Study On The Molecular Pharmacological Mechanism Of NAN-190 Blocking Nav1.7 Sodium Channel To Exert Analgesic Effec

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.MethodsIn 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 NAN190.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.ResultsNAN-190 selectively inhibited the inactivated state of Nav1.7 channel in a dosedependent 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 from59.59±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 NAN190 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.ConclusionWe 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 Ntype 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.