Investigation of the pharmacophore of BK[ca] potassium channel openers.

Large conductance voltage-activated calcium-sensitive potassium channels (BK[ca]) are fundamental in the control of cellular excitability which is critical in the regulation of many physiological processes. Agents that activate the channel (openers) have been proposed to be potential therapeutics for a number of un-met clinical conditions. Several heterogeneous classes of compounds have been described as BK[ca] channel openers and preliminary pharmacophore data has arisen for synthetic molecules based on the structure of benzimidazolones.The project aimed to explore a series of novel compounds based on a benzanilide template for BK[ca] channel opener properties and to further probe the pharmacophore of BK[ca] channel openers. This was achieved by the validation and optimisation of a medium through-put, non-radioactive, rubidium (Rb[+]) efflux assay using recombinant HEK293 cells expressing BK[ca] channel subunits. From which novel benzanilides have been identified as BKca channel openers that display varying degrees of potency, efficacy and co-operativity. The Rb[+] efflux stimulated by each compound was blocked by use of the BKca channel blockers paxilline and iberiotoxin demonstrating specificity to BK[ca] channels in the cell lines studied. Furthermore initial data demonstrates that some of these compounds showed selectivity for the BK[ca] channel alpha-subunit.The benzanilide compounds were also examined using whole cell electrophysiology; all compounds tested, relative to control, produced shifts in V50 values in the hyperpolarising direction, indicative of BK[ca] channel activation. The benzanilides did not affect the kinetics of activation or deactivation. Furthermore, the order of effectiveness determined using whole cell electrophysiology showed good correlation with that obtained using the Rb[+] efflux assay. Using both the electrophysiology and Rb+ efflux techniques BKOEt1 was identified as a novel and potent BKca channel opener and selected as a lead compound.In single channel electrophysiology recordings of cloned cell lines expressing BK[ca] channel subunits application of BKOEt1 promoted significant, paxilline sensitive, BK[ca] channel activation. BKOEt1 increased open pore probability rapidly for small changes in voltage and increased the voltage-sensitivity of the channel. In addition, BKOEt1 did not affect single channel conductance. BKOEt1 activated BKca channels in the near absence of intracellular calcium and its effects were not additive. Furthermore, the compound did not affect the level of intracellular calcium. It was concluded that BKOEt1 acts directly on the channel at a site located on the alpha-subunit. A Hill slope of unity suggested one binding site per tetrameric channel complex and either an intracellular or transmembrane site of interaction was proposed. BKOEt1 also stimulated paxilline sensitive Rb+ efflux from rat bladder myocytes and initial Rb+ efflux studies demonstrated that BKOEt1 could activate K[v] and SK channels.Molecular modelling of the series of benzanildies provided clues as to the chemical or structural features required, in particular for BKOEt1, to retain potent channel opener properties. In addition, physicochemical properties were determined and revealed commonalities for compounds to retain potent BKca channel opener properties. A confirmatory pharmacophore was proposed with compounds requiring two substituted phenyl rings, the presence of an oxygen containing group, an amide group provided by the linker region and hydrophobic moieties.