NS19504: a novel BK channel activator with relaxing effect on bladder smooth muscle spontaneous phasic contractions

Abstract

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

Fig. 1.

FLIPR-based hBK assay. (A) Fluorescence emission from HEK293 cells stably overexpressing BK and loaded with BTC-AM followed over time on a fluorescence-imaging plate reader (FLIPR). In the first addition, cells are stimulated with either Cl⁻-free buffer (full line) or Cl⁻-free buffer containing a BK activator to be tested (dashed line). In the second addition, a Tl⁺-based stimulus buffer containing the Ca²⁺ ionophore A23187 and K₂SO₄ (for slight depolarization, because hBK channels are voltage- as well as Ca²⁺-sensitive) at final concentrations of 1.5 µM and 12.5 mM, respectively, is added (all traces). Note that, under control conditions, there is a minor increase in the fluorescence signal upon addition of stimulus buffer (full line), whereas in the presence of a hBK-positive modulator, there is a marked increase in the response after the second addition (dashed line). (B) A representative example of a screening plate showing the fluorescence traces over time. Wells A1-P22 were exposed to test compound at a concentration of 30 µM. One positive modulator hit (green square) was identified on the illustrated screening plate. Columns 23 and 24 contain control wells: buffer control (blue square, wells A23:H24) and a proprietary BK channel modulator used as positive control (red square, wells I23:P24). (C) Typical example of a concentration-response curve for NS19504 tested in 1/2-log dilutions ranging from 0.316 nM to 31.6 µM. Each compound concentration was tested in four individual wells, and responses were normalized toward the proprietary BK channel modulator. Symbols and error bars depict average responses and S.D. (D) Structure of NS19504. Ctrls, controls.

Fig. 2.

Activation of hBK channels measured in whole-cell patch-clamp experiments. (A) hBK-mediated currents recorded from voltage ramps (top) before (Saline) and in the presence of NS19504 in the concentrations indicated to the right of the traces. Currents were measured at a ramp potential of 25 mV as indicated by arrow. (B) The currents at +25 mV were measured and plotted as a function of time. At the 0.03 µM free Ca²⁺ used in this experiment, activation was prominent from a concentration of 1 µM NS19504. Due to the high numbers of channels in the HEK293 cell line, the currents were too large to test higher concentrations of the compound. (C) Summary bar graph showing percentage BK current (control = 100%) at various concentrations of NS19504. The average values ± S.E.M. are 0.1 µM: 100% (n = 1); 0.32 µM: 121 ± 5.7% (n = 6); 1 µM: 205 ± 34% (n = 6); 3.2 µM: 530 ± 74% (n = 6); and 10 µM: 1834 ± 917 (n = 4).

Fig. 3.

Electrophysiological characterization and activation of BK channels by NS19504. Current was measured in inside-out patches obtained from HEK293 cells stably expressing BK channels. Experiments were conducted at a physiologic K⁺ gradient (4/154 mM K⁺) and at a free intracellular Ca²⁺ concentration of 0.3 µM. (A) Current-voltage (I-V) relationships measured in the absence (Control) or presence of 10 µM NS19504 in the intracellular/bath solution. Currents were elicited by applying linear voltage ramps from −120 to +80 mV from a holding potential of −90 mV. (B) BK current measured at +80 mV depicted as a function of time. NS19504 (10 µM) was applied to the inside of the patch, indicated by the bar. Breaks in the recording indicate periods where voltage step protocols were applied. Data are from a single experiment representative of four independent experiments. (C) BK current activated by membrane potential steps, as illustrated in the drawing to the right. (D) Normalized tail currents depicted as a function of step potential. Data are mean ± S.E.M. of four independent experiments. Peak tail currents were measured by stepping to −120 mV after obtaining steady current activation at depolarized potentials either in the absence or presence of 10 µM NS19504.

Fig. 4.

NS19504 increases BK currents in freshly isolated single USBM cells. (A) Representative whole-cell currents in response to 200-millisecond voltage ramps from −100 to +50 mV (insert in top) before (saline) and in the presence of NS19504 in the concentrations indicated to the right of the traces. BK channel inhibitor paxilline (1 µM, Pax) added at the end of experiment eliminates the effect of NS19504 and decreases the currents below the control (saline) amplitude. (B) Summary of relative to control percent increase of whole-cell BK currents by 0.32, 1.0, and 3.2 µM NS19504 measured at +50 mV. BK currents were obtained by subtraction of the currents in the presence of paxilline (1 µM) from control currents and those in the presence of NS19504. Data are means ± S.E. (n = 7– 8; *P < 0.05, one-way ANOVA).

Fig. 5.

Effect of 1 µM NS19504 on spontaneous contractions of myogenic origin (SPCs, A–D). Representative recordings of SPCs in guinea pig detrusor strips under control conditions (0.1% DMSO) (A), with application of 1 µM NS19504 only (B), with application of 1 µM NS19504 to strips pretreated with 100 nM IbTx (C), and with application of 1 µM NS19504 to strips pretreated with 300 nM Apa (D). Application of 1 µM NS19504 reduced SPCs to 56% ± 4% of control, an effect that was blocked by pretreatment with IbTx, but not by pretreatment with Apa. (E) Summary of data. DMSO, vehicle control; NS19504, 1 µM NS19504; NS19504 (IbTx), 1 µM NS19504 in strips pretreated with IbTx; NS19504 (Apa), 1 µM NS19504 in strips pretreated with Apa (***P < 0.01 compared with NS19504).

Fig. 6.

NS19504 decreases SPC activity in a concentration-dependent manner. (A) Addition of increasing concentrations of NS19504 decreased SPC activity under control conditions (top); pretreatment with 100 nM IbTx inhibited the relaxing effect of NS19504 at concentrations lower than 1 µM (bottom). (B) Concentration-response curve. IbTx shifted the concentration-response curve to the right, with an EC₅₀ for NS19504 of 640 nM in the absence and 4.27 µM in presence of IbTx (*P < 0.05; ***P < 0.01; two-way ANOVA).

Fig. 7.

NS19504 modestly decreases nerve-evoked contractions. (A) Original recording of contractile response to EFS before and after application of 1 µM NS19504. (B) Summary data showing a statistically significant, but small, relaxing effect of NS19504 on EFS-induced contractions (***P < 0.01 compared with control; paired t test).

Fig. 8.

NS19504 does not inhibit depolarization-induced contraction. (A) Original recording of contractile force induced by 60 mM K⁺ PSS. After about 15 minutes, force reached a stable plateau and addition of 1 µM NS19504 had no effect. (B) Summary data (P = 0.200; paired t test, n = 6).