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1.
Prog Chem Org Nat Prod ; 110: 177-238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31621014

RESUMO

Interference with the hERG potassium ion channel may cause cardiac arrhythmia and can even lead to death. Over the last few decades, several drugs, already on the market, and many more investigational drugs in various development stages, have had to be discontinued because of their hERG-associated toxicity. To recognize potential hERG activity in the early stages of drug development, a wide array of computational tools, based on different principles, such as 3D QSAR, 2D and 3D similarity, and machine learning, have been developed and are reviewed in this chapter. The various available prediction tools Similarity Ensemble Approach, SuperPred, SwissTargetPrediction, HitPick, admetSAR, PASSonline, Pred-hERG, and VirtualToxLab™ were used to screen a dataset of known hERG synthetic and natural product actives and inactives to quantify and compare their predictive power. This contribution will allow the reader to evaluate the suitability of these computational methods for their own related projects. There is an unmet need for natural product-specific prediction tools in this field.


Assuntos
Produtos Biológicos/farmacologia , Biologia Computacional , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Química Farmacêutica , Humanos , Aprendizado de Máquina , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade
2.
J Pharmacol Sci ; 140(3): 284-290, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31481348

RESUMO

The human ether-a-go-go-related gene (hERG) encodes the K+ channel that carries the rapid component of the delayed rectifier current in the human heart. Reduction of hERG activity induced by gene mutations or pharmacological inhibition is responsible for the type 2 form of long QT syndrome in patients which can develop into ventricular arrhythmia and sudden cardiac death. Therefore, pharmacological activation of hERG may lead to therapeutic potential for cardiac arrhythmias. In this study we characterized a small and novel compound, N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide, HW-0168, that exhibits potent activation of hERG channel with an EC50 of 0.41 ± 0.2 µM. Using whole-cell patch clamp recording of HEK293 cells stably expressed hERG channels, we found that HW-0168 dramatically increased current amplitude about 2.5 folds and slowed down current inactivation about 4 folds. HW-0168 shifted the voltage-dependent channel activation to hyperpolarizing direction about 3.7 mV and the voltage-dependent channel inactivation to depolarizing direction about 9.4 mV. In addition, recording of guinea-pig ventricular cells confirmed that HW-0168 shortened the action potential duration. In conclusion, we identified a novel hERG channel activator HW-0168 that can be used for studying the physiological role of hERG in cardiac myocytes and may be beneficial for treating long QT syndrome.


Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/metabolismo , Linhagem Celular , Cobaias , Células HEK293 , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Humanos , Síndrome do QT Longo/tratamento farmacológico , Síndrome do QT Longo/metabolismo , Masculino
3.
Elife ; 82019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31490124

RESUMO

Voltage-gated potassium channels (Kvs) are gated by transmembrane voltage sensors (VS) that move in response to changes in membrane voltage. Kv10.1 or Eag1 also has three intracellular domains: PAS, C-linker, and CNBHD. We demonstrate that the Eag1 intracellular domains are not required for voltage-dependent gating but likely interact with the VS to modulate gating. We identified specific interactions between the PAS, CNBHD, and VS that modulate voltage-dependent gating and provide evidence that VS movement destabilizes these interactions to promote channel opening. Additionally, mutation of these interactions renders Eag1 insensitive to calmodulin inhibition. The structure of the calmodulin insensitive mutant in a pre-open conformation suggests that channel opening may occur through a rotation of the intracellular domains and calmodulin may prevent this rotation by stabilizing interactions between the VS and intracellular domains. Intracellular domains likely play a similar modulatory role in voltage-dependent gating of the related Kv11-12 channels.


Assuntos
Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Animais , Células CHO , Calmodulina/metabolismo , Cricetinae , Cricetulus , Canais de Potássio Éter-A-Go-Go/genética , Modelos Moleculares , Proteínas Mutantes/genética , Técnicas de Patch-Clamp , Conformação Proteica
4.
Cancer Invest ; 37(9): 489-500, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496302

RESUMO

Prostate cancer is the most common cancer and leading cause of cancer death for males. Imipramine (IMI), which is a tricyclic antidepressant, has also been shown to has antineoplastic effect. This study was performed to investigate the radiosensitizing effect of IMI on DU145 prostate cancer cell. Cells were divided into 4 groups. Cell index, apoptotic activity, cell cycle arrest, oxidative stress and EAG1 channel currents were determined in all groups. Our findings showed that combined treatment with IMI and radiotherapy (RAD) did not enhance radiosensitivity of DU145 cells but as unexpected finding, treatment of IMI alone was more effective in DU145 cells.


Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Imipramina/farmacologia , Neoplasias da Próstata/metabolismo , Radiossensibilizantes/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Neoplasias da Próstata/terapia , Radioterapia
5.
Eur J Med Chem ; 180: 1-14, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31288149

RESUMO

SAR studies on bicalutamide, enobosarm and enzalutamide analogues, functionalised with polyfluorinated groups, is presented. Among the novel bicalutamide and enobosarm derivatives synthesised, several displayed significantly improved in vitro anticancer activity, with IC50 values in the low micromolar range against four different prostate cancer cell lines (LNCaP, VCaP, DU-145 and 22Rv1), showing up to 48-fold increase in comparison with the parent structures. In particular, SF5 enobosarm analogues were found to be most potent compounds, full AR antagonists and with favourable ADME properties. The most promising compound (48a) was evaluated for its in vivo efficacy in PC xenograft mouse model (22Rv1) with results comparable to the standard-of-care docetaxel.


Assuntos
Anilidas/farmacologia , Antineoplásicos/farmacologia , Nitrilos/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Compostos de Tosil/farmacologia , Anilidas/síntese química , Anilidas/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Células CHO , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/farmacologia , Masculino , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Nitrilos/síntese química , Nitrilos/química , Feniltioidantoína/síntese química , Feniltioidantoína/química , Feniltioidantoína/farmacologia , Neoplasias da Próstata/patologia , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia , Compostos de Tosil/síntese química , Compostos de Tosil/química
6.
BMC Pharmacol Toxicol ; 20(1): 42, 2019 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315662

RESUMO

BACKGROUND: KCNH family of potassium channels is responsible for diverse physiological functions ranging from the regulation of neuronal excitability and cardiac contraction to the regulation of cancer progression. KCNH channels contain a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function of KCNH channels and are important targets for drug development. METHODS: Here we describe a surface plasmon resonance (SPR)-based screening method aimed in identifying small molecule binders of PAS and CNBH domains for three KCNH channel subfamilies: ether-à-go-go (EAG), EAG-related gene (ERG), and EAG-like K+ (ELK). The method involves purification of the PAS and CNBH domains, immobilization of the purified domains on the SPR senor chip and screening small molecules in a chemical library for binding to the immobilized domains using changes in the SPR response as a reporter of the binding. The advantages of this method include low quantity of purified PAS and CNBH domains necessary for the implementation of the screen, direct assessment of the small molecule binding to the PAS and CNBH domains and easiness of assessing KCNH subfamily specificity of the small molecule binders. RESULTS: Using the SPR-based method we screened the Spectrum Collection Library of 2560 compounds against the PAS and CNBH domains of the three KCNH channel subfamilies and identified a pool of small molecules that bind to the PAS or CNBH domains. To further evaluate the effectiveness of the screen we tested the functional effect of one of the identified mEAG PAS domain specific small molecule binders on currents recorded from EAG channels. Undecylenic acid inhibited currents recorded from EAG channels in a concentration-dependent manner with IC50 of ~ 1 µM. CONCLUSION: Our results show that the SPR-based method is well suited for identifying small molecule binders of KCNH channels and can facilitate drug discovery for other ion channels as well.


Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Ácidos Undecilênicos/farmacologia , Animais , Descoberta de Drogas/métodos , Canais de Potássio Éter-A-Go-Go/fisiologia , Humanos , Camundongos , Oócitos/fisiologia , Domínios Proteicos , Bibliotecas de Moléculas Pequenas , Ressonância de Plasmônio de Superfície , Xenopus laevis
7.
J Exp Clin Cancer Res ; 38(1): 324, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31331361

RESUMO

BACKGROUND: The human ether a-go-go-related gene 1 (HERG1) is involved in tumor progression; however, its role in esophageal squamous cell carcinoma (ESCC) is not well studied. This study investigated HERG1 function in ESCC progression and elucidated the underlying mechanisms. METHODS: The prognostic value of HERG1 was determined by immunohistochemistry in ESCC biopsies. Cell growth and proliferation were analyzed by colony formation and methyl thiazolyl tetrazolium assays. Cell migration and invasion were analyzed by wound healing and Boyden transwell assays. Epithelial-mesenchymal transition (EMT) was evaluated by immunoblotting and quantitative polymerase chain reaction (qPCR). A xenograft mouse model was used to validate the tumorigenic and metastatic roles of HERG1 in vivo. RESULTS: HERG1 expression was overall higher in ESCC tissues compared to adjacent non-tumor tissues. A retrospective analysis of 349 patients with ESCC (stages I-IV) confirmed increased HERG1 expression was associated with disease progression and higher mortality rate. The overall survival of the patients was significantly worse when their tumors displayed higher HERG1 expression. HERG1 knockdown reduced tumor growth and metastasis in athymic mice. HERG1 affected the proliferation, migration, and invasion of two ESCC cell lines (TE-1 and KYSE-30). Changes in HERG1 expression affected the expression of cell cycle- and EMT-related proteins; these effects were reversed by altering the expression of thioredoxin domain-containing protein 5 (TXNDC5), which is also associated with the clinicopathological characteristics of patients with ESCC and is relevant to HERG1 in pathological biopsies. Additionally, HERG1 expression altered phosphoinositide 3-kinase (PI3K) and AKT phosphorylation, thereby affecting TXNDC5 expression. CONCLUSIONS: HERG1 contributes to poor prognosis in patients with ESCC by promoting ESCC cell proliferation, migration, and invasion via TXNDC5 through the PI3K/AKT signaling pathway. Our findings provided novel insights into the pathology of ESCC and role of HERG1 in tumor progression, suggesting that targeting HERG1 has potential diagnostic and therapeutic value for ESCC treatment.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas do Esôfago/genética , Canais de Potássio Éter-A-Go-Go/genética , Isomerases de Dissulfetos de Proteínas/genética , Idoso , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Intervalo Livre de Doença , Transição Epitelial-Mesenquimal , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Proteína Oncogênica v-akt/genética , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Mol Pharmacol ; 96(2): 259-271, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31182542

RESUMO

Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K+ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current IKr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.


Assuntos
Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Ivabradina/farmacologia , Lipídeos de Membrana/metabolismo , Sítios de Ligação , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Ivabradina/química , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fenetilaminas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Sulfonamidas/farmacologia
9.
Rev Invest Clin ; 71(3): 186-194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31184333

RESUMO

Background: Expression and activity of the potassium channel ether-à-go-go-1 (EAG1) are strongly related to carcinogenesis and tumor progression, which can be exploited for therapeutic purposes. EAG1 activity may be reduced by preventing its phosphorylation with epidermal growth factor receptor (EGFR) kinase inhibitors and by astemizole, which blocks the channel pore and downregulates its gene expression. Objective: We aimed to study the potential cooperative antiproliferative effect of the EGFR inhibitor gefitinib and the EAG1-blocker astemizole, in breast cancer cells. Materials and Methods: The cells were characterized by immunocytochemistry. Inhibitory concentrations were determined by non-linear regression analysis using dose-response curves. The nature of the pharmacological effect was evaluated by the combination index equation while cell cycle analysis was studied by flow cy-tometry. Results: Astemizole and gefitinib inhibited cell proliferation in a concentration-dependent manner, with inhibitory concentrations (IC 50) values of 1.72 µM and 0.51 µM, respectively. All combinations resulted in a synergistic antiproliferative effect. The combination of astemizole and gefitinib diminished the percentage of cells in G2/M and S phases, while increased accumulation in G0/G1 of the cell cycle. Conclusions: Astemizole and gefitinib synergistically inhibited proliferation in breast cancer cells expressing both EGFR and EAG1. Our results suggest that the combined treatment increased cell death by targeting the oncogenic activity of EAG1.


Assuntos
Antineoplásicos/farmacologia , Astemizol/farmacologia , Neoplasias da Mama/tratamento farmacológico , Gefitinibe/farmacologia , Antineoplásicos/administração & dosagem , Astemizol/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Gefitinibe/administração & dosagem , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia
10.
Eur J Pharmacol ; 855: 262-266, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31082369

RESUMO

Chloroquine (CQ) is an old antimalarial drug currently being investigated for its anti-tumor properties. As chloroquine has been shown to inhibits several potassium channels, we decided to study its effect on the tumor-related Kv10.1 channel by using patch-clamp electrophysiology and cell migration assays. We found that chloroquine inhibited Kv10.1 channels transiently expressed in HEK-293 cells in a concentration- and voltage-dependent manner acting from the cytoplasmic side of the plasma membrane. Chloroquine also inhibited the outward potassium currents from MDA-MB-231 cells, which are mainly carried through Kv10.1 channels as was confirmed using astemizole. Additionally, chloroquine decreased MDA-MB-231 cell migration in the in vitro scratch wound healing assay. In conclusion, our data suggest that chloroquine decreases MDA-MB-231 cell migration by inhibiting Kv10.1 channels. The inhibition of Kv10.1 channels could represent another mechanism of the antitumoral action of chloroquine, besides autophagy inhibition and tumor vessel normalization.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Cloroquina/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Células HEK293 , Humanos
11.
BMC Bioinformatics ; 20(Suppl 10): 250, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31138104

RESUMO

BACKGROUND: Drug candidates often cause an unwanted blockage of the potassium ion channel of the human ether-a-go-go-related gene (hERG). The blockage leads to long QT syndrome (LQTS), which is a severe life-threatening cardiac side effect. Therefore, a virtual screening method to predict drug-induced hERG-related cardiotoxicity could facilitate drug discovery by filtering out toxic drug candidates. RESULT: In this study, we generated a reliable hERG-related cardiotoxicity dataset composed of 2130 compounds, which were carried out under constant conditions. Based on our dataset, we developed a computational hERG-related cardiotoxicity prediction model. The neural network model achieved an area under the receiver operating characteristic curve (AUC) of 0.764, with an accuracy of 90.1%, a Matthews correlation coefficient (MCC) of 0.368, a sensitivity of 0.321, and a specificity of 0.967, when ten-fold cross-validation was performed. The model was further evaluated using ten drug compounds tested on guinea pigs and showed an accuracy of 80.0%, an MCC of 0.655, a sensitivity of 0.600, and a specificity of 1.000, which were better than the performances of existing hERG-toxicity prediction models. CONCLUSION: The neural network model can predict hERG-related cardiotoxicity of chemical compounds with a high accuracy. Therefore, the model can be applied to virtual high-throughput screening for drug candidates that do not cause cardiotoxicity. The prediction tool is available as a web-tool at http://ssbio.cau.ac.kr/CardPred .


Assuntos
Cardiotoxicidade/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Animais , Área Sob a Curva , Bases de Dados Genéticas , Canais de Potássio Éter-A-Go-Go/química , Cobaias , Humanos , Aprendizado de Máquina , Curva ROC
12.
Elife ; 82019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31032798

RESUMO

Most neurons do not simply convert inputs into firing rates. Instead, moment-to-moment firing rates reflect interactions between synaptic inputs and intrinsic currents. Few studies investigated how intrinsic currents function together to modulate output discharges and which of the currents attenuated by synthetic cholinergic ligands are actually modulated by endogenous acetylcholine (ACh). In this study we optogenetically stimulated cholinergic fibers in rat neocortex and find that ACh enhances excitability by reducing Ether-à-go-go Related Gene (ERG) K+ current. We find ERG mediates the late phase of spike-frequency adaptation in pyramidal cells and is recruited later than both SK and M currents. Attenuation of ERG during coincident depolarization and ACh release leads to reduced late phase spike-frequency adaptation and persistent firing. In neuronal ensembles, attenuating ERG enhanced signal-to-noise ratios and reduced signal correlation, suggesting that these two hallmarks of cholinergic function in vivo may result from modulation of intrinsic properties.


Assuntos
Acetilcolina/fisiologia , Adaptação Fisiológica , Canais de Potássio Éter-A-Go-Go/fisiologia , Neocórtex/fisiologia , Potenciais de Ação/fisiologia , Animais , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Feminino , Cinética , Masculino , Potenciais da Membrana , Neurônios , Bloqueadores dos Canais de Potássio/farmacologia , Células Piramidais/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Muscarínicos/metabolismo
13.
Chem Commun (Camb) ; 55(38): 5515-5518, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31020283

RESUMO

A highly sensitive fluorescent probe CBH based on solvatochromism and molecular rotation was designed and developed for imaging of hERG channels by employing a novel targeting group 4-benzylaniline. More importantly, CBH has the potential for the quantitative analysis of the hERG channels expressed in tumor cells.


Assuntos
Compostos de Anilina/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Solventes/química , Animais , Western Blotting , Células CHO , Cricetulus , Canais de Potássio Éter-A-Go-Go/química , Corantes Fluorescentes , Células HeLa , Humanos , Microscopia Confocal
14.
Eur J Pharmacol ; 854: 92-100, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-30954564

RESUMO

Cariprazine is a novel atypical antipsychotic drug that is widely used for the treatment of schizophrenia and bipolar mania/mixed disorder. We used the whole-cell patch-clamp technique to investigate the effects of cariprazine on hERG channels that are stably expressed in HEK cells. Cariprazine inhibited the hERG 1A and hERG 1A/3.1 tail currents at -50 mV in a concentration-dependent manner with IC50 values of 4.1 and 12.2 µM, respectively. The block of hERG 1A currents by cariprazine was voltage-dependent, and increased over a range of voltage for channel activation. Cariprazine shifted the steady-state inactivation curve of the hERG 1A currents in a hyperpolarizing direction and produced a use-dependent block. A fast application of cariprazine inhibited the hERG 1A currents elicited by a 5 s depolarizing pulse to +60 mV to fully inactivate the hERG 1A currents. During a repolarizing pulse wherein the hERG 1A current was deactivated slowly, cariprazine rapidly and reversibly blocked the open state of the hERG 1A current. However, cariprazine did not affect hERG 1A and hERG 1A/3.1 channel trafficking to the cell membrane. Our results indicated that cariprazine concentration-dependently inhibited hERG 1A and hERG 1A/3.1 currents by preferentially interacting with the open states of the hERG 1A channel, but not by the disruption of hERG 1A and hERG 1A/3.1 channel protein trafficking. Our study examined cariprazine's mechanism of action provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.


Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Piperazinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína
15.
Neuroreport ; 30(9): 637-644, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31008829

RESUMO

Ion channels play as a pivotal role in hypertension in the processes of maintenance of vascular tone and sympathetic excitement of hypertension. The Kv10.2 channel (encoded by the Kcnh5 gene) belongs to the EAG voltage-gated superfamily. It is distributed widely in the brain, such as the hippocampus, the cortex, and the olfactory bulb. To date, the expression of Kv10.2 in central nervous system nuclei that regulates cardiovascular function and its inter-relationship with hypertension are still unclear. Here, electric foot-shock stressors with noise were used to establish the stress-induced hypertensive (SIH) rat model. The expression of Kv10.2 in the rostral ventrolateral medulla, the nucleus tractus solitarius, and the paraventricular nucleus (PVN) was examined by immunohistochemical staining and western blots. The following results were obtained: (a) the expression level of Kv10.2 was increased obviously in the paraventricular nucleus of SIH rats, whereas no significant difference was found in the rostral ventrolateral medulla and the nucleus tractus solitarius. (b) Kv10.2 was located in neurons. (c) Vesicular glutamate transporter 1 as a protein mark of glutamate neurons was increased in the paraventricular nucleus of the SIH group. (d) The expression of vesicular glutamate transporter 1 protein in neurons was significantly decreased when the Kcnh5 gene was knocked down by small interfering RNA in vitro. These findings indicate that the changes in Kv10.2 may be related to SIH, which may provide a potential avenue for further investigation of SIH.


Assuntos
Encéfalo/metabolismo , Canais de Potássio Éter-A-Go-Go/biossíntese , Hipertensão/metabolismo , Animais , Hipertensão/etiologia , Masculino , Ratos , Ratos Sprague-Dawley
16.
PLoS One ; 14(4): e0215905, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017964

RESUMO

The alpha subunit of the voltage gated human ether-a-go-go-related (hERG) potassium channel regulates cell excitability in a broad range of cell lines. HERG channels are also expressed in a variety of cancer cells and control cell proliferation and apoptosis. Hypoxia, a common feature of tumors, alters gating properties of hERG currents in SH-SY5Y neuroblastoma cells. In the present study, we examined the molecular mechanisms and physiological significance underlying hypoxia-altered hERG currents in SH-SY5Y neuroblastoma cells. Hypoxia reduced the surface expression of 150kDa form and increased 125kDa form of hERG protein expression in the endoplasmic reticulum (ER). The changes in protein expression were associated with ~50% decrease in hERG potassium conductance. ER retention of hERG 125kDa form by CH was due to defective trafficking and was rescued by exposing cells to hypoxia at low temperatures or treatment with E-4031, a hERG channel blocker. Prolonged association of hERG with molecular chaperone Hsp90 resulting in complex oligomeric insoluble aggregates contributed to ER accumulation and trafficking defect. Hypoxia increased reactive oxygen species (ROS) levels and manganese (111) tetrakis (1methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant prevented hypoxia-induced degradation of 150kDa and accumulation of 125kDa forms. Impaired trafficking of hERG by hypoxia was associated with reduced cell proliferation and this effect was prevented by antioxidant treatment. These results demonstrate that hypoxia through increased oxidative stress impairs hERG trafficking, leading to decreased K+ currents resulting in cell cycle arrest in SH-SY5Y cells.


Assuntos
Pontos de Checagem do Ciclo Celular , Canais de Potássio Éter-A-Go-Go/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Retículo Endoplasmático/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo
17.
J Affect Disord ; 252: 413-420, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31003110

RESUMO

BACKGROUND: Psychological distress symptoms are associated with an increased risk of psychiatric disorders and medical illness. Although psychological distress is influenced by environmental factors, such as socioeconomic status, lifetime events, or interpersonal relationships, substantial interindividual variation also exists. However, heritability and genetic determinants of distress are poorly understood. METHODS: In the Korean Genome and Epidemiology Study sample (n = 12,680), we estimated the heritability of individual psychological distress symptoms using the GCTA-REML method and carried out a genome-wide association study of individual psychological distress symptoms showing significant heritability. RESULTS: We found three psychological distress items showing significant heritability: subjective well-being (9%), fatigue and appetite (11%), and enjoying daily life (8%). Additionally, we found genome-wide significant associations of rs6735649 located between STEAP3 and C1QL2 on chromosome 2 with subjective well-being (P = 1.32 × 10-8, odds ratio [OR] = 1.18, 95% confidence interval [CI]: 1.12-1.25) and rs35543418 located between SYT16 and KCNH5 on chromosome 14 with enjoying daily life (P = 1.33 × 10-8, OR = 1.59, 95% CI: 1.35-1.86). LIMITATIONS: The lack of replication in independent cohorts and longitudinal assessment of distress may limit generalizability. CONCLUSIONS: Our results indicate that distress symptoms are partly heritable. Further analysis in larger cohorts investigating gene-environment interactions is required to identify genetic variants that explain the proportion of variation in distress.


Assuntos
Estresse Psicológico/genética , Adulto , Proteínas de Ciclo Celular/genética , Complemento C1q/genética , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Interação Gene-Ambiente , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Oxirredutases/genética , Sinaptotagminas/genética
18.
Biosens Bioelectron ; 132: 310-318, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30884318

RESUMO

Ion channel is a pore-forming membrane protein that allows ions to pass through the cell membrane, which is essential for the continuation of life. Analysis of ion channel characteristics at the single cell level will help to study the role of cellular heterogeneity in disease progression and cellular signaling processes. In this study, we fabricate a photocleavable and entropy-driven reaction based electrochemical biosensor for ultrasensitive detection of human ether-a-go-go related gene (hERG) potassium channel activity on HEK293 cell. We employed an antibody-DNA1 to conjugate the hERG channel in the cell membrane via antibody-antigen reaction. The release of the DNA1 by photocleavable reaction will trigger an amplification reaction by using the Exonuclease III (Exo III) to generate intermediate DNA. In addition, two hairpin DNA (DNA3 and DNA4) was employed for the signal amplification. We well designed a toehold on DNA3 for intermediate DNA hybridization to form double-strand DNA that opens the DNA3 hairpin. The free DNA3 exposed the relocked toehold domain to open the DNA4. After the entropy-driven toehold-mediated displacement amplification reaction by using intermediate DNA, DNA4 hybridized with DNA3 effectively, making the ferrocene labeled on the 5'-termini DNA4 close to the Au electrode surface to produce the electrochemical response. Then, the displaced intermediate DNA was released from the cell surface into solution for the next entropy-driven reaction. After two steps amplification reaction, one ion channel triggered thousands of DNA3/DNA4 duplex on the biosensor surface. By using this biosensor, electrochemical curve of hERG ion channels on a single cell was obtained.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Canais de Potássio Éter-A-Go-Go/análise , Imunoconjugados/química , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Técnicas Eletroquímicas/métodos , Entropia , Canais de Potássio Éter-A-Go-Go/metabolismo , Compostos Ferrosos/química , Células HEK293 , Humanos , Limite de Detecção , Metalocenos/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Processos Fotoquímicos
19.
J Biol Chem ; 294(16): 6506-6521, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30808709

RESUMO

Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, KV10.1 and human ether-a-go-go-related gene (hERG or KV11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5L) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6T) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5L to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in KV10.2 channels. Using cysteine bridges and S4-S5L-mimicking peptides, we show that the ligand/receptor model is conserved in KV10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in KV10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.


Assuntos
Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Ativação do Canal Iônico , Modelos Moleculares , Animais , Células COS , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Peptídeos/química , Domínios Proteicos
20.
Bioorg Med Chem ; 27(7): 1283-1291, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30792104

RESUMO

The ATP-synthase inhibitor bedaquiline is effective against drug-resistant tuberculosis but is extremely lipophilic (clogP 7.25) with a very long plasma half-life. Additionally, inhibition of potassium current through the cardiac hERG channel by bedaquiline, is associated with prolongation of the QT interval, necessitating cardiovascular monitoring. Analogues were prepared where the naphthalene C-unit was replaced with substituted pyridines to produce compounds with reduced lipophilicity, anticipating a reduction in half-life. While there was a direct correlation between in vitro inhibitory activity against M. tuberculosis (MIC90) and compound lipophilicity, potency only fell off sharply below a clogP of about 4.0, providing a useful lower bound for analogue design. The bulk of the compounds remained potent inhibitors of the hERG potassium channel, with notable exceptions where IC50 values were at least 5-fold higher than that of bedaquiline. Many of the compounds had desirably higher rates of clearance than bedaquiline, but this was associated with lower plasma exposures in mice, and similar or higher MICs resulted in lower AUC/MIC ratios than bedaquiline for most compounds. The two compounds with lower potency against hERG exhibited similar clearance to bedaquiline and excellent efficacy in vivo, suggesting further exploration of C-ring pyridyls is worthwhile.


Assuntos
Antituberculosos/farmacologia , Diarilquinolinas/farmacologia , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Piridinas/farmacologia , Animais , Antituberculosos/síntese química , Antituberculosos/química , Diarilquinolinas/síntese química , Diarilquinolinas/química , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Piridinas/síntese química , Piridinas/química , Relação Estrutura-Atividade
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