Identification of a series of novel n-aryl-benzamide compounds AS BKCa channel openers

1Robert W. Kirby, 1Donna J. Sellers, 1Anne-Marie Harrison, 2Vincenzo Calderone, 1Kim Lawson and 1Neil G. McKay. 1Biomedical Research Centre, Faculty of Health and Wellbeing, Sheffield Hallam University, Sheffield, S1 1WB. 2Faculty of Pharmacy, University of Pisa, I-56126 Pisa.

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability (Lawson and McKay 2006). Thus, compounds that activate BKCa could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system (Calderone 2002). A series of novel N-aryl-benzamide compounds (Calderone et al 2006a,b) compared with the reference NS1619 (Olesen et al 1994), were evaluated for their BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the zero variant of the human BKCa channel alpha subunit.

In a non-radioactive rubidium (Rb+) efflux assay (Kirby et al 2006), cells were incubated in RbCl buffer for 4 hours then washed prior to incubation for 10 minutes in KCl buffer containing either test compounds (0.1-100μM) or vehicle (DMSO, final concentration 0.3%). The supernatant and lysate samples were collected and the Rb+ content was determined by atomic absorption spectrometry and Rb+ efflux (%) was calculated. Data are presented as mean values (±SEM). All novel compounds evoked a concentration related increase in Rb+ efflux. The maximum increase in Rb+ efflux (Emax) evoked by N-aryl-benzamide compounds ranged between 53-91% (n=8) above that the baseline of 22.2±1.1% (n=10-12); that achieved with NS1619 was 62.5±4.8% (n=8). In the presence of the BKCa channel blocker iberiotoxin (0.1μM), the compounds (100μM for 10 min) failed to increase Rb+ efflux. The series of compounds demonstrated a 40-fold potency range (EC40:0.12-2.95μM); EC40 for NS1619 was 0.35±0.40μM (n=8).

Whole-cell voltage clamp electrophysiology currents were recorded using a voltage step protocol (–100 to +100mV) in symmetrical potassium (140mM) with 0.3μM free Ca2+ in the bath and pipette solutions. Normalised conductance vs voltage (G-V) curves were plotted and V50 values (voltage required to produce half-maximal activation) calculated. The novel compounds (30μM), like NS1619, produced significant (p<0.001) leftward-shifts in the G-V curve indicative of BKCa activation. In the presence of the compounds V50 values in the range of 31.32-64.07mV (n=6) were obtained compared to the control value of 79.52±1.37mV (n=48) and that in presence of NS1619 of 13.86±2.19mV (n=6).

In conclusion, the N-aryl-benzamide compounds tested exhibit properties consistent with the activation of BKCa channels. Preliminary data indicate discrimination, based on efficacy and potency, to activate BKCa channels by the series of compounds studied. Such agents will assist in the development of pharmacophore models to give insight into the receptor site for openers within the BKCa protein.

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