Research The Analgesic Mechanism Of XEN402 And Design,Synthesis And Activity Evalation Of Its Derivatives

Voltage-gated sodium channels(Nav)play a pivotal role in electrical activities of neurons and other excitable cells.Defects of Nav channels have been linked to a variety of channlopathies such as pain,epilepsy and arrhythmia.Nav1.7 is the seventh member of nine cloned Nav channels,Nav1.1?Nav1.9.Gain-of-function mutations in Nav1.7 result in distinct pain syndromes such as primary erythromelalgia,paroxysmal extreme pain disorder and small-fibre neuropathy,whereas loss-of-function mutations in this gene produce congenital insensitivity to pain.Notably,Nav 1.7 caused CIP patients experience normal physiological functions except for an impaired sense of smell.The features make Nav1.7 channel a promising target for analgesic drug discovery.Given the fact that a large number of Nav 1.7 inhibitors have been developed,only three molecules moved forward into phase II clinical study.XEN402(TV-45070)is a novel Nav1.7 channel inhibitor developed by Xenon and Teva,and has been granted as an orpHan drug designation for the treatment of erythromelalgia by FDA in 2013.However,the questions how XEN402 produces its inhibitory activity and if different kinds of PE patients are sensitive to it remain unclear.Meanwhile,the low inhibitory efficacy and poor selectivity of the drug might limit its following clinical application.Thus,there is no doubt that a detailed mechanism study of XEN402 and identification of novel derivatives would promote its following clinical usage and benefit the drug discovery based on Nav 1.7 channel.In the present study,we found that XEN402 potently inhibits wild type Nav1.7 channel expressed in Chinese hamster ovary cells with the half maximal inhibitory concentration(IC50)of 4.4 ± 0.1 ?M.Kinetics analysis shown that XEN402 significantly hyperpolarizes the wild type Nav 1.7 channel fast inactivation,depolarizes the channel activation,whereas has no effect on the slow inactivation.Moreover,XEN402 did not affect the time constants of activation and fast inactivation in the wild type Nav1.7 channel.Our study demonstrated that the inhibition on Nav 1.7 channel caused by XEN402 is in a state-and use-dependent manner,which is very similar to the classical sodium channel inhibitors.The properties would make XEN402 exhibits a more potent inhibition on the channels involved in pain sensing than those in the resting state.In addition,we also found that XEN402 promotes the development of the closed-state inactivation and slows the channel recovery from the inactivated state,and thereby trapping the channel in nonfunctional inactivated state.Together,our data suggested that changing Nav1.7 channel activation and inactivation,especially for the fast inactivation,and trapping the channel in inactivated state contribute to XEN402 produced inhibitory activity.According to the kinetics features,the changes caused by Nav1.7 mutations linked to primary erythromelalgia could be divided into three categories:hyperpolarization in the channel activation,depolarization in channel inactivation and change in both.To investigate the pHarmacological effects of XEN402 on these distinct mutants,we constructed 11 mutants using molecular biology technique.Contrast to the modulation on wild type Nav1.7 channel,XEN402 demonstrated distinct effects on these mutants.For the mutants like P1308L which mainly hyperpolarizes channel activation,XEN402 shifted the dampened activation towards depolarization direction.For the mutants like G616R which only depolarizes channel inactivation,XEN402 reversed the impaired channel inactivation and did not affect inactivation.For the mutants like G856D in which both activation and inactivation were changed,XEN402 rescued them simultaneously.Thus,our data furtherly supported that XEN402 might benefit the treatment of PE patients caused by different mutations.Hyperpolarization in the Nav1.7 channel activation was thought to be the mainly pathogenetic reason of PE.Provided the fact that XEN402 exhibits a weak effect on the channel activation under a high concentration,we made structuralmodifications of XEN402 through the optimization of structure-activity relationships.On the one hand,an ethylene group was added to the methylene of the XEN402 at connection area,forming a cyclopropyl ring.The tension of the cyclopropyl group resulted in rearrangement to form allyl group after ring opening reaction.Therefore,we got the new derivatives containing allyl group.On the other hand,a kind of novel structure could be obtained by converting the specific monocyclic aryl group in XEN402 into polycyclic aryl group to investigate the effect of different aryl types towards the activity,for example,furan was replaced by 2,3-dihydrobenzofuran According to the above two methods mentioned above,four derivatives(E1,E2,E3 and E11)were designed and synthesized respectively.using electropHysiological technique,the activities of these 4 derivatives were evaluated and E1-S was selected.E1-S exhibited a much more potent inhibitory effect on Nav1.7 channel than XEN402 with an IC50 value of 1.6 ± 0.1 ?M.Moreover,E1-S caused a larger change in the degree of channel activation for both wild type Nav1.7 and G856D mutant casual to PE.These data shown that E1-S is a potent inhibitor of Nav1.7 channel.In conclusion,our study probed the inhibitory mechanism underlying the analgesic activity of XEN402 and E1-S identified in the current study might provide a novel compound for the following analgesic drug discovery targeted on Nav1.7 channel.