RESUMEN
In vitro screens using cellular preparations expressing human Ether-à-go-go related gene (hERG) potassium channels have become an intrinsic tool for evaluating cardiac liability of compounds during early preclinical stage development. Although hERG channel blocking effects are most reliably evaluated using the low-throughput, manual patch clamp technique, methods and technologies that yield hERG activity data in multiwell format are required to address increased throughput requirements. In most cases, multiwell approaches to measuring hERG activity involve achieving a reasonable balance between throughput and data fidelity. Here we compared two functional multiwell hERG assays: a fluorescence-based fluorometric imaging plate reader (FLIPR(®)) screen measuring thallium (Tl(+)) influx through hERG channels and an automated patch clamp assay using an IonWorks Quattro(®). Mean Z' values for FLIPR-Tl(+) and IonWorks Quattro assays were similar, 0.57 ± 0.09 (±SD; n = 10) versus 0.63 ± 0.11 (n = 12), respectively. IC50 determinations for a set of 17 reference compounds were used to evaluate potency shifts relative to conventional voltage clamp data. The reference compound set included members that are known to exert severe potency shifts in multiwell assays. Mean potency shift values for FLIPR-Tl(+) and IonWorks Quattro assays were 117- and 8-fold, respectively. On the basis of reduced potency shifts and low data variability, we conclude that the IonWorks Quattro screen was a better predictor of hERG activity in conventional whole-cell patch clamp than the Tl(+) influx assay.
Asunto(s)
Evaluación Preclínica de Medicamentos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Talio/metabolismo , Células Cultivadas , Descubrimiento de Drogas , Canales de Potasio Éter-A-Go-Go/fisiología , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Mediciones Luminiscentes , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-ClampRESUMEN
T-type calcium channel antagonists were designed using a protocol involving the program SPROUT and constrained by a ComFA-based pharmacophore model. Scaffolds generated by SPROUT were evaluated based on their ability to be translated into structures that were synthetically tractable. From this exercise, a novel series of potent and selective T-type channel antagonists containing a biaryl sulfonamide core were discovered.
