RESUMEN
Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of µM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the ß-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.
Asunto(s)
Antivirales/efectos adversos , Arritmias Cardíacas/inducido químicamente , Cloroquina/farmacología , Hidroxicloroquina/efectos adversos , Potenciales de Acción/efectos de los fármacos , Bioensayo , Simulación por Computador , Correlación de Datos , Relación Dosis-Respuesta a Droga , Canal de Potasio ERG1/agonistas , Canal de Potasio ERG1/antagonistas & inhibidores , Canal de Potasio ERG1/metabolismo , Células HEK293 , Humanos , Concentración 50 Inhibidora , Canal de Potasio KCNQ1/antagonistas & inhibidores , Canal de Potasio KCNQ1/metabolismo , Cinética , Miocitos Cardíacos/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Medición de Riesgo , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
BACKGROUND AND PURPOSE: TRPM4 is a calcium-activated non-selective cation channel expressed in many tissues and implicated in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and selective inhibitors. We sought to discover a novel series of small-molecule inhibitors by combining in silico methods and cell-based screening assay, with sub-micromolar potency and improved selectivity from previously reported TRPM4 inhibitors. EXPERIMENTAL APPROACH: Here, we developed a high throughput screening compatible assay to record TRPM4-mediated Na+ influx in cells using a Na+ -sensitive dye and used this assay to screen a small set of compounds selected by ligand-based virtual screening using previously known weakly active and non-selective TRPM4 inhibitors as seed molecules. Conventional electrophysiological methods were used to validate the potency and selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied using Western blots and electrophysiology experiments. KEY RESULTS: A series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub-micromolar potency and adequate selectivity. We also showed for the first time that a naturally occurring variant of TRPM4, which displays loss-of-expression and function, is rescued by the most promising compound 5 identified in this study. CONCLUSIONS AND IMPLICATIONS: The discovery of compound 5, a potent and selective inhibitor of TRPM4 with an additional chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates.
Asunto(s)
Amidas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Amidas/química , Animales , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Células HEK293 , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Ratones , Células RAW 264.7 , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Canales Catiónicos TRPM/metabolismoRESUMEN
Voltage-gated sodium channels are pore-forming transmembrane proteins that selectively allow sodium ions to flow across the plasma membrane according to the electro-chemical gradient thus mediating the rising phase of action potentials in excitable cells and playing key roles in physiological processes such as neurotransmission, skeletal muscle contraction, heart rhythm, and pain sensation. Genetic variations in the nine human genes encoding these channels are known to cause a large range of diseases affecting the nervous and cardiac systems. Understanding the molecular effect of genetic variations is critical for elucidating the pathologic mechanisms of known variations and in predicting the effect of newly discovered ones. To this end, we have created a Web-based tool, the Ion Channels Variants Portal, which compiles all variants characterized functionally in the human sodium channel genes. This portal describes 672 variants each associated with at least one molecular or clinical phenotypic impact, for a total of 4,658 observations extracted from 264 different research articles. These data were captured as structured annotations using standardized vocabularies and ontologies, such as the Gene Ontology and the Ion Channel ElectroPhysiology Ontology. All these data are available to the scientific community via neXtProt at https://www.nextprot.org/portals/navmut.
Asunto(s)
Biología Computacional , Bases de Datos Genéticas , Mutación , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Biología Computacional/métodos , Fenómenos Electrofisiológicos/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Anotación de Secuencia Molecular , Fenotipo , Dominios Proteicos , Índice de Severidad de la Enfermedad , Programas Informáticos , Canales de Sodio Activados por Voltaje/química , Navegador WebRESUMEN
Ion channels are membrane proteins that gate the flow of ions into and out of a cell. They are present in the membranes of human, animal, plant, and bacterial cells. They are profoundly involved in diverse tasks ranging from neuronal functions to hormonal secretion and cell division. Biophysical characterization and modulation of ion channel targets are important approaches in modern drug discovery. With the heterologous expression of the nicotinic acetylcholine receptor alpha7 (nAChRalpha7) in a host cell, we show a way to construct and use such a stable cell-based expression system for electrophysiological assays.
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Canales Iónicos/fisiología , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Colinérgicos/metabolismo , Humanos , Técnicas de Placa-Clamp , Ratas , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The aim of this work was to compare hepatocellular toxicity and pharmacological activity of amiodarone (2-n-butyl-3-[3,5 diiodo-4-diethylaminoethoxybenzoyl]-benzofuran; B2-O-Et-N-diethyl) and of eight amiodarone derivatives. Three amiodarone metabolites were studied, namely, mono-N-desethylamiodarone (B2-O-Et-NH-ethyl), di-N-desethylamiodarone (B2-O-Et-NH(2)), and (2-butyl-benzofuran-3-yl)-(4-hydroxy-3,5-diiodophenyl)-methanone (B2) carrying an ethanol side chain [(2-butylbenzofuran-3-yl)-[4-(2-hydroxyethoxy)-3,5-diiodophenyl]-methanone; B2-O-Et-OH]. In addition, five amiodarone analogs were investigated, namely, N-dimethylamiodarone (B2-O-Et-N-dimethyl), N-dipropylamiodarone (B2-O-Et-N-dipropyl), B2-O-carrying an acetate side chain [[4-(2-butyl-benzofuran-3-carbonyl)-2,6-diiodophenyl]-acetic acid; B2-O-acetate], B2-O-Et carrying an propionamide side chain (B2-O-Et-propionamide), and B2-O carrying an ethyl side chain [(2-butylbenzofuran-3-yl)-(4-ethoxy-3,5-diiodophenyl)-methanone; B2-O-Et]. A concentration-dependent increase in lactate dehydrogenase leakage from HepG2 cells and isolated rat hepatocytes was observed in the presence of amiodarone and of most analogs, confirming their hepatocellular toxicity. Using freshly isolated rat liver mitochondria, amiodarone and most analogs showed a dose-dependent toxicity on the respiratory chain and on beta-oxidation, significantly reducing the respiratory control ratio and oxidation of palmitate, respectively. The reactive oxygen species concentration in hepatocytes increased time-dependently, and apoptotic/necrotic cell populations were identified using flow cytometry and annexin V/propidium iodide staining. The effect of the three least toxic amiodarone analogs on the human ether-a-go-go-related gene (hERG) channel was compared with amiodarone. Amiodarone, B2-O-acetate, and B2-O-Et-N-dipropyl (each 10 microM) significantly reduced the hERG tail current amplitude, whereas 10 microM B2-O-Et displayed no detectable effect on hERG outward potassium currents. In conclusion, three amiodarone analogs (B2-O-Et-N-dipropyl, B2-O-acetate, and B2-O-Et) showed a lower hepatocellular toxicity profile than amiodarone, and two of these analogs (B2-O-Et-N-dipropyl and B2-O-acetate) retained hERG channel interaction capacity, suggesting that amiodarone analogs with class III antiarrhythmic activity and lower hepatic toxicity could be developed.
Asunto(s)
Amiodarona/análogos & derivados , Amiodarona/toxicidad , Antiarrítmicos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Adenosina Trifosfato/metabolismo , Amiodarona/farmacología , Animales , Anexina A5 , Antiarrítmicos/síntesis química , Antiarrítmicos/farmacología , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular , Separación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fenómenos Químicos , Química Física , Colorantes , Cricetinae , Electrofisiología , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Técnicas In Vitro , Indicadores y Reactivos , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/patología , Necrosis , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Propidio , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , SolubilidadRESUMEN
1,4-Addition of benzylamine to 2(5H)-furanone followed by dialkylation of the 3-position with allylbromide gave (+/-)-4-benzyl-3,3-diallyl-2(3H)-furanone (8), which served as the intermediate for the synthesis of various N-substituted 4-amino-3,3-dipropyl-2(3H)-furanones (+/-)-9a-l. The compounds were evaluated for their capacity to potentiate or inhibit GABA-evoked currents in Xenopus laevis oocytes expressing recombinant alpha1beta2gamma2 GABA(A) receptors. The benzyl, ethyl, and allyl carbamates ((R)-9a (100 microM), (+/-)-9b (100 microM), (+/-)-9c (200 microM)) stimulated GABA currents by 279 +/- 47%, 426 +/- 8%. and 765 +/- 61%, respectively, while the phenylcarboxamide (+/-)-9f (200 microM) stimulated currents by 420 +/- 33%. Concentration-response studies showed that compound 9c was approximately twice as potent in stimulating GABA currents as alpha-EMTBL (2), the most potent 3,3-dialkylbutyrolactone known to date. On the other hand, the N-sulfonyl analogues were much less active or even inhibited GABA-evoked currents. In vitro radioligand displacement studies on rat brain membranes showed that these compounds did not bind to the benzodiazepine or GABA recognition sites of the GABA(A) receptor. However, these compounds generally weakly displaced [(35)S]-TBPS (approximately 50% displacement at 100 microM), though potencies did not correlate with GABA current potentiation. Results obtained with alpha1beta1 and mutant alpha1beta2N265S receptors, which compared to alpha1beta2 receptors are both much less sensitive to current stimulation produced by the anticonvulsant loreclezole, suggest that at least some of these aminobutyrolactones, (e.g., 9a, 9c), and interestingly also alpha-EMTBL, share stimulatory properties with loreclezole.