A graphite furnace-atomic absorption spectrometry-based rubidium efflux assay for screening activators of the Kv 7.2/3 channel.
Arch Pharm (Weinheim)
; 356(5): e2200585, 2023 May.
Article
em En
| MEDLINE
| ID: mdl-36748851
ABSTRACT
For the characterization of Kv 7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for Kv 7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv 7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of Kv 7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z'-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Rubídio
/
Grafite
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article