Abstract
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.
Introduction
The large-conductance calcium-activated potassium channel (BKCa channel; also called Maxi K, Slo1, or KCa 1.1) is activated by membrane depolarization or increases in intracellular Ca2+ concentration (Barrett et al., 1982; Yang et al., 2015). Potassium ions pass through the channel rapidly from the intracellular side to the extracellular side when the BKCa channel is opened, resulting in membrane hyperpolarization (Salkoff et al., 2006; Cui et al., 2009). The BKCa channel is composed of four pore-forming α subunits and auxiliary β subunits. Four types of β subunits, β1–β4, show tissue-specific distributions and affect the activation of BKCa channels (Orio et al., 2002). The BKCa channel is expressed in the brain, smooth muscle, bladder, cochlea, and other various tissues (Poulsen et al., 2009). The BKCa channel regulates several physiologic processes—for example, neurotransmitter release (Wang, 2008), smooth muscle contraction (Wu and Marx, 2010), hormone secretion from endocrine cells (Marty, 1981; Wang et al., 1994), hearing (Salkoff et al., 2006), and circadian rhythms (Meredith et al., 2006). Based on these roles, malfunction of the BKCa channel is linked to several diseases, such as stroke (Gribkoff et al., 2001), erectile dysfunction (Werner et al., 2005), and overactive bladder (OAB) (Layne et al., 2010). In addition, a gain-of-function mutation of the BKCa channel was linked to epilepsy and paroxysmal dyskinesia (Du et al., 2005).
OAB syndrome makes patients feel frequent urinary urgency both day and night (Cerruto et al., 2012). In the United States, 16.0% of men and 16.9% of women suffer from OAB (Stewart et al., 2003). In the urination pathway, acetylcholine (ACh) released from parasympathetic nerves binds to muscarinic receptors on urinary bladder smooth muscle (UBSM) to induce contraction of UBSM and urination (Hegde and Eglen, 1999). Muscarinic receptor antagonists, which inhibit ACh-induced UBSM contraction, are commonly used to treat OAB (Abrams and Andersson, 2007). However, these induce several side effects, such as blurred vision, dry mouth, constipation, tachycardia, and cognitive impairment (Eglen et al., 1999; Kay and Granville, 2005). Therefore, different therapeutic targets for OAB syndrome that are associated with fewer side effects need to be studied.
As mentioned previously, the BKCa channel controls smooth muscle contraction (Wu and Marx, 2010). The BKCa channel is highly expressed in UBSM cells (Hristov et al., 2011), where it maintains the resting membrane potential and regulates the repolarization phase of the spontaneous action potential that controls UBSM contraction (Petkov, 2014). Cholinergic- and purinergic-induced contractility is decreased by BKCa channel activation, and alterations in BKCa channel expression or function affect OAB symptoms (Werner et al., 2007; Sprossmann et al., 2009). Thus, UBSM relaxation can be induced by BKCa channel activation, suggesting that the BKCa channel is a potential therapeutic target for the treatment of OAB.
In this study, we screened 8364 chemical compounds using a cell-based fluorescence assay to search for a novel BKCa channel activator. A TI+-based fluorescence assay platform, which measures the activity of voltage-gated K+ channels, was used for screening the activity of the compounds against a mutant BKCa channel. The channel has highly increased sensitivity to Ca2+, meaning an increase in intracellular Ca2+ concentration is not required for channel activation (Lee et al., 2013). From the initial screening and the secondary structure-activity relationship study, we identified one chemical compound, 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD), as a potent activator of the BKCa channel. The conductance-voltage (G-V) relationship of the BKCa channel shifted to a more negative value in a concentration-dependent manner when CTIBD was applied to the extracellular side of the channel. In addition, CTIBD significantly decreased ACh-induced contraction of rat UBSM strips (Bo and Burnstock, 1990). When injected intraperitoneally into a rat model of acetic acid (AA)-induced OAB, CTIBD increased the reduced intercontraction interval and voiding volume significantly. Thus, CTIBD and its derivatives may hold potential as OAB therapeutics that target the BKCa channel.
Materials and Methods
Materials.
A chemical library, named the Korea Research Institute of Chemical Technology (KRICT) Diversity Library, which contains 8364 unique chemical compounds, was obtained from the KRICT (Daejeon, South Korea; www.chembank.org). CTIBD and other isoxazolyl benzene derivatives were first purchased from Vitas-M Laboratory (Causeway Bay, Hong Kong; www.vitasmlab.biz). After that, a large amount of CTIBD and its derivatives were synthesized (Supplemental Fig. 1). Those compounds are prepared by dissolving compounds in DMSO (Sigma-Aldrich, St. Louis, MO).
Synthesis of Compound 4 (CTIBD).
Compound 4 (CTIBD) was synthesized as outlined in Scheme 1. The synthetic procedure is as follows for 2-(4-chlorophenyl)-1-(2,4-dihydroxyphenyl)ethan-1-one 3: To a solution of resorcinol 1 (1.42 g, 12.89 mmol) in boron trifluoride diethyl etherate (20 ml) was added 2-(4-chlorophenyl) acetic acid 2 (2 g, 11.72 mmol), and the reaction mixture was heated at 75°C for 4 hours under a nitrogen atmosphere. The mixture was poured into water and extracted with ethyl acetate (EtOAc). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. Purification of the resulting residue by column chromatography using EtOAc/hexane as the eluent provided 2-(4-chlorophenyl)-1-(2,4-dihydroxyphenyl)ethan-1-one 3 (1.7 g, 55% yield). NMR data of 3 is as follows: 1H NMR (DMSO-d6) δ 12.40 (s, 1H), 10.69 (s, 1H), 7.93 (d, J = 8.85 Hz, 1H), 7.37 (d, J = 8.24 Hz, 2H), 7.30 (d, J = 8.54 Hz, 2H), 6.40 (dd, J = 8.85, 2.14 Hz, 1H), 6.26 (d, J = 2.44 Hz, 1H), 4.33 (s, 2H).
4-(4-(4-Chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol 4: To a solution of 2-(4-chlorophenyl)-1-(2,4-dihydroxyphenyl)ethan-1-one 3 (1.7 g, 6.47 mmol) in pyridine (5 ml), trifluoroacetic anhydride (2.8 ml, 19.42 mmol) was added dropwise with cooling in an ice bath, and then the mixture was stirred at room temperature for 48 hours. The mixture was concentrated to dryness to yield 3-(4-chlorophenyl)-7-hydroxy-2-(trifluoromethyl)-4H-chromen-4-one, which was used for the next step. A mixture of 3-(4-chlorophenyl)-7-hydroxy-2-(trifluoromethyl)-4H-chromen-4-one and hydroxylamine hydrochloride (856.7 mg, 12.33 mmol) dissolved in pyridine was heated at reflux for 12 hours and then cooled to room temperature. The reaction mixture was acidified with dilute HCl and extracted with EtOAc. The organic layer was separated and washed with saline solution, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting residue was purified by column chromatography to provide 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol 4 (800 mg, 51% yield over two steps). Characterization of 4 is as follows: 1H NMR (DMSO-d6) δ 9.96 (s, 1H), 9.95 (s,1H), 7.52-7.42 (multiplet peak, 2H), 7.28 (d, J = 8.24 Hz, 2H), 7.10 (d, J = 8.24 Hz, 1H), 6.33 (d, J = 2.14 Hz, 1H), 6.28 (dd, J = 8.54, 2.14 Hz, 1H); liquid chromatography–mass spectrometry (mass-to-charge ratio): 356.1 (M + H).
Fluorescence Assay and Data Analysis.
Modified human embryonic kidney cells (AD-293 cells) that stably express a mutant BKCa channel (G803D/N806K) (Lee et al., 2013) were used for the cell-based assay. Cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT) containing 10% fetal bovine serum (Hyclone) and 1 mg/ml geneticin (Gibco/Life Technologies, Waltham, MA). Approximately 20,000 cells per well were seeded on a 96-well clear-bottom black-wall assay plate (Corning Incorporated, Corning, NY) coated with poly(d-lysine) (Sigma-Aldrich). The cell-based assay used a FluxOR potassium channel assay kit (Invitrogen, Eugene, OR) and was performed using the following steps. First, cell growth medium was removed, and 80 μl of loading buffer containing FluxOR fluorescent dye was added to each well. The plate was then incubated at room temperature in the dark for 1 hour. After incubation, loading buffer was removed, and 100 μl of assay buffer containing varying concentrations of test compounds was added to each well of the plate. The plate was incubated at room temperature for 20–30 minutes. DMSO (1%) was used as the vehicle. DMSO 1% did not affect fluorescence signal significantly. Fluorescence was measured using a FlexStation 3 multimode microplate reader (Molecular Devices, Sunnyvale, CA) and SoftMax Pro software. A hybrid multimode microplate reader (Synergy H1; BioTek Instrument Inc., Winooski, VT) and Gen5 software were used for Supplemental Data. The fluorescence excitation wavelength was 485 nm, and the emission wavelength was 528 nm. The fluorescence signal was measured every 2 seconds for FlexStation 3 and every 10 seconds for Synergy H1. Depolarization of the cell membrane was induced chemically by the addition of 20 μl of stimulus buffer containing a high concentration of TI+ to each well. For FlexStation 3, stimulus buffer is added automatically 20 seconds after initiation of fluorescence measurements. In the case of Synergy H1, stimulus buffer is added manually 40 seconds after initiation of fluorescence measurements. Fluorescence signals were analyzed as relative fluorescence units (RFU or F/F0, F0: minimum value of fluorescence signals of each well). One-way ANOVA was used for statistical analysis (*P value < 0.05, **P value < 0.01, ***P value < 0.001).
Functional Expression of the BKCa Channel in Xenopus Oocytes.
Rat BKCa channel α (Slo1), β1, and β4 subunits were expressed in Xenopus laevis oocytes for electrophysiological recording. An oocyte expression vector (pNBC1.0 or pNBC2.0) was used for rat BKCa channel subcloning and functional expression, which were carried out according to previously described methods (Ha et al., 2006). The cDNA sequence information is listed in GenBank. The accession number of the rat BKCa channel α subunit was AF135265, the β1 subunit was FJ154955.1, and the β4 subunit was AY028605.
Oocytes were taken surgically from the ovarian lobes of X. laevis at stages V–VI (Nasco, Fort Atkinson, WI), placed in Ca2+-free oocyte Ringer’s culture medium [86 mM NaCl, 1.5 mM KCl, 2 mM MgCl2, and 10 mM HEPES (pH 7.6)] containing 3 mg/ml collagenase (Worthington Biochemicals, Freehold, NJ), and incubated for 1.5–2 hours at room temperature to remove the follicular cell layer. After incubation, oocytes were rinsed with Ca2+-free oocyte Ringer’s medium and then ND-96 medium [96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and 50 g/ml gentamicin (pH 7.6)]. Rinsed oocytes were incubated in ND-96 medium at 18°C for at least 24 hours to stabilize. Approximately 50 ng of synthesized Slo1 cRNA prepared in nuclease-free water was injected into each oocyte, after which they were incubated at 18°C for 1–3 days in ND-96 medium. For the coexpression experiments, cRNA of rβ1 and rβ4 subunits were injected into each oocyte with Slo1 cRNA. The molar ratio of β subunit cRNA versus Slo1 cRNA was 12:1. That ratio guarantees sufficient coassembling of α and β subunits of the BKCa channel. The vitelline membrane was removed using fine forceps before electrophysiological recordings.
Electrophysiological Recordings and Data Analysis.
A gigaohm seal patch-clamp method with membranes in an outside-out configuration was used for recording macroscopic currents, as described previously (Ha et al., 2006). Borosilicate glass pipettes (World Precision Instruments, Sarasota, FL) were pulled and fire-polished, and they had a resistance of 3–5 MΩ. To facilitate electrophysiological recordings, the channel current was amplified using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA) and low-pass-filtered at 1 kHz using a four-pole Bessel filter. The channel currents were digitized at a rate of 10 or 20 points per millisecond using a Digidata 1200A (Molecular Devices).
For the recording of currents, BKCa channel currents were activated by voltage-clamp pulses from −80 to 200 mV in 10-mV increments. The resting potential was held at −100 mV. Oocytes were placed in recording solution, which contained 120 mM potassium gluconate, 10 mM HEPES, 4 mM KCl, and 5 mM EGTA (pH 7.2). The MaxChelator program (Patton et al., 2004) was used to calculate the concentration of free Ca2+ in the intracellular solution. The intracellular solution contained 116 mM KOH, 10 mM HEPES, and 4 mM KCl (pH 7). Concentrations of N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid, EGTA, and free Ca2+ are calculated by the MaxChelator program. Solution exchange was completed within 0.5 seconds. Clampex 8.0 and Origin 9.1 software (OriginLab Corporation, Northampton, MA) were used for data acquisition and analysis. Data were summarized as means ± S.D. A two-tailed paired t test was used for statistical analysis (*P value < 0.05; **P value < 0.01; ***P value < 0.001).
Isometric Tension Recording from UBSM.
Experiments recording the isometric tension of UBSM were performed according to a previously described method (Lee et al., 2016). Briefly, bladder strips were isolated from male Sprague-Dawley rats (300–350 g). The isolated strips (approximately 2 × 8 mm) were mounted in 10-ml organ baths containing Krebs’ solution [118.4 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, 2.5 mM CaCl2, and 12.2 mM glucose (pH 7.4), bubbled with a mixture 95% O2 and 5% CO2 at 37°C]. Isometric force measurements were recorded using a Power Laboratory Data Acquisition System (ADInstruments, Australia, NSW) attached to a computer installed with LabChart software (version 7; ADInstruments). Before each experiment, bladder strips were subjected to 1 g of resting tension and allowed to equilibrate for at least 1 hour. After this equilibration period, each strip was repeatedly exposed to 10 μM ACh until constant responses were recorded with a washout period of 30 minutes. The strips were then pretreated for 30 minutes with CTIBD or 0.1% DMSO, and then ACh-induced contractile responses were measured in the presence of CTIBD or DMSO. The relaxant response to CTIBD was expressed as the percent decrease in the ACh-induced contractile tension in the presence of CTIBD. One-way ANOVA was used for statistical analysis (*P value < 0.05; **P value < 0.01; ***P value < 0.001).
In Vivo Cystometry.
Adult female Sprague-Dawley rats weighing 296 ± 15 g (approximately 13 to 14 weeks old) were anesthetized with isoflurane (3%, 3 ml/min), and a polyethylene catheter (PE-50) was implanted into the bladder. Cystometry was performed 3 days after catheter insertion under anesthesia (urethane 1.3 g/kg, s.c.). The anesthetized animal was fixed supine on a vertically positioned table so that the urethra meatus of the animal pointed downward. A plastic cup placed underneath the urethra meatus and connected to a force transducer for weight measurement (FT0314618; Natus) was used to measure voided urine. The temperature of each animal was maintained at 37°C during anesthesia using a heating pad. In all experiments, the bladder was first filled with saline and then infused with saline for 1 hour at room temperature at a rate of 0.05 ml/min. Then, 0.5% AA was infused to the same rat until a voiding contraction occurred or for a maximum duration of 20 minutes, whichever happened first. CTIBD was administered intraperitoneally to the same rat at a single dose of 20 mg/kg 40 minutes after AA administration. Intravesical pressure was recorded for 90 minutes after AA administration. Baseline pressure (millimeters of mercury) in the bladder, maximum pressure of voiding contraction (millimeters of mercury), intercontraction interval (seconds), and voiding volume (milliliters) were averaged for a 30- to 90-minute period postadministration in each animal. Normal saline filling, 0.5% AA infusing, and intraperitoneal injection of CTIBD were continuously performed in the same rat. Voiding volume was measured accumulatively during the experiment. One-way ANOVA was used for statistical analysis (*P value < 0.05; **P value < 0.01; ***P value < 0.001).
Animal Use Approval.
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Samsung Medical Center. Samsung Medical Center is a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and abides by the Institute of Laboratory Animal Resources guide.
Results
Identifying BKCa Channel Activators Using a Cell-Based Fluorescence Assay.
A chemical library containing 8364 chemical compounds was screened using a cell-based fluorescence assay to identify novel BKCa channel activators. One compound (compound 1, at 5 μM) increased the fluorescence signal by almost 6-fold within 100 seconds compared with vehicle (1% DMSO) (Fig. 1A). Since compound 1 was identified as 4-(4-phenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol, we purchased other derivatives containing a 4-phenyl-isoxazol-5-yl benzene skeleton and tested their effects on the BKCa channel using the cell-based fluorescence assay. Among 19 additional compounds tested at 5 μM, eight compounds significantly increased fluorescence at 100 seconds (Supplemental Fig. 2), with compound 4 (or CTIBD) evoking the highest fluorescence (Fig. 2B; Supplemental Fig. 2). At 5 μM, CTIBD-induced fluorescence was 7.9-fold higher than vehicle-induced fluorescence (Fig. 2C). The increase in fluorescence induced by CTIBD was concentration-dependent and blocked completely by 1 μM paxilline (Fig. 3A), a selective blocker of the BKCa channel (Sanchez and McManus, 1996). At 10 μM, CTIBD showed significantly higher levels of fluorescence compared with three other known BKCa activators: NS 1619, NS 11021, and rottlerin (Supplemental Fig. 4).
Effects of CTIBD on the Macroscopic Currents of the BKCa Channel.
To validate the cell-based assay results, we determined the effects of CTIBD on the BKCa channel using electrophysiology. The α subunit of the wild-type rat BKCa channel was expressed in Xenopus oocytes, and the macroscopic current of excised oocyte membranes was measured. The excised membrane configuration was outside-out, and the intracellular Ca2+ concentration was fixed at 3 μM. CTIBD was applied to the extracellular side of the excised membrane at 3 μM, 30 seconds after initiation of current measurements (Fig. 4). CTIBD increased the amplitude of macroscopic currents in a time-dependent manner (Fig. 4, b and c) from basal level (Fig. 4a). After removal of CTIBD, the channel currents decreased almost completely to the basal level (Fig. 4d). CTIBD appeared to mediate the potentiation and depotentiation process of the BKCa channel in two phases. During the potentiation process, for example, the channel current increased rapidly within a few seconds and then increased more steadily and gradually over a few minutes. Thus, the current data were fitted with a double-exponential function, and the time constants were estimated as 5.1 ± 2.6 and 48.3 ± 8.8 seconds for association and 4.3 ± 3.1 and 76.1 ± 32.0 seconds for dissociation.
We then investigated the mechanism of CTIBD-induced BKCa channel potentiation. A series of voltage pulses were applied to activate the BKCa channel, and macroscopic currents were measured under different concentrations of CTIBD. When the extracellular concentration of CTIBD was increased, the channel was activated at a lower voltage, and the tail currents evoked by repolarization were drastically increased (Fig. 5A). Figure 5B represents the G-V relationship of BKCa channel macroscopic currents under varying concentrations of CTIBD. Application of CTIBD shifted the G-V curve to a more negative voltage and increased the maximum conductance (Gmax) in a concentration-dependent manner. The concentration-dependent shift of the G-V relationship was quantified in terms of half-activation voltage (V1/2) (shown in Fig. 5C). As the extracellular concentration of CTIBD was increased from 0.1 to 10 μM, V1/2 decreased by approximately 50 mV from 155.8 ± 5.8 to 105.7 ± 4.4 mV. These results indicate that CTIBD lowers the threshold voltage for activation and increases the maximum open probability of the BKCa channel. A large negative shift of the G-V relationship by CTIBD was also observed at a basal intracellular Ca2+ concentration of 0.1 μM (Supplemental Fig. 5).
Effects of CTIBD on Gating Kinetics of the BKCa Channel.
Since the tail currents of the BKCa channel were increased by CTIBD (Fig. 5A), we further investigated the effects of this compound on gating kinetics. Figure 6 shows the activation and deactivation kinetics of the BKCa channel when analyzed in the absence and presence of 10 μM CTIBD. Figure 6, A and B shows representative current traces of activation (or opening) and deactivation (or closing), respectively, of the BKCa channel in the presence of 10 μM compound. The activation time constant (τactivation) values were obtained by fitting the current traces using a single-exponential function at each voltage (Fig. 6C). In the presence of 10 μM CTIBD, the activation rate was slightly increased at all voltages tested. The effects of CTIBD on channel deactivation were dramatic, and the deactivation time constant (τdeactivation) greatly increased (Fig. 6D). Channel closing greatly slowed in the presence of 10 μM CTIBD at all voltages tested. The deactivation rate decreased by 11.5-fold at 160 mV in the presence of 10 μM CTIBD. These results indicate that CTIBD potentiates BKCa channel activity by slowing channel closing with minimal effects on channel opening and suggest that upon binding to the channel, the compound stabilizes the activation conformation of the BKCa channel.
Effects of β Subunits on CTIBD-Induced Activation of the BKCa Channel.
Functional characteristics of the BKCa channel are altered by auxiliary β subunits. The effects of channel modulators can also be affected by the presence of β subunits. In Figs. 7 and 8, the effect of β subunits on CTIBD-induced potentiation of the BKCa channel was examined. cRNA of rSlo and rat β subunits were injected together to express rSlo/rβ1 or rSlo/rβ4 heteromeric BKCa channels. Vehicle (0.1% DMSO) or 10 μM CTIBD was treated to the extracellular side of the membrane. At 10 μM CTIBD, the rSlo/rβ1 channel shows a slight but significant shift of the G-V relationship. V1/2 values of vehicle-treated channel and 10 μM CTIBD–treated channel were 114.9 ± 6.7 and 98.1 ± 8.1 mV, respectively (Fig. 7B). In the case of the rSlo/rβ4 channel, the V1/2 value was decreased approximately 40 mV from 163.5 ± 13.9 to 121.5 ± 8.4 mV at 10 μM CTIBD (Fig. 8B).
Activation and deactivation time constant are also analyzed in Figs. 7 and 8. The activation time constant of the rSlo/rβ1 coexpressed BKCa channel was slightly increased by CTIBD but did not show a significant difference compared with the vehicle-treated condition (Fig. 7C). On the other hand, the deactivation time constant of the rSlo/rβ1 coexpressed BKCa channel was increased by CTIBD significantly (Fig. 7D). In the case of the rSlo/rβ4 coexpressed BKCa channel, the activation time constant was slightly decreased by CTIBD, but it was only significant at low-voltage pulses between 150 and 170 mV (Fig. 8C). The deactivation time constant of the rSlo/rβ4 coexpressed BKCa channel was dramatically increased by CTIBD (Fig. 8D). The deactivation rate decreased by 7.6-fold at 150 mV in the presence of 10 μM CTIBD. These results indicate that CTIBD activates both rSlo/rβ1 and rSlo/rβ4 coexpressed channels mainly by decreasing the closing rate of the channel, which is similar to the rSlo homomeric channel but occurs to a much lesser extent.
Relaxation Effect of CTIBD on Rat Bladder Smooth Muscle Strips.
Since it is well documented that the activation of the BKCa channel relaxes the smooth muscle in the bladder, we investigated whether CTIBD exerted relaxation effects on excised bladder strips from rats (Fig. 9). Initially, 10 μM ACh was applied to rat detrusor muscle strips, and the isometric tension was measured. ACh treatment caused the tension to rise dramatically. Immediately afterward, tension decreased and plateaued at half of the peak level. Pretreatment with CTIBD concentration-dependently reduced ACh-induced peak contractions (Fig. 9A). Although 3 and 10 μM CTIBD did not produce significant relaxation, significant relaxation by CTIBD was observed at higher concentrations: 42.1% ± 21.1% at 30 μM, 65.3% ± 14.0% at 100 μM, and 81.4% ± 10.2% at 300 μM compared with controls (Fig. 9B). The EC50 value of CTIBD was determined as 28.0 ± 5.0 μM. CTIBD did not show any relaxation effect on rat bladder strips at basal condition without ACh (Supplemental Fig. 6).
Effects of CTIBD on the Micturition Function of OAB Rats.
To observe the therapeutic effects of CTIBD on micturition, CTIBD was injected intraperitoneally into an AA-induced rat model of OAB. Rats infused with normal saline showed three voiding contractions at regular intervals, whereas rats treated with 0.5% AA showed hyperactive frequent voiding contractions (Fig. 10A, middle panel). Pretreatment of rats with 20 mg/kg CTIBD decreased the number of voiding contractions to basal levels (Fig. 10A, right panel). Thus, CTIBD restored the frequency of AA-induced voiding contractions to the basal state. During the in vivo cystometry experiments, basal pressure, maximum pressure of voiding contraction, intercontraction interval, and voiding volume were analyzed (Fig. 10B). CTIBD treatment did not significantly alter basal pressure or maximum pressure of voiding contraction. However, CTIBD treatment increased 0.5% AA-induced significant decreases in the intercontraction interval (Fig. 10D) and voiding volume (Fig. 10E). These results indicate that CTIBD produces anti-OAB effects in rats, which are most likely caused by relaxation of bladder detrusor muscle.
Discussion
In this study, we aimed to identify new chemical activators of the BKCa channel that contained novel skeletons. Initially, a chemical library containing 8364 unique compounds was screened using a cell-based fluorescence assay that we previously established (Lee et al., 2013). From the library, 25 compounds evoked a fluorescence that was at least 1.5-fold higher than vehicle (1% DMSO) at 100 milliseconds (data not shown). A single compound, identified subsequently as 4-(4-phenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (compound 1), was prominent in rapidly and robustly increasing fluorescence (Fig. 1). We then purchased 19 additional compounds containing a phenyl-isoxazolyl benzene skeleton for a structure-activity relationship (SAR) study.
Among the 20 compounds tested, nine compounds increased fluorescence significantly, with P values < 0.05 (Fig. 2; Supplemental Fig. 2). Three compounds were especially potent in the fluorescence assay. EC50 values were determined as 7.7 ± 0.5 μM for compound 1, 3.9 ± 0.6 μM for compound 4, and 9.4 ± 1.7 μM for compound 7. Thus, compound 4 (CTIBD) was chosen for further study (Supplemental Fig. 3). Compound 4 (CTIBD) increased the fluorescence signal most rapidly and highly among all derivatives tested in a concentration-dependent manner (Fig. 3). Results from the SAR study suggest that m-dihydroxy and trifluoromethyl moieties are essential for activity as channel activators. Although compound 4, with a p-chloro substituent of (4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol), was most active, there is clearly room for further optimization of this class of compounds, and thus more extensive SAR studies would be of great value.
We validated the potentiating effects of CTIBD on BKCa channel activity and investigated its mechanism of action. Application of CTIBD to the extracellular side of the channel reversibly and concentration-dependently potentiated macroscopic currents of the BKCa channel (Fig. 4). The potentiation and depotentiation trajectories could be fitted with double-exponential functions, indicating that the channel current comprised two different phases: an early fast phase and a late slow phase. One plausible explanation for this is that CTIBD simply has two different binding sites on the channel, each with distinct affinities: one site with high affinity and the other with lower affinity. Since the BKCa channel is composed of four α subunits, four identical affinity sites should exist in a tetrameric holo-channel with 4-fold symmetry. Thus, it is also conceivable that the binding of CTIBD to a single site of high affinity may convert another site to a lower-affinity binding site. It will be intriguing to determine experimentally the stoichiometry of CTIBD binding to individual channel subunits and also to determine the binding site on the channel that is responsible for current potentiation.
Mechanistically, CTIBD affects both voltage-dependent activation and maximum conductance of the channel. CTIBD concentration-dependently shifted the G-V relationship of the BKCa channel progressively to a negative direction (Fig. 5). Based on extrapolation, the maximum shift of V1/2 value was estimated as −70.3 mV. CTIBD also increases the maximum channelconductance at a given voltage. It is reported that the open probability of the BKCa channel does not reach unity even at extreme positive voltages (Sigg and Bezanilla, 1997; Ma et al., 2006). It will be intriguing to reveal in future research the mechanism of how the binding of CTIBD increases the maximum open probability and thus the maximum conductance of the BKCa channel. Kinetically, CTIBD potentiated the activity of the BKCa channel by dramatically slowing channel closing without significantly affecting the activation rate. At 10 μM CTIBD, the closing rate of the channel was slowed by 11.6-fold at 160 mV. It is likely that CTIBD binds to the open conformation of the channel more tightly, thus stabilizing the open state of the channel and preventing the channel from closing.
Since the functional characteristics are altered by auxiliary β subunits, the effects of β subunits on CTIBD-induced BKCa channel activation were studied. Two main β subunits, β1 and β4 subunits, were expressed together with α (or Slo). It is worth mentioning that these two β subunits are known to express predominantly in the bladder (Poulsen et al., 2009; Petkov, 2014). CTIBD shifted the G-V curve of both rSlo/rβ1 and rSlo/rβ4 channels to negative direction (Figs. 7 and 8). Whereas 10 μM CTIBD shifted the V1/2 value of rSlo/rβ1 approximately −16 mV, the same concentration of CTIBD shifted the V1/2 of rSlo/rβ4 channel as much as −40 mV. Thus, the potentiation of BKCa channel by CTIBD was decreased by coexpression of both β1 and β4 subunits, but the reduced activation was more significant for β1 than β4. The expression of β subunits also differentially affected the gating kinetics of CTIBD-induced BKCa channel activation and deactivation. CTIBD activates both rSlo/rβ1 and rSlo/rβ4 channels mainly by slowing down the channel closure. A similar kinetic effect of CTIBD was also observed for the homomeric rSlo channel but was more significant compared to rSlo/rβ1 and rSlo/rβ4 coexpressed channels.
The BKCa channel plays an important role in the contraction of UBSM by controlling the resting membrane potential and repolarization phase. Thus, we tested the effect of CTIBD on the relaxation of excised rat UBSM strips. As expected, pretreatment with CTIBD concentration-dependently inhibited ACh-induced contraction of rat UBSM strips at 30 μM or higher concentrations. At the highest concentration of 300 μM, CTIBD inhibited ACh-induced contraction by 81.4% compared with the negative control. These results indicate that CTIBD relaxes UBSM. We then tested the efficacy of CTIBD on micturition in a rat model of AA-evoked OAB. Acetic acids enhance sensitivity of neuronal activity of bladder smooth muscle cells and cause OAB in a rat model (Mitobe et al., 2008; Choudhary et al., 2015). Using in vivo cystometry, intraperitoneal injection of CTIBD (20 mg/kg) relaxed AA-induced frequent voiding contraction significantly. It is worth noting and encouraging that other parameters of bladder functions, such as the basal intravesical pressure and the maximum pressure of voiding contraction, were not significantly affected by CTIBD treatment.
In summary, a series of new BKCa channel activators containing a 4-phenyl-isoxazol-5-yl benzene skeleton were identified, and one compound (CTIBD) was characterized as a novel potent activator of the channel. CTIBD potentiated the activity of the channel by directly binding to the channel and stabilizing its open conformation. In rats, CTIBD effectively relaxes UBSM ex vivo and bladder hyperactivity in vivo. Taken together, these results indicate that CTIBD and its derivatives that target the bladder BKCa channel can be considered as strong new candidates for OAB therapeutics.
Acknowledgments
The chemical library used in this study was kindly provided by the Korea Chemical Bank (www.chembank.org) of the Korea Research Institute of Chemical Technology.
Authorship Contributions
Participated in research design: N. Lee, Park.
Conducted experiments: N. Lee, Lim, K.-S. Lee, Shin, S. W. Lee, Kang.
Contributed new reagents or analytic tools: H. S. Pagire, S. H. Pagire, Ahn.
Performed data analysis: N. Lee, Lim, K.-S. Lee, Shin, S. W. Lee, Kang, Park.
Wrote or contributed to the writing of the manuscript: N. Lee, Ahn, S. W. Lee, Kang, Park.
Footnotes
- Received June 30, 2020.
- Accepted November 11, 2020.
This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bio Industry Technology Development Program; funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) [317070-4]; and supported by a Gwangju Institute of Science and Technology (GIST) Research Institute (GRI) Integrated Institute of Biomedical Research (IIBR) grant funded in 2020.
↵This article has supplemental material available at molpharm.aspetjournals.org.
Abbreviations
- τdeactivation
- deactivation time constant
- AA
- acetic acid
- ACh
- acetylcholine
- τactivation
- activation time constant
- BKCa channel
- large-conductance calcium-activated potassium channel
- cRNA
- complementary RNA
- CTIBD
- 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol
- d
- doublet peak
- dd
- doublet of doublet peak
- EtOAc
- ethyl acetate
- Gmax
- maximum conductance
- G-V
- conductance-voltage
- KRICT
- Korea Research Institute of Chemical Technology
- OAB
- overactive bladder
- rβ1
- rat β1 subunit
- rβ4
- rat β4 subunit
- RFU
- relative fluorescence unit
- rSlo
- rat BKCa channel
- s
- singlet peak
- SAR
- structure-activity relationship
- Slo1
- a subunit of large-conductance calcium-activated potassium channel
- UBSM
- urinary bladder smooth muscle
- V1/2
- half-activation voltage
- Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics