas/efectos de la radiación , Tejido Subcutáneo/químicaRESUMEN
Adiponectin secreted from adipocytes binds to adiponectin receptors AdipoR1 and AdipoR2, and exerts antidiabetic effects via activation of AMPK and PPAR-α pathways, respectively. Levels of adiponectin in plasma are reduced in obesity, which causes insulin resistance and type 2 diabetes. Thus, orally active small molecules that bind to and activate AdipoR1 and AdipoR2 could ameliorate obesity-related diseases such as type 2 diabetes. Here we report the identification of orally active synthetic small-molecule AdipoR agonists. One of these compounds, AdipoR agonist (AdipoRon), bound to both AdipoR1 and AdipoR2 in vitro. AdipoRon showed very similar effects to adiponectin in muscle and liver, such as activation of AMPK and PPAR-α pathways, and ameliorated insulin resistance and glucose intolerance in mice fed a high-fat diet, which was completely obliterated in AdipoR1 and AdipoR2 double-knockout mice. Moreover, AdipoRon ameliorated diabetes of genetically obese rodent model db/db mice, and prolonged the shortened lifespan of db/db mice on a high-fat diet. Thus, orally active AdipoR agonists such as AdipoRon are a promising therapeutic approach for the treatment of obesity-related diseases such as type 2 diabetes.
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Diabetes Mellitus Tipo 2/tratamiento farmacológico , Longevidad/efectos de los fármacos , Obesidad/fisiopatología , Piperidinas/farmacología , Receptores de Adiponectina/agonistas , Adenilato Quinasa/metabolismo , Adiponectina/metabolismo , Adiponectina/farmacología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/patología , Administración Oral , Animales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa , Evaluación Preclínica de Medicamentos , Dislipidemias/tratamiento farmacológico , Activación Enzimática/efectos de los fármacos , Intolerancia a la Glucosa/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Resistencia a la Insulina , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculos/citología , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Obesidad/genética , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Piperidinas/administración & dosificación , Piperidinas/metabolismo , Piperidinas/uso terapéutico , Receptores de Adiponectina/deficiencia , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Factores de Transcripción/biosíntesis , Triglicéridos/metabolismoRESUMEN
Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.
Asunto(s)
Presión Intraocular/efectos de los fármacos , Hipertensión Ocular/tratamiento farmacológico , Inhibidores de Fosfodiesterasa/uso terapéutico , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Animales , Humor Acuoso , Línea Celular , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Glaucoma/metabolismo , Glaucoma/fisiopatología , Humanos , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Estructura Molecular , Hipertensión Ocular/inducido químicamente , Inhibidores de Fosfodiesterasa/química , Malla Trabecular/efectos de los fármacosRESUMEN
In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p Ka for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable p Ka and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.
Asunto(s)
Fluorescencia , Colorantes Fluorescentes/química , Neoplasias/diagnóstico por imagen , Imagen Óptica , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Colorantes Fluorescentes/farmacocinética , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Ratones Desnudos , Estructura Molecular , Neoplasias/patología , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/patología , Solubilidad , Agua/químicaRESUMEN
Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azo-based strategy should be widely applicable to design a range of activatable photosensitizers.
Asunto(s)
Compuestos Azo/farmacología , Hipoxia de la Célula/efectos de los fármacos , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Compuestos Azo/síntesis química , Compuestos Azo/química , Línea Celular Tumoral , Humanos , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/químicaRESUMEN
Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.
Asunto(s)
Péptido Hidrolasas/metabolismo , Péptidos/metabolismo , Línea Celular Tumoral , Endopeptidasas/química , Endopeptidasas/metabolismo , Humanos , Conformación Molecular , Especificidad de Órganos , Péptido Hidrolasas/química , Péptidos/químicaRESUMEN
We synthesized a series of 1,2,3,4-tetrahydroisoquinoline-type positive allosteric modulators of prostacyclin receptor (IPPAMs), aiming to improve the metabolic stability of the previously identified hit compound IPPAM-3 (2). Our results indicated that the 3-position of the 2-substituted phenyl ring in this series of IPPAM-3 derivatives is a hot spot for metabolism catalyzed by human hepatic microsomes. This conclusion was confirmed by the finding that 8, in which the 3-position is blocked by a fluorine substituent, exhibited superior metabolic stability (t1/2 21min versus 7min for parent compound 2). The primary route of metabolism of 8 was found to be oxidative defluorination, i.e., ipso-substitution of the fluorine atom to a hydroxyl group, affording catechol derivative 12. The primary metabolite 12 underwent further hydroxylation mainly on the 1,2,3,4-tetrahydroisoquinoline moiety. These findings should be helpful for design of IPPAMs with longer duration of action.
Asunto(s)
Microsomas Hepáticos/metabolismo , Receptores de Epoprostenol/agonistas , Tetrahidroisoquinolinas/metabolismo , Tetrahidroisoquinolinas/farmacología , Animales , Células CHO , Cricetulus , Sistema Enzimático del Citocromo P-450/metabolismo , Halogenación , Humanos , Hidroxilación , Oxidación-Reducción , Receptores de Epoprostenol/metabolismo , Tetrahidroisoquinolinas/químicaRESUMEN
We present a practical synthesis of both enantiomers of 1,2,3,4-tetrahydroisoquinoline derivative IPPAM-1 (1), which is a positive allosteric modulator (PAM) of prostacyclin receptor (IP) and a candidate for treatment of pulmonary arterial hypertension without the side effects caused by IP agonists. Assay of cAMP production by CHO-K1 cells stably expressing human IP clearly demonstrated that the IPPAM activity resides exclusively on the R-form of 1.
Asunto(s)
Antihipertensivos/química , Receptores de Epoprostenol/metabolismo , Tetrahidroisoquinolinas/química , Regulación Alostérica , Animales , Antihipertensivos/síntesis química , Antihipertensivos/uso terapéutico , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Receptores de Epoprostenol/agonistas , Receptores de Epoprostenol/genética , Estereoisomerismo , Tetrahidroisoquinolinas/síntesis química , Tetrahidroisoquinolinas/uso terapéuticoRESUMEN
The Keap1-Nrf2 system is an attractive target for drug discovery regarding various unmet medical needs. Only covalent inhibitors for protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 have been approved or are under clinical trials, but such electrophilic compounds lack selectivity. Therefore, specific non-covalent Keap1-Nrf2 PPI inhibitors are expected to be safer Nrf2 activators. We found a novel class of non-covalent Keap1-Nrf2 PPI inhibitor that has a benzo[g]indole skeleton and an indole-3-hydroxamic acid moiety and that exhibits significant PPI inhibitory activity. Additionally, the benzo[g]indole-3-carbohydrazide derivatives were newly prepared. The benzo[g]indole derivatives showed a stronger Keap1-Nrf2 PPI inhibitory activity than Cpd16, a previously reported non-covalent PPI inhibitor. Moreover, most of the PPI inhibitors showed a high metabolic stability in a human microsome system with a low cytotoxicity against HepG2 cell lines, which suggests that novel benzo[g]indole-type Keap1-Nrf2 PPI inhibitors are expected to be biological tools or lead compounds for Nrf2 activators.
Asunto(s)
Indoles/química , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células Hep G2 , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/toxicidad , Indoles/síntesis química , Indoles/toxicidad , Concentración 50 Inhibidora , Proteína 1 Asociada A ECH Tipo Kelch/antagonistas & inhibidores , Microsomas Hepáticos/metabolismo , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Dominios y Motivos de Interacción de ProteínasRESUMEN
We adopted a spirocyclization-based strategy to design γ-glutamyl hydroxymethyl selenorhodamine green (gGlu-HMSeR) as a photo-inactive compound that would be specifically cleaved by the tumor-associated enzyme γ-glutamyltranspeptidase (GGT) to generate the potent photosensitizer HMSeR. gGlu-HMSeR has a spirocyclic structure and is colorless and does not show marked phototoxicity toward low-GGT-expressing cells or normal tissues upon irradiation with visible light. In contrast, HMSeR predominantly takes an open structure, is colored, and generates reactive oxygen species upon irradiation. The γ-glutamyl group thus serves as a tumor-targeting moiety for photodynamic therapy (PDT), switching on tumor-cell-specific phototoxicity. To validate this system, we employed chick chorioallantoic membrane (CAM), a widely used model for preliminary evaluation of drug toxicity. Photoirradiation after gGlu-HMSeR treatment resulted in selective ablation of implanted tumor spheroids without damage to healthy tissue.
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Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Fármacos Fotosensibilizantes/farmacología , Compuestos de Espiro/farmacología , gamma-Glutamiltransferasa/antagonistas & inhibidores , Células A549 , Antineoplásicos/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Espiro/química , gamma-Glutamiltransferasa/metabolismoRESUMEN
We have established a coupled assay system targeting protein l-isoaspartyl methyltransferase (PIMT), a key enzyme in the metabolism of isoaspartyl peptides and proteins. The system utilizes a fluorogenic peptide probe containing an isoaspartyl residue at the P1' position of the caspase-3 recognition sequence. Following PIMT-catalyzed methyl transfer reaction, the methylated probe is specifically cleaved by caspase-3 to give fluorescence activation. High-throughput screening of our chemical library with this assay system identified PIMT inhibitors that may be useful as leads in the design of chemical probes for controlling PIMT activity.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/farmacología , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/antagonistas & inhibidores , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Caspasa 3/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Péptidos/metabolismo , Especificidad por SustratoRESUMEN
Diacylglycerol kinase (DGK) consists of 10 isozymes. The α-isozyme enhances the proliferation of cancer cells. However, DGKα facilitates the nonresponsive state of immunity known as T-cell anergy; therefore, DGKα enhances malignant traits and suppresses immune surveillance. The aim of this study was to identify a novel small molecule that selectively and potently inhibits DGKα activity. We screened a library containing 9,600 chemical compounds using a newly established high-throughput DGK assay. As a result, we have obtained a promising compound, 5-[(2E)-3-(2-furyl)prop-2-enylidene]-3-[(phenylsulfonyl)amino]2-thioxo-1,3-thiazolidin-4-one) (CU-3), which selectively inhibited DGKα with an IC50 value of 0.6 µM. CU-3 targeted the catalytic region, but not the regulatory region, of DGKα. CU-3 competitively reduced the affinity of DGKα for ATP, but not diacylglycerol or phosphatidylserine. Moreover, this compound induced apoptosis in HepG2 hepatocellular carcinoma and HeLa cervical cancer cells while simultaneously enhancing the interleukin-2 production of Jurkat T cells. Taken together, these results indicate that CU-3 is a selective and potent inhibitor for DGKα and can be an ideal anticancer drug candidate that attenuates cancer cell proliferation and simultaneously enhances immune responses including anticancer immunity.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diacilglicerol Quinasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Rodanina/análogos & derivados , Sulfonamidas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Tiazoles/farmacología , Animales , Células COS , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Concentración 50 Inhibidora , Interleucina-2/biosíntesis , Isoenzimas/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Rodanina/farmacología , Especificidad por Sustrato , Linfocitos T/metabolismoRESUMEN
K(+) is the most abundant metal ion in cells, and changes of [K(+)] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K(+)] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K(+), TLSHalo. TLSHalo is responsive to [K(+)] in the physiological range, with good selectivity over Na(+) and retains its K(+)-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K(+)] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K(+)] change induced by K(+) ionophores or by activation of a native Ca(2+)-dependent K(+) channel (BK channel). Further, we show that K(+) efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K(+) dynamics with high spatiotemporal resolution.
Asunto(s)
Compuestos de Boro/química , Membrana Celular/metabolismo , Éteres Corona/química , Colorantes Fluorescentes/química , Potasio/metabolismo , Cationes Monovalentes , Células HEK293 , Células HT29 , Células HeLa , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Ionóforos de Potasio/farmacologíaRESUMEN
Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450â nm). This allowed us to stimulate neural cells in mouse brain slices.