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7-Bromo-4-chloro-1H-indazol-3-amine is a heterocyclic fragment used in the synthesis of Lenacapavir, a potent capsid inhibitor for the treatment of HIV-1 infections. In this manuscript, we describe a new approach to synthesizing 7-bromo-4-chloro-1H-indazol-3-amine from inexpensive 2,6-dichlorobenzonitrile. This synthetic method utilizes a two-step sequence including regioselective bromination and heterocycle formation with hydrazine to give the desired product in an overall isolated yield of 38-45%. The new protocol has been successfully demonstrated on hundred-gram scales without the need for column chromatography purification. This new synthesis provides a potential economical route to the large-scale production of this heterocyclic fragment of Lenacapavir.
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A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered using CRISPR-Cas9-mediated gene editing technology to carry the D2-I212F variant. Drd2I212F mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212F mice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2 +/+ mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also â¼fourfold slower in midbrain slices from Drd2I212F mice than Drd2 +/+ mice. Furthermore, in contrast to Drd2 +/+ mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gα o versus Gα i, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.
Assuntos
Dopamina , Transtornos dos Movimentos , Receptores de Dopamina D2 , Animais , Humanos , Camundongos , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Marcha/genética , Hipercinese , Mutação , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , SulpiridaRESUMO
The activity of dopamine neurons is dependent on both intrinsic properties and afferent projections. One potent form of inhibition is mediated by the activation of two inhibitory G protein-coupled receptors, D2 and GABAB receptors. Each of these receptors activates G protein-coupled inwardly rectifying potassium (GIRK) channels. Recordings in brain slices have shown that co-activation using saturating concentrations of agonists results in occlusion of the GIRK current. The present study examined the interaction between D2 and GABAB receptors using transient applications of sub-saturating concentrations of agonists where the co-application of one agonist resulted in both facilitation and inhibition (desensitization) of the other. The heterologous facilitation was modelled based on the known cooperative interaction between the G protein ßγ subunits and GIRK channels. The results indicate that a low tonic level of G ßγ results in facilitation of GIRK current and a high level of G ßγ results in occlusion. The kinetics of the current induced by transient receptor activation is prolonged in each case. The results suggest that the cooperative interaction between G ßγ subunits and GIRK channels determines both the amplitude and kinetics of GPCR-dependent current. KEY POINTS: Inhibitory D2 and GABAB receptors modulate dopamine neuron activity through shared G protein-coupled inwardly rectifying potassium (GIRK) channels. This study reports robust bidirectional interactions between these two converging receptor pathways. Coincident activation of D2 and GABAB receptors leads to facilitation of GIRK channel currents, augmenting both amplitude and prolonging the duration of phasic responses. Activation of either D2 or GABAB receptors also acutely desensitized the GIRK channel current induced by D2 receptor activation that rapidly recovers following termination of desensitizing stimulus. Results demonstrate that the activity of either G protein-coupled receptor system must be considered in the context of other G protein-coupled receptors.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Receptores de GABA-B , Receptores de GABA-B/metabolismo , Receptores de Dopamina D2/metabolismo , Potássio/metabolismo , Ácido gama-AminobutíricoRESUMO
We report a photoactivatable agonist of the AMPA subtype of ionotropic glutamate receptors, TMP-CyHQ-AMPA, which was designed to study the fast excitatory transmission between neurons. Upon visible light excitation, TMP-CyHQ-AMPA quantitatively released AMPA in high quantum yield on an ultra-short timescale. Intriguingly, the photolyisis can be carried out using 2-photon excitation (2PE) with remarkable efficiency, giving a two-photon uncaging action cross section (δu) value of 1.71 GM. TMP-CyHQ-AMPA is soluble in pysiological buffer and no hydrolysis was detected in the absence of light. Molecular docking experiments indicated that the photocaging strategy abolishes the affinity of AMPA for the GluR2 receptor and no GABAergic effects (as commonly observed in caged glutamates) are expected. TMP-CyHQ-AMPA can be used to study glutamatergic neuronal transmission with exceptional spatial-temporal resolution in complex tissue preparations.
Assuntos
Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
Photoremovable protecting groups (PPGs) are powerful tools for physiological studies, harnessing light as an on/off switch to provide tight spatio-temporal control over the release of biological effectors through two-photon excitation (2PE) in tissue culture and whole-animal studies. We carried out a series of systematic structural modifications to the (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ) chromophore to conduct an SAR study with the aim of enhancing its photochemical properties, especially its two-photon uncaging action cross section (δu). The best results were obtained when substituents were added at the C4 position, which improved δu for release of acetate up to 7-fold, while retaining all the other excellent properties of the CyHQ PPG, including high quantum yield (Φu), low susceptibility to spontaneous hydrolysis in the dark, and good aqueous solubility. Hammett correlation analysis suggested that photolysis efficiency is favored by electron-rich substituents at C4, giving important insights into the mechanism of the photolysis reaction. The four best CyHQ derivatives were used to mediate the efficient release of homopiperonylic acid in high yield under simulated physiological conditions. Our efforts have led to the development of 2PE-sensitive PPGs with remarkable δu values (up to 2.64 GM), excellent quantum yields (up to 0.88), and high-yielding effector release (up to 92%).
RESUMO
The direct release of dialkylanilines was achieved by controlling the outcome of a photorearrangement reaction promoted by the (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ) photoremovable protecting group. The substrate scope was investigated to obtain structure-activity relationships and to propose a reaction mechanism. Introducing a methyl substituent at the 2-methyl position of the CyHQ core enabled the bypass of the photorearrangement and significantly improved the aniline release efficiency. We successfully applied the strategy to the photoactivation of mifepristone (RU-486), an antiprogestin drug that is also used to induce the LexPR gene expression system in zebrafish and the gene-switch regulatory system based on the pGL-VP chimeric regulator in mammals.
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Compostos de Anilina/química , Quinolinas/química , Estrutura Molecular , Processos Fotoquímicos , FotóliseRESUMO
Maritime transport has been playing a decisive role in global trade. Its contribution to the air pollution of the sea and coastal areas has been widely recognized. The air pollutant emission inventories of several harbors in China have already been established. However, the emission factors of local ships have not been addressed comprehensively, and thus are lacking from the emission inventories. In this study, on-board emission tests of eight diesel-powered offshore fishing ships were conducted near the coastal region of the northern Yellow Bo Sea fishing ground of Dalian, China. Results show that large amounts of fine particles (<0.5µm, 90%) were found in maneuvering mode, which were about five times higher than those during cruise mode. Emission rates as well as emission factors based on both distance and fuel were determined during the cruise and maneuvering modes (including departure and arrival). Average emission rates and distance-based emission factors of CO, HC and PM were much higher during the maneuvering mode as compared with the cruise mode. However, the average emission rate of Nitrous Oxide (NOx) was higher during the cruise mode as compared with the maneuvering modes. On the contrary, the average distance-based emission factors of NOx were lower during the cruise mode relative to the maneuvering mode due to the low sailing speed of the latter.
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Poluentes Atmosféricos/análise , Monitoramento Ambiental , Material Particulado/análise , Navios/estatística & dados numéricos , Emissões de Veículos/análise , Poluição do Ar/estatística & dados numéricos , ChinaRESUMO
Representative tertiary amines were linked to the 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting group (PPG) to create photoactivatable forms suitable for use in studying cell physiology. The photoactivation of tamoxifen and 4-hydroxytamoxifen, which can be used to activate Cre recombinase and CRISPR-Cas9 gene editing, demonstrated that highly efficient release of bioactive molecules could be achieved through one- and two-photon excitation (1PE and 2PE). CyHQ-protected anilines underwent a photoaza-Claisen rearrangement instead of releasing amines. Time-resolved spectroscopic studies revealed that photorelease of the tertiary amines was extremely fast, occurring from a singlet excited state of CyHQ on the 70 ps time scale.
Assuntos
Aminas/química , Fenômenos Fisiológicos Celulares , Processos Fotoquímicos , Aminas/síntese química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Solubilidade , Análise Espectral/métodos , Água/químicaRESUMO
The generation of common and stereochemically rich medium-sized benzo-fused sultams via complementary pairing of heretofore-unknown (o-fluoroaryl)sulfonyl aziridine building blocks with an array of amino alcohols/amines in a modular one-pot, sequential protocol using an aziridine ring opening and intramolecular nucleophilic aromatic substitution is reported. The strategy employs a variety of amino alcohols/amines and proceeds with 6 + 4/6 + 5 and 6 + 1 cycloetherification pathways in a highly chemo- and regioselective fashion to obtain skeletally and structurally diverse, polycyclic, 10- to 11- and 7-membered benzo-fused sultams for broad-scale screening.
Assuntos
Aziridinas/química , Naftalenossulfonatos/síntese química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Naftalenossulfonatos/químicaRESUMO
Phenols confer bioactivity to a plethora of organic compounds. Protecting the phenolic functionality with photoremovable protecting groups (PPGs) sensitive to two-photon excitation (2PE) can block the bioactivity and provide controlled release of these compounds in a spatially and temporally restricted manner by photoactivation with IR light. To develop an efficient 2PE-sensitive PPG for releasing phenols, the (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ) chromophore was functionalized at the C4 position with methyl, morpholine, methoxy, para-tolyl, and 3,4,5-trimethoxyphenyl groups to provide 4-methyl-CyHQ (Me-CyHQ), 4-morpholino-CyHQ (Mor-CyHQ), 4-methoxy-CyHQ (MeO-CyHQ), 4-(p-tolyl)-CyHQ (pTol-CyHQ), and 4-(3,4,5-trimethoxyphenyl)-CyHQ (TMP-CyHQ) PPGs. The probes possess attributes useful for biological use, including high quantum yield (Φu), hydrolytic stability, and good aqueous solubility in physiological conditions. The MeO-CyHQ PPG enhanced the two-photon uncaging action cross section (δu) of dopamine 3.5-fold (0.85 GM) compared to CyHQ (0.24 GM) at 740 nm and 1.49 GM at 720 nm. MeO-CyHQ was used to mediate photoactivation via 2PE of serotonin, rotigotine, N-vanillyl-nonanoylamide (VNA) (a capsaicin analogue), and eugenol. The constructs except rotigotine showed excellent efficiency in 2PE with δu ranging from 0.75 to 1.01 GM at 740 nm and from 1.31 to 1.36 GM at 720 nm high yielding release of the payloads. These probes also performed well by using conventional single photon excitation (1PE). The spatially and temporally controlled release of dopamine from CyHQ-DA and MeO-CyHQ-DA and serotonin (5-HT) from MeO-CyHQ-5HT was quantified in cell culture by using genetically encoded sensors for dopamine and serotonin, respectively. Calcium imaging was employed to quantify the release of VNA and eugenol (EG) from MeO-CyHQ-VNA and MeO-CyHQ-EG, respectively. These tools will enable experiments to understand the intricate mechanisms involved in neurological signaling and the roles played by neurotransmitters, such as dopamine and serotonin, in the activation of their respective receptors.
Assuntos
Fenóis , Serotonina , Fenóis/farmacologia , Eugenol , Preparações de Ação Retardada , DopaminaRESUMO
Two after treatment units, selective catalytic reduction (SCR) and continuously regenerating trap (CRT), were independently retrofitted to a diesel engine, with the objective to investigate their impact on the conversion/reduction (CR) of polycyclic aromatic hydrocarbons (PAHs). The experiments were conducted under the European steady state cycle (ESC) first without any retrofits to get baseline emissions, and then with SCR and CRT respectively, on the same engine. The particulate matter (PM)-phase PAHs were trapped in fiberglass filters, whereas gas-phase PAHs were collected in cartridges, and then analyzed using a gas chromatograph-mass spectrometer (GC-MS). Both PM-phase and gas-phase PAHs were greatly reduced with CRT showing respective CR of 90.7% and above 80%, whereas only gas-phase PAHs were abated in the case of SCR, with CR of above 75%. Lower molecular weight (LMW) PAHs were in abundance, while naphthalene exhibited a maximum relative contribution (RC) to LMW-PAHs for all three cases. Further, the CR of naphthalene and anthracene were increased with increasing catalyst temperature of SCR, most likely due to their conversion to solid particles. Moreover, the Benzo[a]Pyrene equivalent (BaP(eq)) of PAHs was greatly reduced with CRT, owing to substantial reduction of total PAHs.
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Hidrocarbonetos Policíclicos Aromáticos/análise , Meios de Transporte/instrumentação , Emissões de Veículos/análise , Catálise , Peso MolecularRESUMO
Two continuously regenerating diesel particulate filter (CRDPF) with different configurations and one particles oxidation catalyst (POC) were employed to perform experiments in a controlled laboratory setting to evaluate their effects on NO2, smoke and particle number emissions. The results showed that the application of the after-treatments increased the emission ratios of NO2/NOx significantly. The results of smoke emissions and particle number (PN) emissions indicated that both CRDPFs had sufficient capacity to remove more than 90% of total particulate matter (PM) and more than 97% of solid particles. However, the POC was able to remove the organic components of total PM, and only partially to remove the carbonaceous particles with size less than 30 nm. The negligible effects of POC on larger particles were observed due to its honeycomb structure leads to an inadequate residence time to oxidize the solid particles or trap them. The particles removal efficiencies of CRDPFs had high degree of correlations with the emission ratio of NO2/NOx. The PN emission results from two CRDPFs indicated that more NO2 generating in diesel oxidation catalyst section could obtain the higher removal efficiency of solid particles. However this also increased the risk of NO2 exposure in atmosphere.
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Automóveis , Gasolina/análise , Dióxido de Nitrogênio/análise , Material Particulado/química , Emissões de Veículos/análise , Monóxido de Carbono/análise , Catálise , Nitratos/análise , Nitritos/análise , Oxirredução , Tamanho da PartículaRESUMO
The photolysis reactions of (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ)-caged amines have been investigated using time-resolved spectroscopy methods. Unexpectedly, an unconventional Hofmann-Martius rearrangement reaction with high yield and regioselectivity occurred during the photolysis of some CyHQ-protected dialkylanilines (such as compounds 1a and 2a). To have more insights into the mechanism of this unexpected photorearrangement reaction, we characterized the reaction intermediates directly using time-resolved spectroscopy. Our new results showed that the anionic form of compound 1a was photoexcited to the singlet excited state, then a heterolytic cleavage of the C-N bond took place to give CyHQ+ and the corresponding aniline. Thereafter, the recombined intermediate 6 was found to appear in about 19.7 and 44.3 ps for 1a (A) and 2a (A), respectively, before the generation of an ortho-substituted aniline (1b and 2b) via the excited-state deprotonation of 6. Thus, a logical photodynamic mechanism of this photoinduced Hofmann-Martius rearrangement reaction was deduced. This new insight into the reaction mechanisms may be helpful for the design of novel related photoactivatable aniline molecules and for understanding other similar photorearrangement reaction mechanisms.
Assuntos
Quinolinas , Aminas , Compostos de Anilina/química , Fotólise , Análise EspectralRESUMO
On-road emission and fuel consumption (FC) levels for Euro III and IV buses fueled on diesel and compressed natural gas (CNG) were compared, and emission and FC characteristics of buses were analyzed based on approximately 28,700 groups of instantaneous data obtained in Beijing using a portable emissions measurement system (PEMS). The experimental results revealed that NOx and PM emissions from CNG buses were decreased by 72.0% and 82.3% respectively, compared with Euro IV diesel buses. Similarly, these emissions were reduced by 75.2% and 96.3% respectively, compared with Euro III diesel buses. In addition, CO2, CO, HC, NOx, PM emissions and FC of Euro IV diesel buses were reduced by 26.4%, 75.2%, 73.6%, 11.4%, 79.1%, and 26.0%, respectively, relative to Euro III diesel buses. The CO2, CO, HC, NOx, PM emissions and FC factors all decreased with bus speed increased, while increased as bus acceleration increased. At the same time, the emission/FC rates as well as the emission/FC factors exhibited a strong positive correlation with the vehicle specific power (VSP). They all were the lowest when VSP < 0, and then rapidly increased as VSP increased. Furthermore, both the emission/FC rates and emission/FC factors were the highest at accelerations, higher at cruise speeds, and the lowest at decelerations for non-idling buses. These results can provide a base reference to further estimate bus emission and FC inventories in Beijing.
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Poluentes Atmosféricos/química , Cidades , Combustíveis Fósseis , Gasolina , Emissões de Veículos , China , Monitoramento Ambiental/métodos , Humanos , Veículos AutomotoresRESUMO
The effects of continuously regenerating diesel particulate filter (CRDPF) systems on regulated gaseous emissions, and number-size distribution and mass of particles emanated from a diesel engine have been investigated in this study. Two CRDPF units (CRDPF-1 and CRDPF-2) with different specifications were separately retrofitted to the engine running with European steady-state cycle (ESC). An electrical low pressure impactor (ELPI) was used for particle number-size distribution measurement and mass estimation. The conversion/reduction rate (R(CR)) of hydrocarbons (HC) and carbon monoxide (CO) across CRDPF-1 was 83% and 96.3%, respectively. Similarly, the R(CR) of HC and CO and across CRDPF-2 was 91.8% and 99.1%, respectively. The number concentration of particles and their concentration peaks; nuclei mode, accumulation mode and total particles; and particle mass were highly reduced with the CRDPF units. The nuclei mode particles at downstream of CRDPF-1 and CRDPF-2 decreased by 99.9% to 100% and 97.8% to 99.8% respectively; and the particle mass reduced by 73% to 92.2% and 35.3% to 72.4%, respectively, depending on the engine conditions. In addition, nuclei mode particles increased with the increasing of engine speed due to the heterogeneous nucleation initiated by the higher exhaust temperature, while accumulation mode particles were higher at higher loads due to the decrease in the air-to-fuel ratio (A/F) at higher loads.
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Filtração/instrumentação , Gasolina/análise , Veículos Automotores , Tamanho da Partícula , Material Particulado/química , Emissões de Veículos/análise , Ar , Poluentes Atmosféricos/análise , Dióxido de Carbono/análise , Monóxido de Carbono/análise , Europa (Continente) , Hidrocarbonetos/análise , Peso Molecular , Nitratos/análiseRESUMO
Neuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition.
Assuntos
Dopamina/metabolismo , Receptores de Dopamina D2/metabolismo , Sinapses/metabolismo , Animais , Espaço Extracelular/metabolismo , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Cinética , Luz , Masculino , Camundongos Endogâmicos C57BL , Fotólise , Fatores de TempoRESUMO
A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. Compared to the wild type D2 receptor, the novel allelic variant D2-I212F activates a Gαi1ß1γ2 heterotrimer with higher potency and modestly enhanced basal activity in human embryonic kidney (HEK) 293 cells and has decreased capacity to recruit arrestin3. We now report that omitting overexpressed G protein-coupled receptor kinase-2 (GRK2) decreased the potency and efficacy of quinpirole for arrestin recruitment. The relative efficacy of quinpirole for arrestin recruitment to D2-I212F compared to D2-WT was considerably lower without overexpressed GRK2 than with added GRK2. D2-I212F exhibited higher basal activation of GαoA than Gαi1 but little or no increase in the potency of quinpirole relative to D2-WT. Other signs of D2-I212F constitutive activity for G protein-mediated signaling, in addition to basal activation of Gαi/o, were enhanced basal inhibition of forskolin-stimulated cyclic AMP accumulation that was reversed by the inverse agonists sulpiride and spiperone and a â¼4-fold increase in the apparent affinity of D2-I212F for quinpirole, determined from competition binding assays. In mouse midbrain slices, inhibition of tonic current by the inverse agonist sulpiride in dopamine neurons expressing D2-I212F was consistent with our hypothesis of enhanced constitutive activity and sensitivity to dopamine relative to D2-WT. Molecular dynamics simulations with D2 receptor models suggested that an ionic lock between the cytoplasmic ends of the third and sixth α-helices that constrains many G protein-coupled receptors in an inactive conformation spontaneously breaks in D2-I212F. Overall, these results confirm that D2-I212F is a constitutively active and signaling-biased D2 receptor mutant and also suggest that the effect of the likely pathogenic variant in a given brain region will depend on the nature of G protein and GRK expression.
Assuntos
Receptores de Dopamina D2 , Transdução de Sinais , Animais , AMP Cíclico , Agonistas de Dopamina/farmacologia , Células HEK293 , Humanos , Camundongos , Quimpirol/farmacologia , Receptores de Dopamina D2/genéticaRESUMO
The present work is aimed at the study of number-size distribution of particles, volatile organic compounds (VOCs), and carbonyl compounds (CC) or carbonyls emitted from a 4-cylinder turbocharged diesel engine equipped with a vanadium-based urea selective catalytic reduction catalyst. The engine was run on an electric dynamometer in accordance with the European steady-state cycle. Pollutants were analyzed using an electric low pressure impactor, a gas chromatograph/mass spectrometer, and a high performance liquid chromatography system for the number-size distribution of particles, VOCs, and CC emissions, respectively. Experimental results revealed that total number of particles were decreased, and their number-size distributions were moved from smaller sizes to larger sizes in the presence of the catalyst. The VOCs were greatly reduced downstream of the catalyst. There was a strong correlation between the conversion of styrene and ethyl benzene. The conversion rate of benzene increased with increase of catalyst temperature. Formaldehyde, acetaldehyde, acrolein and acetone were significantly reduced, resulting in a remarkable abatement in carbonyls with the use of the vanadium-based urea-SCR system.
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Gasolina/análise , Ureia/química , Vanádio/química , Emissões de Veículos/análise , Poluentes Atmosféricos/química , Catálise , Tamanho da Partícula , Compostos Orgânicos Voláteis/químicaRESUMO
The chemosensory system of any animal relies on a vast array of detectors tuned to distinct chemical cues. Odorant receptors and the ion channels of the TRP family are all uniquely expressed in olfactory tissues in a species-specific manner. Great effort has been made to characterize the molecular and pharmacological properties of these proteins. Nevertheless, most of the natural ligands are highly hydrophobic molecules that are not amenable to controlled delivery. We sought to develop photoreleasable, biologically inactive odorants that could be delivered to the target receptor or ion channel and effectively activated by a short light pulse. Chemically distinct ligands eugenol, benzaldehyde, 2-phenethylamine, ethanethiol, butane-1-thiol, and 2,2-dimethylethane-1-thiol were modified by covalently attaching the photoremovable protecting group (8-cyano-7-hydroxyquinolin-2-yl)methyl (CyHQ). The CyHQ derivatives were shown to release the active odorant upon illumination with 365 and 405 nm light. We characterized their bioactivity by measuring activation of recombinant TRPV1 and TRPA1 ion channels expressed in HEK 293 cells and the electroolfactogram (EOG) response from intact mouse olfactory epithelium (OE). Illumination with 405 nm light was sufficient to robustly activate TRP channels within milliseconds of the light pulse. Photoactivation of channels was superior to activation by conventional bath application of the ligands. Photolysis of the CyHQ-protected odorants efficiently activated an EOG response in a dose-dependent manner with kinetics similar to that evoked by the vaporized odorant amyl acetate (AAc). We conclude that CyHQ-based, photoreleasable odorants can be successfully implemented in chemosensory research.
Assuntos
Benzaldeídos/farmacologia , Eugenol/farmacologia , Hidroxiquinolinas/química , Odorantes , Fenetilaminas/farmacologia , Compostos de Sulfidrila/farmacologia , Animais , Benzaldeídos/síntese química , Eugenol/síntese química , Feminino , Células HEK293 , Humanos , Hidroxiquinolinas/síntese química , Hidroxiquinolinas/efeitos da radiação , Masculino , Camundongos , Mucosa Olfatória/efeitos dos fármacos , Fenetilaminas/síntese química , Compostos de Sulfidrila/síntese química , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismo , Raios UltravioletaRESUMO
Kinetic analysis of dopamine receptor activation and inactivation and the study of dopamine-dependent signaling requires precise simulation of the presynaptic release of the neurotransmitter dopamine and tight temporal control over the release of dopamine receptor antagonists. The 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting group was conjugated to dopamine and the dopamine receptor antagonist sulpiride to generate "caged" versions of these neuromodulators (CyHQ-O-DA and CyHQ-sulpiride, respectively) that could release their payloads with 365 or 405 nm light or through 2-photon excitation (2PE) at 740 nm. These compounds are stable under physiological conditions in the dark, yet photolyze rapidly and cleanly to yield dopamine or sulpiride and the caging remnant CyHQ-OH. CyHQ-O-DA mediated the light activation of dopamine-1 (D1) receptors on the breast cancer cell line MDA-MB-231 in culture. In mouse brain slice from the substantia nigra pars compacta, localized flash photolysis of CyHQ-O-DA accurately mimicked the natural presynaptic release of dopamine and activation of dopamine-2 (D2) receptors, causing a robust, concentration-dependent, and repeatable G protein-coupled inwardly rectifying potassium channel-mediated outward current in whole-cell voltage clamp recordings that was amplified by cocaine and blocked by sulpiride. Photolysis of CyHQ-sulpiride rapidly blocked synaptic activity, enabling measurement of the unbinding rates of dopamine and quinpirole, a D2 receptor agonist. These tools will enable more detailed study of dopamine receptors, their interactions with other GPCRs, and the physiology of dopamine signaling in the brain.