Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

Unconventional role of the inwardly rectifying potassium channel Kir2.2 as a constitutive activator of RelA in cancer.

The constitutive activation of NF-κB is a major event leading to the initiation, development, and progression of cancer. Recently, we showed that the size of preestablished tumors was reduced after the depletion of Kir2.2, an inwardly rectifying potassium channel. To determine the precise mechanism of action of Kir2.2 in the control of tumor growth, we searched for interacting proteins. Notably, NF-κB p65/RelA was identified as a binding partner of Kir2.2 in a yeast two-hybrid analysis. Further analyses revealed that Kir2.2 directly interacted with RelA in vitro and coimmunoprecipitated with RelA from cell lysates. Kir2.2 increased RelA phosphorylation at S536 and facilitated its translocation from the cytoplasm to the nucleus, thereby activating the transcription factor and increasing the expression level of NF-κB targets, including cyclin D1, matrix metalloproteinase (MMP)9, and VEGF. Kir2.2 was overexpressed in human cancer and the expression level was correlated with increased colony formation and tumor growth in mouse tumor models. On the basis of these findings, we propose an unconventional role for Kir2.2 as a constitutive RelA-activating protein, which is likely to contribute to tumor progression in vivo.

The high throughput screening of neuropeptide FF2 receptor ligands from Korean herbal plant extracts.

We have screened 356 libraries of Korean herbal plant extracts to find potential anti-obesity drugs. We employed the recently developed fluorescence polarization high throughput screening (FP HTS) assays of human neuropeptide FF (NPFF) receptors in 384-well microtiter plates. The primary hits were cherry-picked from the libraries and further analyzed by secondary displacement curve assays, in vitro GTPgammaS binding assays and cell-based CRE luciferase reporter assays. Agonists of NPFF receptors showed biphasic affinity curves while the antagonist, BIBP 3226, gave a monophasic affinity curve in competitive binding assays. We isolated and characterized two agonists of human NPFF2 receptor, PC 314 with K(i) of 1.42 microM, and PC 315 with K(i) of 2.17 microM from Schizandra chinensis. PC 314 and PC 315 have been characterized as benzoylgomisin Q (M.W. 552) and gomisin G (M.W. 536). We report that PC 314 and PC 315 are the first non-peptide, natural compounds, which bind to human NPFF2 receptors with good affinity. PC 314 and PC 315 inhibit forskolin-stimulated luciferase expression when CHO cells are co-transfected with NPFF2 receptor and CRE reporter vector. They possess the pharmacological and functional profiles of full agonists. The FP HTS system provides a specific, sensitive and reproducible methodology for studying and screening NPFF receptor ligands.