RESUMO
The development of cell-based fluorescent assays has resulted in an incredible tool for searching new ion channels' modulators with a biophysical and clinical profile. Among all the ion channels, potassium (K+)-permeable channels represent the most diverse and relevant for cell function, making them attractive targets for drug discovery. Some of the cell-based assays for K+ channels take advantage of a thallium-sensitive dye whose fluorescence increased upon the binding of thallium (Tl+), an ion able to move through K+ channels. We optimize the FLIPR Potassium Assay Kit based on thallium influx to measure the Kv10.1 activity.
Assuntos
Tálio , Tálio/metabolismo , Humanos , Corantes Fluorescentes/química , Células HEK293 , Fluorescência , Canais de Potássio Éter-A-Go-GoRESUMO
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.
Assuntos
Canais de Potássio Éter-A-Go-Go , Relação Quantitativa Estrutura-Atividade , Humanos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Medição de Risco , Análise de Regressão , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/químicaRESUMO
The diagnosis of cardiovascular disease (CVD) is still limited. Therefore, this study demonstrates the presence of human ether-a-go-go-related gene 1 (hERG1) and heat shock protein 47 (Hsp47) on the surface of small extracellular vesicles (sEVs) in human peripheral blood and their association with CVD. In this research, 20 individuals with heart failure and 26 participants subjected to cardiac stress tests were enrolled. The associations between hERG1 and/or Hsp47 in sEVs and CVD were established using Western blot, flow cytometry, electron microscopy, ELISA, and nanoparticle tracking analysis. The results show that hERG1 and Hsp47 were present in sEV membranes, extravesicularly exposing the sequences 430AFLLKETEEGPPATE445 for hERG1 and 169ALQSINEWAAQTT- DGKLPEVTKDVERTD196 for Hsp47. In addition, upon exposure to hypoxia, rat primary cardiomyocytes released sEVs into the media, and human cardiomyocytes in culture also released sEVs containing hERG1 (EV-hERG1) and/or Hsp47 (EV-Hsp47). Moreover, the levels of sEVs increased in the blood when cardiac ischemia was induced during the stress test, as well as the concentrations of EV-hERG1 and EV-Hsp47. Additionally, the plasma levels of EV-hERG1 and EV-Hsp47 decreased in patients with decompensated heart failure (DHF). Our data provide the first evidence that hERG1 and Hsp47 are present in the membranes of sEVs derived from the human cardiomyocyte cell line, and also in those isolated from human peripheral blood. Total sEVs, EV-hERG1, and EV-Hsp47 may be explored as biomarkers for heart diseases such as heart failure and cardiac ischemia.
Assuntos
Biomarcadores , Doenças Cardiovasculares , Vesículas Extracelulares , Proteínas de Choque Térmico HSP47 , Miócitos Cardíacos , Humanos , Vesículas Extracelulares/metabolismo , Biomarcadores/sangue , Masculino , Doenças Cardiovasculares/metabolismo , Feminino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Pessoa de Meia-Idade , Animais , Proteínas de Choque Térmico HSP47/metabolismo , Ratos , Canal de Potássio ERG1/metabolismo , Idoso , Adulto , Canais de Potássio Éter-A-Go-Go/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/sangueRESUMO
INTRODUCTION: Long QT Syndrome (LQTS) is an inherited disease with an abnormal electrical conduction system in the heart that can cause sudden death as a result of QT prolongation. LQT2 is the second most common subtype of LQTS caused by loss of function mutations in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. Although more than 900 mutations are associated with the LQTS, many of these mutations are not validated or characterized. METHODS AND RESULTS: Sequencing analyses of genomic DNA of a family with LQT2 identified a putative mutation. i.e., KCNH2(NM_000238.3): c.3099_3112del, in KCNH2 gene which appeared to be a definite pathogenic mutation. The family pedigree information showed a gender difference in clinical features and T-wave morphology between male and female patients. The female with mutation exhibited recurring ventricular arrhythmia and syncope, while two male carriers did not show any symptoms. In addition, T-wave in females was much flatter than in males. The female proband showed a positive reaction to the lidocaine test. Lidocaine injection almost completely blocked ventricular arrhythmia and shortened the QT interval by ≥30 ms. Treatment with propranolol, mexiletine, and implantation of cardioverter-defibrillators prevented the sustained ventricular tachycardia, ventricular fibrillation, and syncope, as assessed by a 3-year follow-up evaluation. CONCLUSIONS: A putative mutation c.3099_3112del in the KCNH2 gene causes LQT2 syndrome, and the pathogenic mutation mainly causes symptoms in female progeny.
Assuntos
Canais de Potássio Éter-A-Go-Go , Síndrome do QT Longo , Humanos , Masculino , Feminino , Canais de Potássio Éter-A-Go-Go/genética , Canal de Potássio ERG1/genética , Fatores Sexuais , Mutação/genética , Síndrome do QT Longo/genética , Síndrome do QT Longo/diagnóstico , Síncope , LidocaínaRESUMO
The first conotoxin affecting the voltage-gated potassium channels of the EAG family was identified and characterized from the venom of the vermivorous species Conus spurius from the Gulf of Mexico. This conopeptide, initially named Cs68 and later designated κO-SrVIA, is extremely hydrophobic and comprises 31 amino acid residues, including six Cysteines in the framework VI/VII, and a free C-terminus. It inhibits the currents mediated by two human EAG subtypes, Kv10.1 (IC50 = 1.88 ± 1.08 µM) and Kv11.1 (IC50 = 2.44 ± 1.06 µM), and also the human subtype Kv1.6 (IC50 = 3.6 ± 1.04 µM). Despite its clear effects on potassium channels, it shares a high sequence identity with δ-like-AtVIA and δ-TsVIA. Also, κO-SrVIA is the third conopeptide from the venom of C. spurius with effects on potassium channels, and the seventh conotoxin that blocks Kv1.6 channels.
Assuntos
Conotoxinas , Caramujo Conus , Canais de Potássio Éter-A-Go-Go , Animais , Humanos , Sequência de Aminoácidos , Conotoxinas/farmacologia , Conotoxinas/química , Caramujo Conus/química , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/metabolismo , Canais de Potássio Éter-A-Go-Go/toxicidade , Peptídeos/químicaRESUMO
Cervical cancer is the fourth most common cancer among women worldwide. The main factor associated with the onset and progression of this neoplasia is the human papillomavirus (HPV) infection. The HPV-oncogenes E6 and E7 are critical drivers of cellular transformation, promoting the expression of oncogenes such as KCNH1. The phytochemical α-mangostin (AM) is a potent antineoplastic and antiviral compound. However, its effects on HPV oncogenes and KCNH1 gene expression remain unknown. This study evaluated the effects of AM on cell proliferation, cell cycle distribution and gene expression, including its effects on tumor growth in xenografted mice. AM inhibited cell proliferation in a concentration-dependent manner, being the most sensitive cell lines those with the highest number of HPV16 copies. In addition, AM promoted G1-cell cycle arrest in CaSki cells, while led to cell death in SiHa and HeLa cells. Of interest was the finding of an AM-dependent decreased gene expression of E6, E7 and KCNH1 both in vitro and in vivo, as well as the modulation of cytokine expression, Ki-67, and tumor growth inhibition. On these bases, we suggest that AM represents a good option as an adjuvant for the treatment and prevention of cervical cancer.
Assuntos
Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Humanos , Feminino , Animais , Camundongos , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Células HeLa , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Repressoras/genética , Oncogenes , Proliferação de Células , Expressão Gênica , Canais de Potássio Éter-A-Go-Go/genéticaRESUMO
Carcinogenesis is a multistage process involving the dysregulation of multiple genes, proteins, and pathways that make any normal cell acquire a cancer cell phenotype. Therefore, it is no surprise that numerous ion channels could be involved in this process. Since their discovery and subsequent cloning, ion channels have been established as therapeutic targets in excitable cell pathologies (e.g., cardiac arrhythmias or epilepsy); however, their involvement in non-excitable cell pathologies is relatively recent. Among all ion channels, the voltage-gated potassium channels Kv10.1 have been established as a promising target in cancer treatment due to their high expression in tumoral tissues compared to low levels in healthy tissues.
Assuntos
Epilepsia , Neoplasias , Carcinogênese/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Neoplasias/genéticaRESUMO
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.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Loperamida/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Relação Dose-Resposta a Droga , Fluorescência , Células HEK293 , Humanos , Loperamida/administração & dosagem , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/administração & dosagem , Reprodutibilidade dos Testes , Tálio/metabolismoRESUMO
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.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/farmacologia , Animais , Colômbia , Canais de Potássio Éter-A-Go-Go/fisiologia , Peptídeos/isolamento & purificação , Bloqueadores dos Canais de Potássio/isolamento & purificação , Venenos de Escorpião/isolamento & purificação , EscorpiõesRESUMO
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.