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For the characterization of Kv7.2/3 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 Kv7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv7.2/3 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 Kv7.2/3-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.
The potassium channel opening drugs flupirtine and retigabine have been withdrawn from the market due to occasional drug-induced liver injury (DILI) and tissue discoloration, respectively. While the mechanism underlying DILI after prolonged flupirtine use is not entirely understood, evidence indicates that both drugs are metabolized in an initial step to reactive ortho- and/or para-azaquinone diimines or ortho- and/or para-quinone diimines, respectively. Aiming to develop safer alternatives for the treatment of pain and epilepsy, we have attempted to separate activity from toxicity by employing a drug design strategy of avoiding the detrimental oxidation of the central aromatic ring by shifting oxidation toward the formation of benign metabolites. In the present investigation, an alternative retrometabolic design strategy was followed. The nitrogen atom, which could be involved in the formation of both ortho- or para-quinone diimines of the lead structures, was shifted away from the central ring, yielding a substitution pattern with nitrogen substituents in the meta position only. Evaluation of KV7.2/3 opening activity of the 11 new specially designed derivatives revealed surprisingly steep structure–activity relationship data with inactive compounds and an activity cliff that led to the identification of an apparent “magic methyl” effect in the case of N-(4-fluorobenzyl)-6-[(4-fluorobenzyl)amino]-2-methoxy-4-methylnicotinamide. This flupirtine analogue showed potent KV7.2/3 opening activity, being six times as active as flupirtine itself, and by design is devoid of the potential for azaquinone diimine formation.