The Mechanism Study On A Novel Antiepileptic Drug And The Discovery Of Selective Activators Targeting KCNQ Channels

The voltage-gated potassium channel KCNQ?Kv7?consists of five subtypes of?KCNQ1^KCNQ5?and plays an important role in the regulation of excitability of nerves and other tissues.Different KCNQ subtypes vary in terms of tissue distribution,physiological and pathological functions.That lays the foundation of their different pharmacological properties and application prospects.The KCNQ2 and KCNQ3subtypes distributed in the central nervous system are recognized anti-epileptic drug targets,and the KCNQ4 and KCNQ5 subtypes distributed in visceral smooth muscle and vascular smooth muscle are considered to be a therapeutic target for smooth muscle related diseases.Therefore,it is important to study the mechanism of action of drugs targeting KCNQ channels and to develop subtype-selective activators for drug discovery and clinical applications targeting KCNQ.Being the only approved anti-epileptic drug that targets KCNQ channels,Retigabine has been withdrawn from the market due to serious adverse reactions and commercial reasons in 2017.Collaborating the medicinal chemists,we have discovered a new antiepileptic drug candidate named Pynegabine?HN37?,which has better chemical stability,better blood-brain distribution and better efficacy.HN37 has been approved for clinical trials.However,the mechanism underly the potent agonistic activity of HN37 remain elusive.In the current study,combining electrophysiology and mutagenesis methods,we have examined the effects of HN37on the mutants that may cause human epilepsy and those mutants that have been reported to be critical for Retigabine.We found that the mutation of the key residue for Retigabine,W236L,abolished the potentiation activity of HN37 also,supporting that HN37 may act on a same binding pocket with that of Retigabine.However,the action mode of the HN37 might be slightly different from that of Retigabine.For example,the effects of HN37 and Retigabine show significant difference on the four mutants of L243V,L275V,L299V,and I238A in the binding pocket.These results suggest that HN37 may act on a same binding pocket with that of Retigabine but with a slightly different action mode from that of Retigabine.In the second part of the study,initially,we screened a compound libray consisting of 80,000 small molecules using a high-throughput screening method and validated using the manual patch clamp technique.A series of KCNQ activator s with novel structural backbone were identified.Then,according the revealed structural characteristics of both the newly identified activators and Retigabine,new derivatives of Retigabine were designed and evaluated.We found some of these derivatives displayed improved selectivity to KCNQ4 and KCNQ5.For example,compound 6e and 10g potentiated KCNQ4 and KCNQ5 with I/I ₀ value varied between 3 and 8.In contrast,these two compounds hardly potentiated KCNQ1,KCNQ2 and KCNQ3.Furthermore,using the compound 10g as a probe,whether KCNQ4 and KCNQ5could act as a therapeutic target for visceral pain was investigated.We found that compound 10g showed significant analgesic activity in a mustard oil-induced colorectal pain mouse model.This part of the work reported a series of newly identified selective KCNQ4 and KCNQ5 activiators and provided evidences that these two subtype KCNQ channels could be used as therapeutic targets for visceral pain.