Identification, Characterization, and Optimization of BK(Ca) Channel Activators Containing Tricyclic Quinazoline Ring

Abstract

The large-conductance Ca2+-activated K+ channel (BKCa channel) is involved in repolarizing the membrane potential and has a variety of cellular functions. The BKCa channel is highly expressed in bladder smooth muscle and mediates muscle relaxation. Compounds that activate the BKCa channel have therapeutic potential against pathological symptoms associated with the overactivity of bladder smooth muscle. In this regard, I screened a chemical library of 9,938 compounds to identify novel BKCa channel activators. A cell-based fluorescence assay identified a structural family of compounds containing a tricyclic quinazoline ring that activated the BKCa channel. The most potent compound TTQC-1 (7-bromo-N-(3-methylphenyl)-5-oxo-1-thioxo-4,5-dihydro[1,3]thiazolo[3,4-a]quinazoline-3-carboxamide) directly and reversibly activated the macroscopic current of BKCa channels expressed in Xenopus oocytes from both sides of the cellular membrane. TTQC-1 increased the maximum conductance and shifted the half activation voltage to the left. The apparent half-maximal effective concentration and dissociation constant were 2.8 μM and 7.95 μM, respectively. TTQC-1 delayed the kinetics of channel deactivation without affecting channel activation. The activation effects were observed over a wide range of intracellular Ca2+ concentrations and dependent on the co-expression of β1 and β4 auxiliary subunits. In the isolated smooth muscle cells of rat urinary bladder, TTQC-1 increased the K+ currents, and oral administration of TTQC-1 to hypertensive rats decreased the urination frequency. Therefore, TTQC-1 is a BKCa channel activator with a novel structure that has potential therapeutic effects for BKCa channel-related diseases, such as overactive bladder syndrome. Furthermore, I verified the pharmacophore of TTQC-1 by testing 19 more derivatives. Among them, halide substituted compounds had significant enhancement in vitro efficacy. Finally, one binding site was suggested on the transmembrane domain of the channels through molecular docking study.