RESUMEN

The human Ether-à-go-go-Related Gene (hERG) is a transmembrane protein that regulates cardiac action potential, and its inhibition can induce a potentially deadly cardiac syndrome. In vitro tests help identify hERG blockers at early stages; however, the high cost motivates searching for alternative, cost-effective methods. The primary goal of this study was to enhance the Pred-hERG tool for predicting hERG blockage. To achieve this, we developed new QSAR models that incorporated additional data, updated existing classificatory and multiclassificatory models, and introduced new regression models. Notably, we integrated SHAP (SHapley Additive exPlanations) values to offer a visual interpretation of these models. Utilizing the latest data from ChEMBL v30, encompassing over 14,364 compounds with hERG data, our binary and multiclassification models outperformed both the previous iteration of Pred-hERG and all publicly available models. Notably, the new version of our tool introduces a regression model for predicting hERG activity (pIC50). The optimal model demonstrated an R2 of 0.61 and an RMSE of 0.48, surpassing the only available regression model in the literature. Pred-hERG 5.0 now offers users a swift, reliable, and user-friendly platform for the early assessment of chemically induced cardiotoxicity through hERG blockage. The tool provides versatile outcomes, including (i) classificatory predictions of hERG blockage with prediction reliability, (ii) multiclassificatory predictions of hERG blockage with reliability, (iii) regression predictions with estimated pIC50 values, and (iv) probability maps illustrating the contribution of chemical fragments for each prediction. Furthermore, we implemented explainable AI analysis (XAI) to visualize SHAP values, providing insights into the contribution of each feature to binary classification predictions. A consensus prediction calculated based on the predictions of the three developed models is also present to assist the user's decision-making process. Pred-hERG 5.0 has been designed to be user-friendly, making it accessible to users without computational or programming expertise. The tool is freely available at http://predherg.labmol.com.br.

Asunto(s)

Canales de Potasio Éter-A-Go-Go , Relación Estructura-Actividad Cuantitativa , Humanos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Medición de Riesgo , Análisis de Regresión , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Potasio/química

RESUMEN

Pamabrom is a diuretic that is effective in treating premenstrual syndrome and primary dysmenorrhea. The aim of this study was to examine the effect of metformin and modulators of the opioid receptor-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP)-K+ channel pathway on the local antinociception induced by pamabrom. The rat paw 1% formalin test was used to assess the effects. Rats were treated with local administration of pamabrom (200-800 µg/paw) or indomethacin (200-800 µg/paw). The antinociception of pamabrom or indomethacin was evaluated with and without the local pretreatment of the blockers. Local administration of pamabrom and indomethacin produced dose-dependent antinociception during the second phase of the test. Local pretreatment of the paws with naloxone (50 µg/paw), l-nitro-arginine methyl ester (10-100 µg/paw), or 1H-(1,2,4)-oxadiazolo[4,2-a]quinoxalin-1-one (10-100 µg/paw) reverted the antinociception induced by local pamabrom, but not of indomethacin. Similarly, the K+ channel blockers glibenclamide, glipizide, 4-aminopyridine, tetraethylammonium, charybdotoxin, or apamin reverted the pamabrom-induced antinociception, but not of indomethacin. Metformin significantly blocked the antinociception of pamabrom and indomethacin. Our data suggest that pamabrom could activate the opioid receptor-NO-cGMP-K+ channel pathway to produce its peripheral antinociception in the formalin test. Likewise, a biguanide-dependent mechanism could be activated by pamabrom and indomethacin to generate antinociception.

Asunto(s)

Metformina , Naloxona , Femenino , Ratas , Animales , Naloxona/farmacología , GMP Cíclico/metabolismo , Ratas Wistar , Óxido Nítrico/metabolismo , Diuréticos , Metformina/farmacología , Indometacina , Receptores Opioides , Analgésicos/farmacología , Bloqueadores de los Canales de Potasio/farmacología

RESUMEN

Hydrogen sulfide (H2S) is a gasotransmitter that modulates neurotransmission. Indeed, it has been recently demonstrated that H2S inhibits the sympathetic outflow in male rats, although the mechanisms remain elusive. Thus, this study evaluated the role of potassium channels on NaHS-induced sympathoinhibition. For this purpose, male and female Wistar rats were anesthetized, pithed, and cannulated. After that, animals received selective electrical stimulation of the vasopressor sympathetic outflow (T7-T9). Prior to 310 µg/kg·min NaHS i.v. continuous infusion animals received: (1) bidistilled water (tetraethylammonium, TEA; 4-aminopyridine, 4-AP; and barium chloride, BaCl2; vehicle; 1 ml/kg); (2) TEA (non-selective K+ channels blocker; 16.5 mg/kg); (3) 4-AP (non-selective voltage-dependent K+ channels blocker; 5 mg/kg); (4) BaCl2 (inward rectifier K+ channels blocker; 65 µg/kg); (5) DMF 5%, glucose 10% and NaOH 0.1 N (glibenclamide vehicle; 1 ml/kg); (6) glibenclamide (ATP-dependent K+ channels blocker; 10 mg/kg); (7) DMSO 4% (paxilline vehicle; 1 ml/kg); and (8) paxilline (large-conductance voltage- and Ca2+-activated K+ channel blocker; 90 µg/kg). The NaHS-induced sympathoinhibition was: (1) equally observed in male and female rats; (2) unaffected by vehicles; (3) reversed by the potassium channel blockers. Taken together, our results suggest that NaHS-induced sympathoinhibition does not depend on sex and it is mediated by the activation of several potassium channels.

Asunto(s)

Sulfuro de Hidrógeno , 4-Aminopiridina/farmacología , Animales , Femenino , Gliburida/farmacología , Sulfuro de Hidrógeno/farmacología , Masculino , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio , Ratas , Ratas Wistar , Vasoconstrictores/farmacología

RESUMEN

The objective of the present study was to scrutinize the effect of nitric oxide (NO), cyclic GMP (cGMP), potassium channel blockers, and metformin on the citral-produced peripheral antinociception. The rat paw 1% formalin test was used to assess nociception and antinociception. Rats were treated with local peripheral administration of citral (10-100 µg/paw). The antinociception of citral (100 µg/paw) was evaluated with and without the local pretreatment of naloxone, NG-L-nitro-arginine methyl ester (L-NAME, a NO synthesis inhibitor), 1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor), metformin, opioid receptors antagonists, and K+ channel blockers. Injection of citral in the rat paw significantly decreased the nociceptive effect of formalin administration during the two phases of the test. Local pretreatment of the paws with L-NAME and ODQ did not reduced the citral-induced antinociception. Glipizide or glibenclamide (Kir6.1-2; ATP-sensitive K+ channel blockers), tetraethylammonium or 4-aminopyridine (KV; voltage-gated K+ channel blockers), charybdotoxin (KCa1.1; big conductance calcium-activated K+ channel blocker), apamin (KCa2.1-3; small conductance Ca2+-activated K+ channel antagonist), or metformin, but not the opioid antagonists, reduced the antinociception of citral. Citral produced peripheral antinociception during both phases of the formalin test. These effects were due to the activation of K+ channels and a biguanide-dependent mechanism.

Asunto(s)

GMP Cíclico , Metformina , Monoterpenos Acíclicos , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , GMP Cíclico/metabolismo , Metformina/farmacología , Óxido Nítrico/metabolismo , Nocicepción , Dimensión del Dolor , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Wistar , Receptores Opioides/metabolismo

RESUMEN

The Colombian scorpion Centruroides margaritatus produces a venom considered of low toxicity. Nevertheless, there are known cases of envenomation resulting in cardiovascular disorders, probably due to venom components that target ion channels. Among them, the humanether-à-go-go-Related gene (hERG1) potassium channels are critical for cardiac action potential repolarization and alteration in its functionality are associated with cardiac disorders. This work describes the purification and electrophysiological characterization of a Centruroides margaritatus venom component acting on hERG1 channels, the CmERG1 toxin. This novel peptide is composed of 42 amino acids with a MW of 4792.88 Da, folded by four disulfide bonds and it is classified as member number 10 of the γ-KTx1 toxin family. CmERG1 inhibits hERG1 currents with an IC50 of 3.4 ± 0.2 nM. Despite its 90.5% identity with toxin É£-KTx1.1, isolated from Centruroides noxius, CmERG1 completely blocks hERG1 current, suggesting a more stable plug of the hERG channel, compared to that formed by other É£-KTx.

Asunto(s)

Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/farmacología , Animales , Colombia , Canales de Potasio Éter-A-Go-Go/fisiología , Péptidos/aislamiento & purificación , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Venenos de Escorpión/aislamiento & purificación , Escorpiones

RESUMEN

BACKGROUND: Ion channels have been proposed as therapeutic targets for different types of malignancies. One of the most studied ion channels in cancer is the voltage-gated potassium channel ether-à-go-go 1 or Kv10.1. Various studies have shown that Kv10.1 expression induces the proliferation of several cancer cell lines and in vivo tumor models, while blocking or silencing inhibits proliferation. Kv10.1 is a promising target for drug discovery modulators that could be used in cancer treatment. This work aimed to screen for new Kv10.1 channel modulators using a thallium influx-based assay. METHODS: Pharmacological effects of small molecules on Kv10.1 channel activity were studied using a thallium-based fluorescent assay and patch-clamp electrophysiological recordings, both performed in HEK293 stably expressing the human Kv10.1 potassium channel. RESULTS: In thallium-sensitive fluorescent assays, we found that the small molecules loperamide and amitriptyline exert a potent inhibition on the activity of the oncogenic potassium channel Kv10.1. These results were confirmed by electrophysiological recordings, which showed that loperamide and amitriptyline decreased the amplitude of Kv10.1 currents in a dose-dependent manner. Both drugs could be promising tools for further studies. CONCLUSIONS: Thallium-sensitive fluorescent assay represents a reliable methodological tool for the primary screening of different molecules with potential activity on Kv10.1 channels or other K+ channels.

Asunto(s)

Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Loperamida/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Relación Dosis-Respuesta a Droga , Fluorescencia , Células HEK293 , Humanos , Loperamida/administración & dosificación , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/administración & dosificación , Reproducibilidad de los Resultados , Talio/metabolismo

RESUMEN

The aim of this study was to examine if the peripheral antinociception of α-bisabolol involves the participation of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) synthesis followed by K+ channel opening in the formalin test. Wistar rats were injected in the dorsal surface of the right hind paw with formalin (1%). Rats received a subcutaneous injection into the dorsal surface of the paw of vehicles or increasing doses of α-bisabolol (100-300 µg/paw). To determine whether the peripheral antinociception induced by α-bisabolol was mediated by either the opioid receptors or the NO-cGMP-K+ channels pathway, the effect of pretreatment (10 min before formalin injection) with the appropriate vehicles, naloxone, naltrexone, NG-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]-oxadiazolo[4,2-a]quinoxalin-1-one (ODQ), glibenclamide, glipizide, apamin, charybdotoxin, tetraethylammonium, or 4-aminopyridine on the antinociceptive effects induced by local peripheral α-bisabolol (300 µg/paw) were assessed. α-Bisabolol produced antinociception during both phases of the formalin test. α-Bisabolol antinociception was blocked by L-NAME, ODQ, and all the K+ channels blockers. The peripheral antinociceptive effect produced by α-bisabolol was not blocked by the opioid receptor inhibitors. α-Bisabolol was able to active the NO-cGMP-K+ channels pathway to produce its antinoceptive effect. The participation of opioid receptors in the peripheral local antinociception induced by α-bisabolol is excluded.

Asunto(s)

Analgésicos/farmacología , GMP Cíclico/metabolismo , Sesquiterpenos Monocíclicos/farmacología , Óxido Nítrico/metabolismo , Nocicepción/efectos de los fármacos , Canales de Potasio/metabolismo , Receptores Opioides/metabolismo , Animales , Masculino , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/química , Canales de Potasio/genética , Ratas , Ratas Wistar , Receptores Opioides/química , Receptores Opioides/genética

RESUMEN

Riluzole is an anticonvulsant drug also used to treat the amyotrophic lateral sclerosis and major depressive disorder. This compound has antiglutamatergic activity and is an important multichannel blocker. However, little is known about its actions on the Kv4.2 channels, the molecular correlate of the A-type K+ current (IA) and the fast transient outward current (Itof). Here, we investigated the effects of riluzole on Kv4.2 channels transiently expressed in HEK-293 cells. Riluzole inhibited Kv4.2 channels with an IC50 of 190 ± 14 µM and the effect was voltage- and frequency-independent. The activation rate of the current (at +50 mV) was not affected by the drug, nor the voltage dependence of channel activation, but the inactivation rate was accelerated by 100 and 300 µM riluzole. When Kv4.2 channels were maintained at the closed state, riluzole incubation induced a tonic current inhibition. In addition, riluzole significantly shifted the voltage dependence of inactivation to hyperpolarized potentials without affecting the recovery from inactivation. In the presence of the drug, the closed-state inactivation was significantly accelerated, and the percentage of inactivated channels was increased. Altogether, our findings indicate that riluzole inhibits Kv4.2 channels mainly affecting the closed and closed-inactivated states.

Asunto(s)

Bloqueadores de los Canales de Potasio/farmacología , Riluzol/farmacología , Canales de Potasio Shal/antagonistas & inhibidores , Células HEK293 , Humanos , Activación del Canal Iónico , Potenciales de la Membrana , Canales de Potasio Shal/genética , Canales de Potasio Shal/metabolismo , Factores de Tiempo

RESUMEN

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey's excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4-5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.

Asunto(s)

Conotoxinas/metabolismo , Canales de Potasio de la Superfamilia Shaker/metabolismo , Animales , Conotoxinas/química , Oocitos , Concentración Osmolar , Bloqueadores de los Canales de Potasio/farmacología , Unión Proteica , Canales de Potasio de la Superfamilia Shaker/química , Xenopus laevis

RESUMEN

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

Asunto(s)

Venenos de Moluscos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Animales , Caracol Conus , Activación del Canal Iónico , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.4/antagonistas & inhibidores , Canal de Potasio Kv1.4/genética , Canal de Potasio Kv1.4/metabolismo , Canal de Potasio Kv1.5/antagonistas & inhibidores , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/metabolismo , Canal de Potasio Kv1.6/antagonistas & inhibidores , Canal de Potasio Kv1.6/genética , Canal de Potasio Kv1.6/metabolismo , Potenciales de la Membrana , Oocitos , Canales de Potasio de la Superfamilia Shaker/genética , Canales de Potasio de la Superfamilia Shaker/metabolismo , Xenopus laevis

RESUMEN

Adenosine triphosphate (ATP)-dependent potassium channels openers (KATP) protect skeletal muscle against function impairment through the activation of the mitochondrial KATP channels (mitoKATP). Previous reports suggest that modulators of the mitochondrial KATP channels have additional effects on isolated mitochondria. To determine whether the KATP channel opener nicorandil has non-specific effects that explain its protective effect through the mitochondrial function, chicken muscle mitochondria were isolated, and respiration rate was determined pollarographically. The activity of the electron transport chain (ETC) complexes (I-IV) was measured using a spectrophotometric method. Reactive oxygen species (ROS) levels and lipid peroxidation were assessed using flow cytometry and thiobarbituric acid assay, respectively. Both KATP channel opener nicorandil and KATP channel blocker 5-hydroxydecanoate (5-HD) decreased mitochondrial respiration; nicorandil increased complex III activity and decreased complex IV activity. The effects of nicorandil on complex III were antagonized by 5-HD. Nicorandil increased ROS levels, effect reverted by either 5-HD or the antioxidant N-2-mercaptopropionyl glycine (MPG). None of these drugs affected lipid peroxidation levels. These findings suggest that KATP channel opener nicorandil increases mitochondrial ROS production from complex III. This results by partially blocking electron flow in the complex IV, setting electron carriers in a more reduced state, which is favored by the increase in complex III activity by nicorandil. Overall, our study showed that nicorandil like other mitochondrial KATP channel openers might not act through mitoKATP channel activation.

Asunto(s)

Complejo III de Transporte de Electrones/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Nicorandil/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Respiración de la Célula/efectos de los fármacos , Pollos , Transporte de Electrón/efectos de los fármacos , Canales KATP/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Oxidación-Reducción , Consumo de Oxígeno , Bloqueadores de los Canales de Potasio/farmacología

RESUMEN

Withaferin A (WFA), a C5,C6-epoxy steroidal lactone isolated from the medicinal plant Withania somnifera (L.) Dunal, inhibits growth of tumor cells in different cancer types. However, the mechanisms underlying the effect of WFA on tumor cells are not fully understood. In the present study, we evaluated the blockade of TASK-3 channels by WFA in TASK-3-expressing HEK-293 cells. Explore if the WFA-mediated TASK-3 blockade can be used as a pharmacological tool to decrease the cell viability in cancer cells. A combination of functional experiments (patch-clamp, gene downregulation, overexpression and pharmacological inhibition) and molecular docking analysis were used to get insights into the mechanism by which the inhibition of TASK-3 by WFA affects the growth and viability of cancer cells. Withaferin A was found to inhibit the activity of TASK-3 channels. The inhibitory effect of Withaferin A on TASK-3 potassium currents was dose-dependent and independent of voltage. Molecular modeling studies identified putative WFA-binding sites in TASK-3 channel involved the channel blockade. In agreements with the molecular modeling predictions, mutation of residues F125 to A (F125A), L197 to V (L197 V) and the double mutant F125A-L197 V markedly decreased the WFA-induced inhibition of TASK-3. Finally, the cytotoxic effect of WFA was tested in MDA-MB-231 human breast cancer cells transfected with TASK-3 or shRNA that decreases TASK-3 expression. Together, our results show that the cytotoxic effect of WFA on fully transformed MDA-MB-231 cells depends on the expression of TASK-3. Herein, we also provide insights into the mechanism of TASK-3 inhibition by WFA.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Witanólidos/farmacología , Antineoplásicos Fitogénicos/metabolismo , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Potenciales de la Membrana , Bloqueadores de los Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Unión Proteica , Transducción de Señal , Witanólidos/metabolismo

RESUMEN

The mechanisms underlying atrial-selective prolongation of effective refractory period (ERP) and suppression of atrial fibrillation (AF) by NS8593 and UCL1684, small conductance calcium-activated potassium (SK) channel blockers, are poorly defined. The purpose of the study was to confirm the effectiveness of these agents to suppress AF and to probe the underlying mechanisms. Transmembrane action potentials and pseudoelectrocardiograms were recorded from canine isolated coronary-perfused canine atrial and ventricular wedge preparations. Patch clamp techniques were used to record sodium channel current (INa) in atrial and ventricular myocytes and human embryonic kidney cells. In both atria and ventricles, NS8593 (3-10 µM) and UCL1684 (0.5 µM) did not significantly alter action potential duration, suggesting little to no SK channel inhibition. Both agents caused atrial-selective: (1) prolongation of ERP secondary to development of postrepolarization refractoriness, (2) reduction of Vmax, and (3) increase of diastolic threshold of excitation (all are sodium-mediated parameters). NS8593 and UCL1684 significantly reduced INa density in human embryonic kidney cells as well as in atrial but not in ventricular myocytes at physiologically relevant holding potentials. NS8593 caused a shift of steady-state inactivation to negative potentials in atrial but not ventricular cells. NS8593 and UCL1684 prevented induction of acetylcholine-mediated AF in 6/6 and 8/8 preparations, respectively. This anti-AF effect was associated with strong rate-dependent depression of excitability. The SK channel blockers, NS8593 and UCL1684, are effective in preventing the development of AF due to potent atrial-selective inhibition of INa, causing atrial-selective prolongation of ERP secondary to induction of postrepolarization refractoriness.

Asunto(s)

1-Naftilamina/análogos & derivados , Alcanos/farmacología , Antiarrítmicos/farmacología , Fibrilación Atrial/prevención & control , Atrios Cardíacos/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.5/efectos de los fármacos , Compuestos de Quinolinio/farmacología , Bloqueadores de los Canales de Sodio/farmacología , 1-Naftilamina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/fisiopatología , Perros , Femenino , Células HEK293 , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Periodo Refractario Electrofisiológico/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo

RESUMEN

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

Asunto(s)

Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Óxido Nítrico/metabolismo , Venas Umbilicales/metabolismo , Adulto , Arginina/metabolismo , Transportador de Aminoácidos Catiónicos 1/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Insulina/farmacología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Péptidos/farmacología , Placenta/efectos de los fármacos , Placenta/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Embarazo , Pirazoles/farmacología , Venas Umbilicales/efectos de los fármacos , Adulto Joven

RESUMEN

Snake venom serine proteases (SVSPs) are complex and multifunctional enzymes, acting primarily on hemostasis. In this work, we report the hitherto unknown inhibitory effect of a SVSP, named collinein-1, isolated from the venom of Crotalus durissus collilineatus, on a cancer-relevant voltage-gated potassium channel (hEAG1). Among 12 voltage-gated ion channels tested, collinein-1 selectively inhibited hEAG1 currents, with a mechanism independent of its enzymatic activity. Corroboratively, we demonstrated that collinein-1 reduced the viability of human breast cancer cell line MCF7 (high expression of hEAG1), but does not affect the liver carcinoma and the non-tumorigenic epithelial breast cell lines (HepG2 and MCF10A, respectively), which present low expression of hEAG1. In order to obtain both functional and structural validation of this unexpected discovery, where an unusually large ligand acts as an inhibitor of an ion channel, a recombinant and catalytically inactive mutant of collinein-1 (His43Arg) was produced and found to preserve its capability to inhibit hEAG1. A molecular docking model was proposed in which Arg79 of the SVSP 99-loop interacts directly with the potassium selectivity filter of the hEAG1 channel.

Asunto(s)

Hemostasis , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Serina Proteasas/toxicidad , Venenos de Serpiente/toxicidad , Secuencia de Aminoácidos , Antineoplásicos/farmacología , Catálisis , Línea Celular , Diseño de Fármacos , Fenómenos Electrofisiológicos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/química , Humanos , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Bloqueadores de los Canales de Potasio/química , Canales de Potasio/química , Proteínas Recombinantes , Serina Proteasas/química , Venenos de Serpiente/química , Relación Estructura-Actividad

RESUMEN

Kv10.1 (Eag1, or KCNH1) is a human potassium-selective channel associated with tumor development. In this work, we study the interaction of the drug dronedarone with Kv10.1. Dronedarone presents two chemical modifications aimed to lessen side effects produced by its parent molecule, the antiarrhythmic amiodarone. Hence, our observations are discussed within the framework of a previously reported interaction of amiodarone with Kv10.1. Additionally, we show new data regarding the interaction of amiodarone with the channels. We found that, unexpectedly, the effect of dronedarone on Kv10.1 differs both quantitatively and qualitatively to that of amiodarone. Among other observations, we found that dronedarone seems to be an open-pore blocker, in contrast to the reported behavior of amiodarone, which seems to inhibit from both open and closed states. Additionally, herein we provide evidence showing that, in spite of their chemical similarity, these molecules inhibit the K+ conductance by binding to non-overlapping, independent (non-allosterically related) sites. Also, we show that, while amiodarone inhibits the Cole-Moore shift, dronedarone is unable to inhibit this voltage-dependent characteristic of Kv10.1.

Asunto(s)

Dronedarona/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Potenciales de Acción , Dronedarona/química , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Relación Estructura-Actividad

RESUMEN

4-Aminopyridine (4AP) is a specific blocker of voltage-gated potassium channels (KV1 family) clinically approved for the symptomatic treatment of patients with multiple sclerosis (MS). It has recently been shown that [18F]3F4AP, a radiofluorinated analog of 4AP, also binds to KV1 channels and can be used as a PET tracer for the detection of demyelinated lesions in rodent models of MS. Here, we investigate four novel 4AP derivatives containing methyl (-CH3), methoxy (-OCH3) as well as trifluoromethyl (-CF3) in the 2 and 3 position as potential candidates for PET imaging and/or therapy. We characterized the physicochemical properties of these compounds (basicity and lipophilicity) and analyzed their ability to block Shaker K+ channel under different voltage and pH conditions. Our results demonstrate that three of the four derivatives are able to block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Me4AP) was found to be approximately 7-fold more potent than 4AP and 3F4AP; 3-methoxy- and 3-trifluoromethyl-4-aminopyridine (3MeO4AP and 3CF34AP) were found to be about 3- to 4-fold less potent than 4AP; and 2-trifluoromethyl-4-AP (2CF34AP) was found to be about 60-fold less active. These results suggest that these novel derivatives are potential candidates for therapy and imaging.

Asunto(s)

4-Aminopiridina/análogos & derivados , Proteínas de Drosophila/metabolismo , Bloqueadores de los Canales de Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , 4-Aminopiridina/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Concentración de Iones de Hidrógeno , Cinética , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Relación Estructura-Actividad , Xenopus laevis/crecimiento & desarrollo

RESUMEN

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Asunto(s)

Potenciales de Acción , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Receptores de Glutamato Metabotrópico/metabolismo , Potenciales Sinápticos , Cuerpo Trapezoide/metabolismo , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Amilorida/farmacología , Animales , Maleato de Dizocilpina/farmacología , Femenino , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/antagonistas & inhibidores , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Masculino , Metoxihidroxifenilglicol/análogos & derivados , Metoxihidroxifenilglicol/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/metabolismo , Pirimidinas/farmacología , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Cuerpo Trapezoide/citología , Cuerpo Trapezoide/efectos de los fármacos , Cuerpo Trapezoide/fisiología

RESUMEN

TASK-3 is a two-pore domain potassium (K2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC50 = 56.8 ± 3.9 µM. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC50 = 14.2 ± 3.4 µM. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol.

Asunto(s)

Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Diseño de Fármacos , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Bloqueadores de los Canales de Potasio/farmacocinética

RESUMEN

Rational drug design targeting ion channels is an exciting and always evolving research field. New medicinal chemistry strategies are being implemented to explore the wild chemical space and unravel the molecular basis of the ion channels modulators binding mechanisms. TASK channels belong to the two-pore domain potassium channel family and are modulated by extracellular acidosis. They are extensively distributed along the cardiovascular and central nervous systems, and their expression is up- and downregulated in different cancer types, which makes them an attractive therapeutic target. However, TASK channels remain unexplored, and drugs designed to target these channels are poorly selective. Here, we review TASK channels properties and their known blockers and activators, considering the new challenges in ion channels drug design and focusing on the implementation of computational methodologies in the drug discovery process.

Asunto(s)

Diseño de Fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/química , Canales de Potasio/metabolismo , Animales , Descubrimiento de Drogas , Humanos , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/metabolismo