RESUMEN
In the current study effects of fungal extracts on the G-protein-activated inwardly rectifying potassium channel (GIRK1/4) were screened using the automated patch-clamp method. 40 organic (n-hexane, chloroform, and 50% methanol) and aqueous extracts were prepared from 10 mushroom species native to Hungary. Among the examined fungal fractions of different polarities some n-hexane and chloroform extracts exerted considerable ion channel activity. One of the most active fungal species, Hypholoma lateritium was selected for further detailed examination to determine the compounds responsible for the observed pharmacological property. Evaluation of the ion channel activity of mushroom metabolites 1-10 revealed that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) demonstrates remarkable blocking activity on GIRK current (IC50 395.1⯱â¯31.8â¯nM). Investigation of the selectivity of the GIRK inhibitory effect proved that lanosta-7,9(11)-diene-12ß,21α-epoxy-2α,3ß,24ß,25-tetraol (5) has only weak inhibitory activity on hERG channel (7.9⯱â¯2.8% at 100⯵M), exerting more than three orders of magnitude lower blocking activity on hERG channel than on GIRK channel.
Asunto(s)
Agaricales/química , Canal de Potasio ERG1/antagonistas & inhibidores , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Células HEK293 , Humanos , Hungría , Estructura Molecular , Técnicas de Placa-ClampRESUMEN
The proarrhythmic potency of drugs is usually attributed to the IKr current block. During safety pharmacology testing analysis of IKr in cardiomyocytes was replaced by human ether-a-go-go-related gene (hERG) test using automated patch-clamp systems in stable transfected cell lines. Aim of this study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch-clamp methods on HEK293 cells. The native ion currents (IKr, INaL, ICaL) were recorded from rabbit ventricular myocytes by manual patch-clamp technique. Action potentials in rabbit ventricular muscle and undiseased human donor hearts were studied by conventional microelectrode technique. Dofetilide, cisapride, sotalol, terfenadine, and verapamil blocked hERG channels at 37°C with an IC50 of 7 nM, 18 nM, 343 µM, 165 nM, and 214 nM, respectively. Using manual patch-clamp, the IC50 values of sotalol and terfenadine were 78 µM and 31 nM, respectively. The IC50 values calculated from IKr measurements at 37°C were 13 nM, 26 nM, 52 µM, 54 nM, and 268 nM, respectively. Cisapride, dofetilide, and sotalol excessively lengthened, terfenadine, and verapamil did not influence the action potential duration. Terfenadine significantly inhibited INaL and moderately ICaL, verapamil blocked only ICaL. Automated hERG assays may over/underestimate proarrhythmic risk. Manual patch-clamp has substantially higher sensitivity to certain drugs. Action potential studies are also required to analyze complex multichannel effects. Therefore, manual patch-clamp and action potential experiments should be a part of preclinical safety tests.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Antiarrítmicos/toxicidad , Ventrículos Cardíacos/efectos de los fármacos , Canales Iónicos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Bloqueadores de los Canales de Potasio/toxicidad , Animales , Evaluación Preclínica de Medicamentos , Canal de Potasio ERG1/metabolismo , Femenino , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Fenetilaminas/toxicidad , Conejos , Sotalol/toxicidad , Sulfonamidas/toxicidad , Terfenadina/toxicidad , Donantes de Tejidos , Verapamilo/toxicidadRESUMEN
Aconitum diterpene alkaloids are known for their remarkable toxicity, which is due to their effect on ion channels. Activation of voltage-gated Na+ channels is the major cause of their cardiotoxicity, however, influence on K+ channels may also play a role in the overall effect.Here we report the synthesis of a series of lipo-alkaloids, including four new compounds, based on the 14-benzoylaconine structure, which is the core of a vast number of diterpene alkaloids naturally occurring in Aconitum species. The activities of these compounds were measured in vitro on K+ ion channels using the whole-cell patch-clamp technique. Structure-activity analysis was carried out based on the data of 51 compounds (32 genuine diterpene alkaloids, 5 fatty acids, and 14 lipo-alkaloids). Depending on their substitution, these compounds exert different activities on GIRK (G protein-coupled inwardly-rectifying potassium channel) and hERG (human ether-à-go-go-related gene) channels. Fatty acids and diterpene alkaloids show lower activity on the GIRK channel than lipo-alkaloids. Lipo-alkaloids also have less pronounced hERG inhibitory activity compared to the cardiotoxic aconitine. Considering the GIRK/hERG selectivity as an indicator of perspective therapeutic applicability, lipo-alkaloids are significantly more selective than the genuine diterpene alkaloids. 14-Benzoyl-8-O-eicosa-8Z,11Z,14Z-trienoate and 14-benzoyl-8-O-eicosa-11Z,14Z,17Z-trienoate are strong and selective inhibitors of GIRK channels, thus, they are promising subjects for further studies to develop diterpene alkaloid-based antiarrhythmic pharmacons.
Asunto(s)
Aconitum/química , Alcaloides/farmacología , Diterpenos/farmacología , Corazón/efectos de los fármacos , Extractos Vegetales/farmacología , Canales de Potasio/efectos de los fármacos , Alcaloides/síntesis química , Diterpenos/síntesis química , Células HEK293 , Humanos , Técnicas de Placa-Clamp , Plantas Medicinales/química , Relación Estructura-ActividadRESUMEN
Chelidonium majus or greater celandine is spread throughout the world, and it is a very common and frequent component of modern phytotherapy. Although C. majus contains alkaloids with remarkable physiological effect, moreover, safety pharmacology properties of this plant are not widely clarified, medications prepared from this plant are often used internally. In our study the inhibitory effects of C. majus herb extracts and alkaloids on hERG potassium current as well as on cardiac action potential were investigated. Our data show that hydroalcoholic extracts of greater celandine and its alkaloids, especially berberine, chelidonine and sanguinarine have a significant hERG potassium channel blocking effect. These extracts and alkaloids also prolong the cardiac action potential in dog ventricular muscle. Therefore these compounds may consequently delay cardiac repolarization, which may result in the prolongation of the QT interval and increase the risk of potentially fatal ventricular arrhythmias.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Alcaloides/farmacología , Chelidonium/química , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Corazón/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Perros , Femenino , Células HEK293 , Humanos , Masculino , Técnicas de Placa-ClampRESUMEN
The G protein-activated inwardly rectifying K+ channel-modulatory activities of Polygonum persicaria extracts were investigated by using an automated patch-clamp method, with the aim of identifying natural sources of promising ion channel-blocking compounds. The chloroform extract of the whole plant at 0.1 mg/mL exhibited high G protein-activated inwardly rectifying K+ channel-inhibitory activity. Fractionation of this extract by vacuum liquid chromatography on RP-silica gel resulted in 6 fractions, which were evaluated for G protein-activated inwardly rectifying K+ channel-modulatory activity. RP-HPLC of the most active fractions afforded the main compounds 1-4 in pure form and a mixture containing the minor constituents. The structures were identified by means of UV, HRMS, and advanced NMR methods as 3-O-senecioyl-isorhamnetin (1), 3-O-angeloyl-isorhamnetin (2), 5,3',4',5'-tetramethoxy-6,7-methylenedioxyflavone (3), and 3,5,3',4',5'-pentamethoxy-6,7-methylenedioxyflavone (4). Compounds 1-4 are new natural products, though 4 was reported earlier as a synthetic compound. Neither the individual, nor the combined application of compounds 1-4 modified the G protein-activated inwardly rectifying K+ channel activity. However, a marked G protein-activated inwardly rectifying K+ current-inhibitory effect was detected on use of the HPLC eluates containing the minor compounds. These results indicate the presence of electrophysiologically active agents among the minor compounds.
Asunto(s)
Flavonoides/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Extractos Vegetales/farmacología , Polygonum/química , Cloroformo , Ésteres/química , Ésteres/aislamiento & purificación , Ésteres/farmacología , Flavonoides/química , Flavonoides/aislamiento & purificación , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Proteínas de Unión al GTP/metabolismo , Estructura Molecular , Técnicas de Placa-Clamp , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Plantas MedicinalesRESUMEN
Diterpene alkaloids neoline (1), napelline (2), isotalatizidine (3), karakoline (4), senbusine A (5), senbusine C (6), aconitine (7) and taurenine (8) were identified from Aconitum napellus L. subsp. firmum, four (2-4, 6) of which are reported for the first time from this plant. The structures were determined by means of LC-MS, 1D and 2D NMR spectroscopy, including (1)H-(1)H COSY, NOESY, HSQC and HMBC experiments. Electrophysiological effects of the isolated compounds, together with nine diterpene alkaloids previously obtained from Aconitum toxicum and Consolida orientalis were investigated on stable transfected HEK-hERG (Kv11.1) and HEK-GIRK1/4 (Kir3.1 and Kir3.4) cell lines using automated patch clamp equipment. Significant blocking activity on GIRK channel was exerted by aconitine (7) (45% at 10 µM), but no blocking activities of the other investigated compounds were detected. The tested compounds were inactive on hERG channel in the tested concentration. The comparison of the previously reported metabolites of A. napellus subsp. firmum and compounds identified in our experiment reveals substantial variability of the alkaloid profile of this taxon.