脊髓背根神经节（Dorsal root ganglion，DRG）作为将外周疼痛信号整合传递给中枢神经系统的初级神经元，在疼痛的传导过程中发挥着重要作用。调节DRG兴奋性也成为治疗疼痛的一个关键思路，而神经元的兴奋性与表达在神经元上的离子通道关系密切，其中电压门控钠通道（Voltage-gated sodium channel， VGSC或Nav）是神经元动作电位产生的基础。中小直径DRG主要传导痛觉，高表达TTX-S钠通道，其中Nav1.7贡献电流近80%^[1]。临床研究发现SCN9A（Nav1.7编码基因）的突变与多种先天性疼痛感知异常疾病有关，在动物疼痛模型中也发现DRG上Nav1.7表达发生改变，提示Nav1.7可作为治疗疼痛的靶点。现阶段止疼药治疗效果有限，不能满足临床治疗需求，且不良反应严重。本文以Nav1.7 为镇痛靶点，通过筛选，发现化合物1-[2-甲氧苯基]-4-[4-(2-酞酰亚胺基)-丁基]哌嗪（NAN-190）对该靶点有明显的阻断作用，并进一步通过全细胞膜片钳技术研究了NAN-190对Nav1.7的抑制作用，并探讨了其阻断钠通道的分子机制。

研究方法：

本研究采用全细胞膜片钳记录方法，研究NAN-190对电压门控Nav1.7的作用机制。通过改变刺激脉冲参数，在体外重组Nav1.7-Flp-In-HEK细胞上研究了NAN-190的可能作用状态（关闭态、开放态及失活态）及对Nav1.7通道动力学参数的影响，主要包括稳态激活曲线、稳态失活曲线和失活恢复曲线。同时，通过点突变（Nav1.7-F1737A、Nav1.7-WCW、Nav1.7-F1737A-WCW）研究NAN-190作用位点以及阻断Nav1.7作用机制。Nav1.7对于初级痛觉传入DRG的活化起到关键作用，是DRG上主要的河豚毒素（Tetrodotoxin， TTX）敏感通道。本文在体外分离培养的DRG上测试了NAN-190对电压门控钠通道（包括河豚毒素敏感型和河豚毒素不敏感型钠通道）的影响。为验证化合物对电压门控钠通道及其他疼痛相关离子通道选择性，还测试了NAN-190对钠通道家族成员中Nav1.1、Nav1.5和Nav1.8亚型的作用，以及对疼痛相关靶点瞬时感受器电位离子通道香草素受体1(transient receptor potential vanilloid 1, TRPV1) 和Cav2.2是否存在阻断作用。最后采用完全弗氏佐剂（Complete Freund’s adjuvant, CFA）在小鼠成功构建炎症疼痛模型，研究NAN-190对疼痛的缓解作用。

研究结果：

NAN-190对Nav1.7通道失活态半数抑制浓度（Half maximal inhibitory concentration, IC₅₀）为2.92±0.19 μM，而对通道静息态没有明显抑制作用。通过测试Nav1.7通道电生理特性，发现30 μM NAN-190使Nav1.7激活曲线的半数激活电压从-27.29±0.64 mV左移至-30.53±0.86 mV，没有统计学差异，说明NAN-190对Nav1.7激活曲线没有明显影响，但使Nav1.7快失活曲线的半数失活电压从 -59.59±0.65 mV 左移至-68.66±0.38 mV ，慢失活曲线的半数失活电压从-41.75±0.73 mV 左移至 -80.31±0.95 mV，即NAN-190使Nav1.7快失活与慢失活的半数失活电压向超极化方向分别移动了9.07 mV和 38.56 mV。同时，NAN-190使Nav1.7快失活与慢失活后的恢复曲线时间常数分别从2.45±0.21 ms 延长至80.62±8.31 ms，从308.5±13.85 ms 延长至957.8±62.25 ms。NAN-190对局麻药（Local anesthetics，LA）结合位点突变型F1737A失活态IC₅₀值为26.56±2.27 μM，对钠通道快失活缺失突变型WCW、双突变型F1737A-WCW峰电流和稳态电流IC₅₀值分别为大于30 μM 和18.93±2.53 μM，大于30 μM 和 4.19±0.47 μM。NAN-190 对急性分离的大鼠DRG神经元上TTX敏感型钠电流具有明显抑制作用，而对TTX不敏感型钠电流无明显抑制作用。采用CFA构建炎症疼痛模型，腹腔注射NAN-190可以减轻热痛刺激和机械刺激诱发的疼痛行为，即缩爪时间缩短与阈值降低。同时，对NAN-190对VGSC家族亚型选择性进行了探究。NAN-190对Nav1.1和Nav1.5失活态具有明显抑制作用，IC₅₀值分别为3.60±0.78 μM 和1.69±0.13 μM，但对两个通道静息态无明显抑制作用。在对其他疼痛相关离子通道活性研究中发现，NAN-190对Cav2.2钙通道和TRPV1通道的抑制作用较弱，如NAN-190 30 μM对Cav2.2通道静息态和失活态的抑制率分别为19.91±2.02%和57.57±2.06%，但对辣椒素激起的TRPV1电流无明显阻断作用。

研究结论：

通过以上研究，我们证明了化合物NAN-190以剂量依赖的方式选择性作用于Nav1.7通道失活态，对静息态抑制率低。NAN-190使通道失活曲线向超极化方向移动，延长通道从失活态的恢复时间，但对通道激活曲线没有明显影响。NAN-190主要结合在LA作用位点调控Nav1.7通道开放态。NAN-190主要抑制DRG上TTX敏感型钠通道，对CFA所诱导的炎症疼痛模型具有良好的缓解作用。同时，NAN-190对其他疼痛靶点的作用较弱或者无活性，如NAN-190在较高浓度30μM仅抑制一半的Cav2.2通道活性，但对TRPV1通道却无明显阻断作用。综上所述，NAN-190作为Nav1.7通道阻断剂，可作为一个潜在的候选化合物为镇痛化合物的开发提供思路。

DRG plays an important role in pain transmission as primary neurons that integrate and transmit peripheral pain signals to the center nervous system. The small and medium diameter of DRG mainly conducts nociception and highly expresses TTX-S sodium channels, of which Nav1.7 contributes nearly 80% of the sodium channel current. Clinical studies have found that mutations in SCN9A (the gene encoding Nav1.7) are associated with a variety of congenital pain perception abnormalities, and up-regulation of Nav1.7 expression on the DRG has been found in animal pain models, suggesting that Nav1.7 could be a target for pain treatment. At the present stage, the therapeutic effect of painkillers is limited and the side effects are serious, which cannot meet the clinical treatment needs. In this paper, Nav1.7 was treated as an analgesic target, and the compound [1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl] piperazine] (NAN-190) was found to have a significant blocking effect on this target through screening test, and the inhibitory effect of NAN-190 on Nav1.7 was further verified by whole-cell patch clamp technique.

Methods

In this study, whole-cell electrophysiological patch clamp recording method was used to study the mechanism of NAN-190 on Nav1.7 sodium channels. By changing the stimulation pulse parameters, the possible action states (closed state, open state and inactivated state) of NAN-190 and its effects on kinetic parameters were studied in recombinant Nav1.7-Flp-In-HEK cells in vitro, including steady-state activation curve, steady-state inactivation curve and recovery from inactivation. Meanwhile, the action site and mechanism of NAN-190 blocking Nav1.7 was investigated by point mutations (Nav1.7-F1737A, Nav1.7-WCW, Nav1.7-F1737A-WCW). As the primary afferent neurons of peripheral pain, DRG mediates the generation and transmission of pain after activation. Nav1.7 plays a key role in the activation of DRG and is the major tetrodotoxin (TTX)-sensitive channel in DRG. The effects of NAN-190 on voltage-gated sodium channels (including TTX-sensitive and TTX-resistant sodium channels) were tested on isolated DRG neurons. The inflammatory pain model was successfully established in mice with CFA to study the pain-relieving effect of intraperitoneal injection of 10 mg/kg NAN-190. The selectivity of NAN-190 was also verified on other voltage-gated sodium channels Nav1.1, Nav1.5 and Nav1.8, and pain-related ion channels TRPV1 and Cav2.2.

Results

NAN-190 selectively inhibited the inactivated state of Nav1.7 channel in a dose-dependent manner with IC₅₀ at 2.92 ± 0.19 μM, while no significant inhibition of the resting state of the channel was observed. By testing the electrophysiological properties of Nav1.7 channel, it was found that 30 μM NAN-190 left-shifted the half activation voltage of Nav1.7 activation curve from -27.29 ± 0.64 mV to -30.53 ± 0.86 mV without statistical difference, indicating that NAN-190 had no significant effect on Nav1.7 activation curve. The half-inactivation voltage of Nav1.7 fast inactivation curve was left-shifted left from -59.59 ± 0.65 mV to -68.66 ± 0.38 mV and the half-inactivation voltage of slow inactivation curve was left-shifted from -41.75 ± 0.73 mV to -80.31 ± 0.95 mV. NAN-190 shifted the half-inactivation voltage of Nav1.7 fast and slow inactivation toward hyperpolarization by 9.07 mV and 38.56 mV, respectively. At the same time, NAN-190 prolonged the time constants of recovery from fast inactivation and slow inactivation of Nav1.7 from 2.45 ± 0.21 ms to 80.62 ± 8.31 ms, from 308.5 ± 13.85 ms to 957.8 ± 62.25 ms, respectively. The IC₅₀ values of NAN-190 for F1737A inactivation state are 26.56 ± 2.27μM, and the IC₅₀ for fast inactivation-deficient mutant WCW, F1737A-WCW peak current and steady state current are more than 30μM and 18.93 ± 2.53 μM, more than 30 μM and 4.19 ± 0.47 μM, respectively. NAN-190 10 μM significantly inhibited TTX-sensitive currents on isolated small-diameter of DRG neurons, but had no obvious inhibitory effect on TTX insensitive currents. Using CFA to construct an inflammatory pain model, intraperitoneal injection of NAN-190 attenuated pain behaviors induced by thermal and mechanical stimulation, shortening claw reduction time and lowered threshold. The subtype selectivity of NAN-190 was also explored. NAN-190 had a significant inhibitory effect on Nav1.1 and Nav1.5 inactivated states with IC₅₀ values at 3.60 ± 0.78 μM and 1.69 ± 0.13 μM, respectively, but no obvious effect on Nav1.1 and Nav1.5 channel resting states. 30μM NAN-190 inhibited the resting and inactivated states of Cav2.2 channels by 19.91 ± 2.02% and 57.57 ± 2.06%, respectively, but had no significant blocking effect on capsaicin-induced TRPV1 currents.

Conclusion

We proved that compound NAN-190 selectively acted on the inactivated state of Nav1.7 in a dose-dependent manner, without obvious effect on rest state. NAN-190 shifted the inactivation curve to hyperpolarization, and prolonged the recovery from inactivation, but had no obvious effect on the activated state. Point mutation experiments demonstrated that NAN-190 regulated the open state of the channel mainly by binding to the LA action site. NAN-190 mainly inhibited TTX-sensitive sodium channels in DRG and alleviated CFA-induced inflammatory pain behaviors. However, NAN-190 was less active on other pain targets such as transient receptor potential vanilloid type 1 receptor (TRPV1) and N-type calcium channel Cav2.2. In summary, NAN-190, as a Nav1.7 channel blocker, could be a potential candidate compound to provide ideas for the development of analgesic compounds.