Study On The Structure-activity Relationship And Mechanism Of Activation Of KCNQ2/3 Channels By Novel Pyrazolo"1,5-a"Pyrimidin-7(4H)-one Derivativs

M channels are slow-activation and non-inactivation voltage-dependent potassium channels that were first described in bull frog superior cervical ganglion by Brown and Adams in 1980s. Activation of M-currents by depolarization functions as a brake on repetitive action potential discharges and therefore plays a key role in controlling the excitability of central and peripheral neurons including sympathetic neurons, hippocampal pyramidal cells and striatal neurons. M-channels are composed of the subunits of KCNQ (Kv7) family of K+ channels (KCNQ2 to KCNQ5). KCNQ2/3 heterotetramers were originally identified as being the components of the classical M channels. Mutations in KCNQ2 and KCNQ3 genes lead to a form of neonatal epilepsy in humans termed BFNC (Benign Familial Neonatal Convulsions) characterized by KCNQ/M current reduction and resultant neuronal hyperexitability. It is recently found that M channels are also expressed in sensory neurons involved in pain transmission.In light of the critical role of KCNQ/M currents in nervous system diseases, augmentation of KCNQ/M currents by KCNQ activators or openers is expected to be conductive to the treatment of neuronal diseases such as epilepsy and pain. This notion is supported by the finding that the antiepileptic agent retigabine [D-23129; N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid], can activate potassium channels in neuronal cells. Later it is found that the potassium channel activated by retigabine are KCNQ/M channels. A number of recent studies have revealed that retigabine can also relieve pain-like behaviors in animal models of neuropathic pain. Therefore, KCNQ channels as a therapeutic target become increasingly the focus of drug development for the treatment of seizures and pain. Besides retigabine, ICA-27243 [N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide], a more selective KCNQ2/3 channel activator, shows anticonvulsant activity in experimental models of epilepsy.Targeting at KCNQ channels, we have designed and synthesized series of novel compounds and examined their activity on KCNQ channels by high-throughput screening using atomic absorption Rb+ efflux assay. We found that one series of compounds pyrazolo[1,5-a]pyrimidin-7(4H)-one (PPO) derivatives with different groups substituted at C-2, C-3 and C-5 position showed activity on K+ efflux. Here, we examined the activity of compounds QO-26, QO-28, QO-40 and QO-41 on the activity of KCNQ2/3 channels stably expressed in Chinese hamster ovary (CHO) cells by patch-clamp technique and study the structure-activity relationship and mechanism of PPO derivatives on activation of KCNQ2/3 channels.Part 1 The characteristics of KCNQ2/3 currents expressed in CHO cellsObjective:To characterize the KCNQ2/3 currents stably expressed in the Chinese hamster ovary (CHO) cell lines for further study on PPO compounds modulation of KCNQ2/3 function.Methods:Western blot was used to examine the protein expression of KCNQ2 and KCNQ3 channels in CHO cells. The perforated patch-clamp technique was used to study electrophysiological characteristics of KCNQ2/3 currents in CHO cells. Pharmacological blockers were also applied to validate the characteristics of KCNQ2/3 currents.Results:(1) KCNQ2 and KCNQ3 channel proteins with molecular weight of 97 kDa were found only in CHO cell lines stably transfected with KCNQ2 and KCNQ3 genes but not in untransfected CHO cells. Typical KCNQ2/3 currents were detected in CHO cell lines stably expressing KCNQ2/3 channels but not in untransfected CHO cells. (2) The KCNQ2/3 currents were recorded using following voltage protocol:The cells were held at -80 mV and the membrane potential was stepped to a series of voltage levels ranging from -130 mV to +60 mV (in 10-mV increments) for a 1000-ms pulse duration, followed by a 1100-ms step to -120 mV. The recorded currents showed the characteristics of KCNQ2/3 currents with low-threshold, slow-activation, non-inactivation, voltage-dependent properties. The normalized tail current (at -120 mV) -voltage curve was fitted by Boltzmann equation, and the half-maximal activation voltage (V1/2) and slope factor (k) of the curve were -2.2±1.5 mV and 21.7±1.2, respectively. For channel activation kinetics analysis, the time constants for channel activation (Tauact) were calculated from the single exponential fitting of the activated current traces. Tauact showed prominent voltage-dependency at membrane potential from -40 mV to +30 mV. Tauact decreased with the increasing depolarizing voltage steps. The channel deactivation kinetics were measured by the time constants for channel deactivation (Taudeact), which were calculated from the single exponential fitting of the current traces recorded at potentials from -140 mV to-20 mV preceded by a depolarization to +40 mV. Taudeact also showed voltage-dependency at membrane potential from -140 mV to -20 mV; channel deactivation at potentials between -90 mV to -20 mV became slower than that at potentials between -140 mV to -100 mV. (3) Linopirdine, a specific blocker of KCNQ/M currents, and TEA caused prominent concentration-dependent inhibition of KCNQ2/3 currents in CHO cells with IC50s of 0.8±0.08μM (k=0.91±0.06) and 1.76±0.08 mM (k=1.01±0.05), respectively. The maximal inhibition of KCNQ2/3 currents by linopirdine and TEA were 95.5±1.1% and 92.92±1.6%, respectively.Conclusion:Western blot and eletrophysiological results suggest that KCNQ2 and KCNQ3 are co-expressed in CHO cells; the electrophysiological and pharmacological characteristics of the recorded currents are consistent with that of KCNQ2/3 currents. This stable KCNQ2/3 CHO cell lines can be used for studying PPO activity on KCNQ2/3.Part 2 Structure-activity relationship study of PPO derivatives on KCNQ2/3 currentsObjective:To examine the potency and efficacy of PPO compounds QO-26, QO-28, QO-40 and QO-41 on KCNQ2/3 currents in CHO cells and on M currents in native dorsal root ganglia (DRG) neurons. To analyze the structure-activity relationship of these PPO compounds on KCNQ2/3 currents. Retigabine was used as positive control.Methods:Perforated patch-clamp was used to investigate the effects of PPO derivatives on KCNQ2/3 currents.Results:(1) The four PPO derivatives (QO-26, QO-28, QO-40 and QO-41) all potentiated KCNQ2/3 currents in CHO cells verified by the channel specific opener retigabine and the channel specific blocker linopirdine. (2) The four compounds augmented KCNQ2/3 currents in a concentration-dependent manner and with different potency and efficacy. For a quantitative comparison, the half-maximal concentrations and the maximal effects of PPO compounds on KCNQ2/3 currents recorded at -40 mV and -20 mV were analyzed. The EC50s for QO-26 were 47.93±5.04μM and 21.35±2.24μM, respectively; the maximal folds of current increase were 4.76±0.67 and 1.04±0.16, respectively. The EC50s for QO-28 were 31.51±6.66μM and 12.57±4.21μM, respectively; the maximal folds of current increase were 13.36±2.07 and 2.68±0.4, respectively. The EC50s for QO-40 were 15.4±0.56μM and 6.10±1.83μM, respectively; the maximal folds of current increase were 6.60±1.73 and 0.78±0.07, respectively. The EC50s for QO-41 were 9.04±1.53μM and 3.27±0.47μM, respectively; the maximal folds of current increase were 8.44±1.55和1.50±0.12, respectively. The EC50s for retigabine were 2.93±0.25μM and 1.90±0.06μM, respectively; the maximal folds of current increase were 11.86±0.95 and 2.95±0.32, respectively. (3) QO-41 also enhanced M currents in rat small DRG neurons with EC5o of 7.09±1.8μM.Conclusions:(1) QO-26, QO-28, QO-40 and QO-41 potentiate KCNQ2/3 currents in a concentration-dependent manner and with different potency and efficacy. (2) QO-41 shows the highest potency among PPO compounds but is weaker than that of retigabine. (3) QO-28 shows the highest efficacy among PPO compounds and is similar to the efficacy of retigabine. (4) QO-41 also enhances M currents in rat small DRG neurons. (5) Structure-activity relationship study suggests that CF3 at C-2, benzene or Nap at C-3 and CF3 or CH2C1 at C-5 is essential for the activity of PPO derivatives.Part 3 Characteristics and mechanism for the effects of PPO derivatives activation of KCNQ2/3 currentsObjective:To characterize the effects of PPO derivatives on KCNQ2/3 activation and deactivation kinetics and explore the molecular mechanism by which the compounds activate KCNQ2/3 channels.Methods:Perforated patch-clamp was used to investigate the effects of PPO derivatives on the currents of wild-type KCNQ2/3 and mutant KCNQ2(W236L) channels.Results:(1) The current-voltage curves suggested that the four PPO derivatives augmented KCNQ2/3 current amplitude across a range of membrane potentials and shifted the voltage threshold for channel activation towards more negative potentials. (2) Application of the four PPO derivatives with maximal concentration shifted the activation curves to the negative direction. The different PPO compounds maximally shifted the V1/2 of KCNQ2/3 currents with the order of:200μM QO-28 (-34.2 mV)>100μM QO-40 (-21.11 mV)>300μM QO-26 (-18.14 mV)>30μM QO-41 (-16.92 mV). As a positive control,30μM retigabine produced a significant leftward shift of V1/2 by -40.06 mV. The PPO-induced shifts in the activation curves were concentration-dependent. (3) Time constants for channel activation (Tauact) showed prominent voltage-dependency both before and after application of the PPO compounds.300μM QO-26,200μM QO-28 and 100μM QO-41 significantly slowed the channel activation at all potentials tested between -40 mV and +30 mV; on the other hand, QO-40 did not affect the activation kinetics of the channel. Retigabine at 30μM accelerated the channel activation only at potentials negative to -30 mV. The effects of PPO derivatives and retigabine on the channel activation kinetics were also concentration-dependent. (4) The channel deactivation also showed voltage-dependency at membrane potential from -140 mV to -20 mV in control. While after the application of PPO compounds,300μM QO-26,200μM QO-28,100μM QO-40 and 100μM QO-41 significantly slowed channel deactivation. The time constant for channel deactivation (Taudeact) presented a bell-shape change at membrane potential from -140 mV to -20 mV with the maximal Taudeact at -60 mV.30μM retigabine also slowed the channel deactivation in the same manner. The effects of PPO derivatives and retigabine on the channel deactivation kinetics were also concentration-dependent. (5) When PPO compounds and retigabine were applied together they had additive effects on KCNQ2/3 currents. Thus the combined application of PPO derivatives and retigabine potentiated KCNQ2/3 currents to a greater extent across a range of membrane potentials, and shifted the threshold for channel activation to a more negative potential than separate application of either of them. (6) Co-application of PPO derivatives and retigabine also dramatically slowed deactivation. The addition of 300μM QO-26,200μM QO-28,100μM QO-40, and 100μM QO-41 along with 30μM retigabine significantly slowed deactivation. (7) The currents of the mutant KCNQ2(W236L) were still enhanced by the PPO derivatives but not by retigabine.200μM QO-28 augmented mutant KCNQ2(W236L) current amplitude across a range of membrane potentials and decreased the threshold for channel activation.200μM QO-28 shifted the channel activation curve in the hyperpolarizing direction. The V1/2 of KCNQ2(W236L) current under control, retgabine and 200μM QO-28 were -9.0±1.7 mV,-3.2±2.2 mV and-38.5±1.0 mV, respectively.Conclusions:(1) The current-voltage curves suggest that the four PPO derivatives augment KCNQ2/3 current amplitude across a range of membrane potentials and reduce the threshold for channel activation. (2) QO-26, QO-28, QO-40 and QO-41 shift the half-maximal activation voltage (V1/2) for the KCNQ2/3 channels in the hyperpolarizing direction and in a concentration-dependent manner. (3) The compounds markedly slow KCNQ2/3 channel activation and deactivation kinetics in a concentration-dependent manner. (4) The additive effects of the PPO derivatives and retigabine on KCNQ2/3 currents as well as the effects on mutant KCNQ2(W236L) channel indicate that these two types of agents modulate KCNQ2/3 channels by different mechanisms.SUMMARY1 KCNQ2 and KCNQ3 are co-expressed in CHO cells; the electrophysiological and pharmacological characteristics of the recorded currents are consistent with that of KCNQ2/3 currents.2 QO-26, QO-28, QO-40 and QO-41 potentiate KCNQ2/3 currents in a concentration-dependent manner and with different potency and efficacy. QO-41 shows the highest potency among PPO compounds but is weaker than that of retigabine. QO-28 shows the highest efficacy among PPO compounds and is similar to the efficacy of retigabine. QO-41 also enhances M currents in rat small DRG neurons. The structure-activity relationship study suggests that CF3 at C-2, benzene or Nap at C-3 and CF3 or CH2C1 at C-5 are essential for the activity of PPO derivatives.3 The current-voltage curves suggest that the four PPO derivatives augmented KCNQ2/3 current amplitude across a range of membrane potentials and reduce the threshold for channel activation. QO-26, QO-28, QO-40 and QO-41 shift the half-maximal activation voltage (V1/2) for the KCNQ2/3 channel in the hyperpolarizing direction and in a concentration-dependent manner. The compounds markedly slow KCNQ2/3 channel activation and deactivation kinetics. Therefore, PPO derivatives-induced potentiation of KCNQ2/3 currents may be through the increase in the steady-state activation current, negative shifts of activation and the prolonged channel deactivation.4 The additive effects of the PPO derivatives and retigabine on KCNQ2/3 currents as well as the effects on mutant KCNQ2(W236L) channel indicate that these two types of agents modulate KCNQ2/3 channels by different mechanisms.