Small Molecule Drug Design Targeting K_V7 And Its Regulatory Mechanism In Neuropathic Pain

Chronic pain is severe to treat because of the limited therapeutic options,among which Neuropathic Pain（NP）is a stubborn type of chronic pain.Authoritative studies have reported that chronic pain widey affects people’s quality of life even causes anxiety and depression.Recently,Kv7（KCNQ）potassium channel has been found to be a very potent pain target for pain therapy.We used computer-aided drug design and patch-clamp electrophysiological screening to reconstruct the structure of the Retigabine（RTG）.The active compounds（QO series）were found to have good opening activity on the Kv7 channel,and the optimized compound QO-83 was systematically studied in vivo and in vitro.The results showed that QO-83 is a potent opener with good selectivity for Kv7.2.The compound can well treat NP caused by CCI surgery,thalamus pain and NP caused by chemotherapy,and is expected to be opened as a new drug for the treatment of NP.Part One Design and activity screening of small molecule drugs targeting Kv7 channelObjective:To find a stable and efficient Kv7.2 channel opener by structural modification given the chemical instability and selectivity of RTG.Methods:Using DS software,QSAR design was carried out for the RTG-bound Kv7.2 active pocket.The structural activity was observed by replacing the-F on the RTG benzyl group with-CF₃,replacing the alkane of 1-imine group,adding the-F group on the aromatic ring,and different substitutions on the amide side chain.The most effective modified active compounds were verified by manual patch clamp to confirm structural stability,and the compounds with high selectivity of Kv7.2 channel subtypes were further studied.The selected compounds were simulated by computer molecular dynamics.Results:1.A total of more than 100 compounds were designed,including two categories:one was modified with RTG skeleton as the parent nucleus,and the other was based on pyrazolpyrimidine（PPOs）as the skeleton.A total of 16compounds with high scores were screened out.2.Two compounds QO-83 and QO-58 were found to have the best activity on open Kv7.2 channel.The multiples of opening Kv7.2 channel at 30 m V were2.54±0.02 and 2.13±0.11,respectively.The EC₅₀ value of QO-83 was 0.56±0.09μM,which showed stronger biological activity and more stable chemical properties than RTG.3.The molecular dynamics analysis of QO-83 showed that the introduction of cyclohexyl increased the hydrophobicity of the C region,increased the interaction betweenπ～πbonds and surrounding amino acids,and the introduction of trifluoromethyl enhanced the interaction with the active pocket of the Kv7.2channel protein.Conclusions:The designed and screened compound QO-83 has stable properties,better Kv7.2 channel opening activity,and higher free energy of binding to the active pocket than RTG.Part Two Analgesic effect and mechanism of Kv7 channel opener on CCI model miceObjective:To observe the effects of compound QO-83 on CCI model mice pain behavior,dorsal root nerve discharge,DRG neuronal excitability,and calcium signal in chronic compression,and Kv7.2 channel protein expression.Methods:1.The effect of compound QO-83 on the pain behavior of C57 model mice was observed by using the sciatic nerve chronic constriction injury model.2.CCI model was established,and dorsal root nerve fibers were exposed on the 7th day.The spontaneous discharge frequency of dorsal root nerve fibers was recorded by Spike2 software,and the effect of compound QO-83 on CCI model dorsal root nerve fibers was observed.3.Using current patch-clamp electrophysiology,the effect of QO-83 on the action potential of DRG neurons was recorded.In addition,the effect of QO-83 on M current was recorded in DGR neurons by voltage patch-clamp.4.In order to observe the effect of QO-83 on calcium signal of excitatory neurons in the VPL region,the adenovirus AAV2-Ca MKⅡα-GCa MP6f to observe the glutamatergic neurons,AAV2-Ca MKⅡα-GCa MP6f was injected into the VPL region of CCI mice.The effect of QO-83 on the calcium signal of excitatory neurons in the VPL region by free-moving mice was observed by single-channel fiber recording.5.The m RNA changes of KCNQ2 and G9a in CCI model mice treated with QO-83 before and after administration 14 days were detected by Q-PCR.Secondly,the expression levels of Kv7.2 and G9a ion channel proteins in DRG of CCI model mice were detected by the western blot（WB）technique.Results:1.Compound QO-83 can significantly increase the thermal paw withdrawal threshold of CCI model mice,from 8.16±0.26 s to 10.05±0.25 s;at the same time,it can up-regulate the mechanical paw withdrawal threshold of CCI model mice from 0.12±0.02 g to 0.58±0.05 g.2.Intraperitoneal injection of 1 mg/kg compound QO-83 significantly inhibited the discharge frequency of dorsal root nerve fibers,which decreased from 13.74±3.46 Hz（n=9）to 2.56±1.18 Hz（n=6）compared with the model control group.Compound QO-83 significantly reduced the discharge amplitude from 99.72±21.49μV to 53.53±5.57μV.3.Compound QO-83（1μM）can significantly reduce the number of action potential in normal DRG neurons.When injected with a current of 1000 PA,compound QO-83 reduced the number of action potential firings from 15.0±1.2 to 1.9±0.21μM compound QO-83 significantly increased the threshold of action potential from-25.2±2.0 m V to-11.4±5.2 m V.1μM compound QO-83 significantly increased the rheobase from 122.2±14.7 p A to 266.7±12.4p A.1μM compound QO-83 significantly increased the resting membrane potential from-62.6±4.2 m V to-71.3±7.07 m V.DRG neuron recording results for phasic firing are as follows:Action potential threshold increased from-23.7±3.3 m V to-18.9±5.0 m V（P<0.05）,basal intensity increased from 176.2±77.7 p A to 362.5±131.9 p A（P<0.05）,cell resting membrane potential increased from-61.9±0.05 1.0 m V decreased to-70.1±1.8 m V（P<0.05）,all of above were statistically significant different.The effect of compound QO-83 on the amplitude and peak width in DRG action potential was had no effect.Hill fitting was used to calculate the EC₅₀ value of compound QO-83opening activities for the M channel（0.81±0.003μM）.Compared to control group,compound QO-83 at 1μM,2.5μM,and 5μM significantly increased M currents on DRG neurons in a concentration-dependent manner.4.Based on the free-moving CCI model mice implanted with fibers in the VPL region,the changes of VPL calcium signal events induced by noxious stimulation in the hind paw were observed.Compound QO-83 can significantly reduce the frequency and amplitude of calcium signal events after nociceptive stimulation.In addition,compound QO-83 significantly increased the thermal threshold and cold threshold in CCI model mice.WB experiment found that compound QO-83 could significantly increase the expression of Kv7.2 channel protein in the VPL brain region of CCI model mice.5.The results of Q-PCR showed that compound QO-83 significantly increased the level of KCNQ2 m RNA and corresponding Kv7.2 channel protein of DRG neurons in CCI model mice,and down-regulated the m RNA level of G9a involved in gene expression regulation.Conclusions:Compound QO-83 can significantly increase the mechanical threshold and thermal withdrawal latency in CCI mice.This mechanism is related to enhancing the function and expression of the M channel and inhibiting the excitability of DRG and thalamic VPL neurons.Part Three Mechanism of analgesic effect of Kv7 channel opener on thalamic pain model miceObjective:To observe the effects of QO-83 on pain behavior,VPL neuronal spontaneous discharge,M current,and Kv7.2 ion channel protein expression in the thalamic pain model mice.Methods:The central thalamic pain model was established by injecting typeⅣcollagenase into VPL,and the effects of compound QO-83 on pain behavior and free movement of animals were observed.Spontaneous firing and M currents of VPL neurons were recorded by patch-clamp electrophysiology.The expression changes of KCNQ2 m RNA and Kv7.2 protein in VPL brain area were detected by Q-PCR and WB.HE pathological section staining was used to observe the effect of QO-83 on the morphology of neurons in the VPL brain region.Results:1.1.5 mg/kg compound QO-83 could significantly increase the ther mal pain threshold of C57 mice in the thalamic pain model,which fro m 15.45±0.21 s to 19.00±0.71 s.At the same time,1.5 mg/kg com pound QO-83 could up-regulate the mechanical threshold from 0.85±0.09 to 1.32±0.10.1.5 mg/kg compound QO-83 could also up-regulate t he cold threshold of thalamic pain model mice from 14.38±0.86 s（n=10）to 28.60±4.24 s（n=10,P<0.05）.2.Local injection of compound QO-83 in the VPL brain region can also increase the thermal pain and mechanical pain thresholds of C57 mice,and compound QO-83 does not cause abnormal open field autonomic activity in mice.3.1μM compound QO-83 can significantly inhibit the frequency and amplitude of spontaneous firing of VPL neurons and prolong the ISI of VPL neuron firing,thereby inhibiting the excitability of VPL neurons.At the same time,M currents were recorded on VPL neurons,and it was found that compound QO-83 could significantly increase the M currents amplitude of VPL neurons.4.The expression of the KCNQ2 m RNA and the Kv7.2 protein in the VPL brain region of thalamic pain model mice were significantly down-regulated,and the compound QO-83 significantly up-regulated the KCNQ2 m RNA and the level of Kv7.2 protein level.5.A large amount of fibrosis was observed in the VPL area after establishing the thalamic pain model mice utilizing HE staining.1.5 mg/kg compound QO-83 can significantly alleviate the fibrosis in the VPL area.Conclusions:Compound QO-83 can significantly alleviate the pain behavior of thalamic pain model mice,and its mechanism is related to opening the M channel in the VPL neuron,inhibiting the expression of the Kv7.2 channel protein.QO-83 can also reduce the fibrosis in the VPL brain region and play a neuroprotective role.Part Four Analgesic effect and mechanism of QO-83 and GAS in CINP modelObjective:Gastrodin（GAS）is the main bioactive ingredient of Gastrodia,a famous Chinese herbal medicine widely used as an analgesic,but the underlying analgesic mechanism is still unclear.To observe the effects of small molecule compound GAS and compound QO-83 on DRG neurons in the vincristine-induced neuropathic pain model,and to explore the potential mechanism of their effects on the excitability of DRG neurons.Methods:1.The effect of GAS combined with compound QO-83 on the action potential of vincristine model rat DRG neurons was recorded by patch clamp electrophysiological technique.The effects of GAS and compound QO-83 on M current/TTX-S current/Na v1.8 channels current were observed.2.The expression levels of Na v1.7 and Na v1.8 ion channel proteins in DRG of vincristine model rats were detected by the western blot（WB）technique.3.The effect of GAS on the expression of Na v1.7 and Na v1.8 ion channel proteins on DRG neurons of model rats was observed by immunofluorescence technique.Results:1.Compound QO-83 and GAS,alone or in combination,can significantly inhibit the pain hypersensitivity behavior in CINP rats.2.Half doses of gastrodin and compound QO-83 could significantly inhibit the amplitude,threshold,rheobase and number of action potential release of DRG neurons in vincristine model.Compound QO-83 can significantly increase the M current in model DRG neurons,and gastrotin can significantly inhibit TTX-S and Na v1.8 current.3.In addition,gastrodin significantly reduced the protein expression of Na v1.7 and Na v1.8 ion channels in DRG neurons of vincristine model rats.Conclusions:This study suggested that gastrodin alleviates neuropathic pain by regulating Na v1.7 and Na v1.8 sodium channels.Simultaneously,the combination of GAS and compound QO-83 can play a synergistic role by regulating M and TTX-S sodium channels and Na v1.8 channels to inhibit the ectopic discharge of model DRG neurons.