RESUMEN
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.
Asunto(s)
Grafito , Rubidio , Humanos , Espectrofotometría Atómica/métodos , Talio , Células HEK293 , Relación Estructura-ActividadRESUMEN
KV 7 channel openers have proven their therapeutic value in the treatment of pain as well as epilepsy and, moreover, they hold the potential to expand into additional indications with unmet medical needs. However, the clinically validated but meanwhile discontinued KV 7 channel openers flupirtine and retigabine bear an oxidation-sensitive triaminoraryl scaffold, which is suspected of causing adverse drug reactions via the formation of quinoid oxidation products. Here, we report the design and synthesis of nicotinamide analogs and related compounds that remediate the liability in the chemical structure of flupirtine and retigabine. Optimization of a nicotinamide lead structure yielded analogs with excellent KV 7.2/3 opening activity, as evidenced by EC50 values approaching the single-digit nanomolar range. On the other hand, weighted KV 7.2/3 opening activity data including inactive compounds allowed for the establishment of structure-activity relationships and a plausible binding mode hypothesis verified by docking and molecular dynamics simulations.
Asunto(s)
Aminopiridinas , Canales de Potasio KCNQ , Canales de Potasio KCNQ/metabolismo , Relación Estructura-Actividad , Aminopiridinas/químicaRESUMEN
The KV 7 potassium channel openers flupirtine and retigabine have been valuable options in the therapy of pain and epilepsy. However, as a result of adverse reactions, both drugs are currently no longer in therapeutic use. The flupirtine-induced liver injury and the retigabine linked tissue discolouration do not appear related at first glance; nevertheless, both events can be attributed to the triaminoaryl scaffold, which is affected by oxidation leading to elusive reactive quinone diimine or azaquinone diimine metabolites. Since the mechanism of action, i. e. KV 7 channel opening, seems not to be involved in toxicity, this study aimed to further develop safer replacements for flupirtine and retigabine. In a ligand-based design strategy, replacing amino substituents of the triaminoaryl core with alkyl substituents led to carba analogues with improved oxidation resistance and negligible risk of quinoid metabolite formation. In addition to these improved safety features, some of the novel analogues exhibited significantly improved KV 7.2/3 channel opening activity, indicated by an up to 13-fold increase in potency and an efficacy of up to 176 % compared to flupirtine, thus being attractive candidates for further development.
Asunto(s)
Carbamatos , Fenilendiaminas , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Carbamatos/farmacología , Canales de Potasio KCNQ/metabolismo , Fenilendiaminas/farmacologíaRESUMEN
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.