RESUMEN
Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-µL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/normas , COVID-19/diagnóstico , Dispositivos Laboratorio en un Chip , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , SARS-CoV-2/genética , Benchmarking , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19/instrumentación , Prueba de Ácido Nucleico para COVID-19/métodos , Canadá/epidemiología , Humanos , Límite de Detección , Nasofaringe/virología , Pruebas en el Punto de Atención , Juego de Reactivos para Diagnóstico/provisión & distribuciónRESUMEN
A high-throughput screening (HTS) assay was developed for cotransporter, NKCC1, which is a potential target for the treatment of diverse disorders. This nonradioactive rubidium flux assay coupled with ion channel reader series provides a working screen for this target expressed in human embryonic kidney (HEK) cell line. An eightfold window of detection was achieved with the optimized assay. This new functional assay offered a robust working model for NKCC1 in determining reliable and concordant rank orders of the test compounds supporting its sensitivity and specificity. The robustness of manual assay indicated by Z' of 0.9 qualified its amenability to automation. The Z' of 0.7 was displayed by automated assay employed in high-throughput screening of compound libraries against this target. Being electrically neutral, the NKCC1 screening is difficult to achieve by both manual and automated electrophysiological techniques. These techniques, although considered gold standard, suffer from their inherent problems of being too slow to be in high-throughput format and with high running costs. In addition to being a functional assay for NKCC1, it is nontoxic as compared with thallium flux assay, which is prone to generate high number of false-positive/false-negative rates because of its innate fluorescence issues.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Rubidio/análisis , Miembro 2 de la Familia de Transportadores de Soluto 12/análisis , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Rubidio/química , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Nine years ago Aurora Biomed Inc. (Vancouver, Canada) committed to gathering the brightest minds and the most innovative research companies at one conference. The Ion Channel Retreat provides a podium for scientific discourse spanning a wide range of ion channel disciplines. This conference has consistently provided a venue for people to share knowledge, exchange ideas, and establish partnerships. This conference continues to expand and grow each year, demonstrating the value of such a conference. Attendees at the 2011 Ion Channel retreat presented ion channel research from 12 different countries, representing research groups located on 5 of the 7 continents. Aurora Biomed's 2011 Retreat covered a variety of topics including Ion Channels as Disease Targets, Ion Channels as Pain Targets, TRP-channels, Ion Channel Screening Technologies, Cardiac Function and Pharmacology, Cardiac Safety and Toxicology, and Structure and Function of Ion Channels.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales Iónicos/fisiología , Analgésicos/administración & dosificación , Analgésicos/química , Analgésicos/metabolismo , Colombia Británica , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Sistemas de Liberación de Medicamentos/tendencias , Humanos , Canales Iónicos/química , Dolor/tratamiento farmacológico , Dolor/metabolismoRESUMEN
Eight years ago Aurora Biomed Inc. (Vancouver, Canada) committed to gathering the brightest minds and the most innovative research companies at one conference. We sought to provide a podium for scientific discourse spanning a wide range of ion channel disciplines. Since then, researchers from both academia and industry have come together each year to share their knowledge. With attendees from 17 different countries at the 2010 Ion Channel Retreat, this conference continues to grow, and is a testimony to the importance of ion channel research. Aurora Biomed's 2010 Retreat covered a variety of topics, including Ion Channels as Disease Targets, Ion Channels as Pain Targets, K-Channels, TRP-channels, Ion Channel Screening Technologies, Ion Channels in Safety Pharmacology, and Structure and Function of Ion Channels.
Asunto(s)
Canales Iónicos , Diseño de Fármacos , Descubrimiento de Drogas , Humanos , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Terapia Molecular Dirigida , Dolor/tratamiento farmacológico , Dolor/metabolismoRESUMEN
Human Na+, K+ ATPase, an ATP-driven ion transporter, is an emerging drug target for heart-related conditions. Three types of assays including purified enzyme, radiotracer flux, and cold Rb+ flux have been used to determine the activity of this transporter. As an alternative to primary cardiomyocytes, mouse embryonic stem cells-derived cardiomyocytes with functional expression of essential cardiac ion channels were used in the present studies. The results on its pharmacology with digitoxin and ouabain, the 2 well-known cardioglycosides, imply that these cardiomyocytes can be used as a predictive model for the identification of modulators of Na+, K+ ATPase in HTS format.
Asunto(s)
Miocitos Cardíacos/metabolismo , Rubidio/farmacocinética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Células Madre/citología , Animales , Células Cultivadas , Digitoxina/farmacología , Ensayos Analíticos de Alto Rendimiento , Ratones , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
The Kv1.3 channels expressed by human T lymphocytes are emerging as important therapeutic targets. Peptides like agitoxin and margatoxin in scorpion venom and some non-peptide small molecules are known to inhibit this channel. Since such blockers cannot be used as drugs, pharma has a need to discover effective blockers. The major limiting factor for such development has been the lack of a reliable high-throughput screening (HTS) technology. A cell-based HTS assay for this target was developed in 96-well format to facilitate screening of many candidates. The assay incorporates rubidium ion as a tracer for potassium ion, which can be analyzed by the atomic absorption spectroscopy. The assay provided a Z' factor of 0.813 with more than a 4.5-fold window of detection. The two known blockers agitoxin and margatoxin gave a 50% inhibitory concentration (IC(50)) of 1.52 and 2 nM, respectively. These values are about five- and 2.8-fold higher than their IC(50) values obtained from patch clamp. Some non-peptide compounds like tamoxifen, nifedipine, and fluoxetine also inhibited the efflux through these channels, whereas astemizole and pimozide (potent human ether-a-go-go-related gene blockers) did not block Kv1.3 activity.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Canal de Potasio Kv1.3/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Rubidio/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , Fluoxetina/farmacología , Concentración de Iones de Hidrógeno , Canal de Potasio Kv1.3/fisiologíaRESUMEN
An atomic absorption spectroscopy-based detection system was employed to develop a new non-radioactive flux assay for chloride (Cl-) channels in a high throughput format. Cl- flux is assayed by measuring the extent to which Cl- precipitates an excess amount of silver ions (Ag+). A linear correlation was observed between theoretical and determined Cl- concentration with an r2 value of 0.996. The assay was found to be free from interference from various ions and proteins. The assay was used to study the physiology of endogenously expressed Cl- channels in a Chinese hamster ovary-K1 cell line. Cl- efflux was activated in response to an increased concentration of K+ (100 mM), Ca2+ (4 mM), and ionomycin (10 microM) as calcium ionophore. The efflux was also sensitive to pH as slightly higher efflux of Cl- was observed at an acidic pH of 3.2 in comparison to the neutral pH of 7.4. The Cl- efflux was inhibited by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 500 microM 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) but not by tolbutamide, niflumic acid, or glybenclamide, indicating that the channel current is not sensitive to other cystic fibrosis transmembrane conductance regulator inhibitors. The 50% inhibitory concentration (IC50) values of DIDS at pH 7.4 and pH 3.2 were 17 microM and 19 microM, respectively. An IC50 of 26 microM was observed for NPPB. The assay had a Z' factor of 0.678.
Asunto(s)
Canales de Cloruro/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Células CHO , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/genética , Cloruros/farmacología , Cricetinae , Concentración de Iones de Hidrógeno , Cinética , Modelos Biológicos , Potasio/farmacología , Reproducibilidad de los Resultados , Plata/farmacologíaRESUMEN
INTRODUCTION: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery. METHODS: Competitive binding assays were developed using (3)H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points. RESULTS: Binding assays using HEK293-hERG membranes and (3)H-dofetilide were robust (Z'=0.69+/-0.015, mean+/-S.E.M.), highly reproducible (test-retest slope=1.04, r(2)=0.98), and correlated well with IC(50) values obtained by patch clamp (slope=0.98, r(2)=0.89). Binding assays using IMR-32 membranes were less sensitive (Z'=0.4+/-0.03, mean+/-S.E.M., false negative rate=0.4) but still correlated well with patch clamp data (slope=1.06, r(2)=0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC(50)<0.1 muM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate=0.5). Finally, the rubidium efflux assay gave highly reproducible results (Z'=0.80+/-0.02, mean+/-S.E.M.) that correlated with patch clamp IC(50) values (slope=0.87, r(2)=0.73). DISCUSSION: The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Ensayo de Unión Radioligante/métodos , Proteínas Recombinantes/metabolismo , Animales , Células CHO , Línea Celular , Cricetinae , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/biosíntesis , Humanos , Unión Proteica/fisiología , Proteínas Recombinantes/biosíntesisRESUMEN
A high-throughput screening (HTS) assay was developed for the Na(+),K(+)-ATPase channel in order to study rubidium uptake as a measure of the functional activity and modulation of this exchanger. The assay uses elemental rubidium as a tracer for K(+) ions. Three cell lines were used to study the exchanger, and the assay was performed in a 96-well microtiter plate format. Rb(+) uptake was carried by the CHO-K1 cells at 37 degrees C; the maximum ion influx was at 80 min of incubation of the cell line in the medium containing 5.4 mM RbCl. The cells were incubated in Rb(+) uptake buffer (5.4 mM) and with the pump blocker ouabain for 1, 2, and 3 h, respectively. A complete block of the Rb(+) uptake was observed with a 5 mM concentration of ouabain for all the three time intervals. The ouabain 50% inhibitory concentration (IC(50)) value for CHO-K1 cell line ATPase was observed to be 298 microM after 3 h of incubation. In addition, IC(50) values of 94 and 89 microM were observed at 30 min of incubation, indicating that the protocol shows reproducible results. A Z' factor higher than 0.7 was observed in the assays. These studies extend the profile of Na(+),K(+)-ATPases and demonstrate the feasibility of this HTS assay system to screen for compounds that pharmacologically modulate the function of Na(+),K(+)-ATPase.
Asunto(s)
Rubidio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrofotometría Atómica/métodos , Animales , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Ouabaína/farmacología , Sensibilidad y Especificidad , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
Ion channels have been identified as therapeutic targets in various disorders, such as cardiovascular disease, neurological disease, and cystic fibrosis. Flux assays to detect functional ionic flux through ion channels are becoming increasingly popular as tools for screening compounds. In an optimized flux assay, modulation of ion channel activity may produce readily detectable changes in radiolabeled or nonradiolabeled ionic flux. Technologies based on flux assays are currently available in a fully automated high throughput format for efficient screening. This application offers sensitive, precise, and reproducible measurements giving accurate drug rank orders matching those of patch clamp data. Conveniently, the flux assay is amenable to adaptation for different ion channels, such as potassium, sodium, calcium, and chloride channels, by using suitable tracer ions. The nonradiolabeled rubidium-based flux assay coupled with the ion channel reader (ICR) technology has become very successful in ion channel activity analysis and is emerging as a popular technique in modern drug discovery.