Dysregulation of NLRP3 inflammasome results in uncontrolled inflammation,which participates in various chronic diseases.TWIK2 potassium channel mediates potassium efflux that has been reported to be an essential upstream mechanism for ATP-induced NLRP3 inflammasome activation.Thus,TWIK2 potassium channel could be a potential drug target for NLRP3-related inflammatory diseases.In the present study we investigated the effects of known K2P channel modulators on TWIK2 channel expressed in a heterologous system.In order to increase plasma membrane expression and thus TWIK2 currents,a mutant channel with three mutations(TWIK21289A/L290A/Y308A)in the C-terminus was expressed in COS-7 cells.TWIK2 currents were assessed using whole-cell voltage-clamp recording.Among 6 known K2P channel modulators tested(DCPIB,quinine,fluoxetine,ML365,ML335,and TKDC),ML365 was the most potent TWIK2 channel blocker with an IC50 value of 4.07±1.5 μM.Furthermore,ML365 selectively inhibited TWIK2 without affecting TWIK1 or THIK1 channels.We showed that ML365(1,5 μM)concentration-dependently inhibited ATP-induced NLRP3 inflammasome activation in LPS-primed murine BMDMs,whereas it did not affect nigericin-induced NLRP3,or non-canonical,AIM2 and NLRC4 inflammasomes activation.Knockdown of TWIK2 significantly impaired the inhibitory effect of ML365 on ATP-induced NLRP3 inflammasome activation.Moreover,we demonstrated that pre-administration of ML365(1,10,25 mg/kg,ip)dose-dependently ameliorated LPS-induced endotoxic shock in mice.In a preliminary pharmacokinetic study conducted in rats,ML365 showed good absolute oral bioavailability with F value of 22.49％.In conclusion,ML365 provides a structural reference for future design of selective TWIK2 channel inhibitors in treating related inflammatory diseases.

Xiao-yan Wu;Jin-yan Lv;Shi-qing Zhang;Xin Yi;Zi-wei Xu;Yuan-xing Zhi;Bo-xin Zhao;Jian-xin Pang;Ken Kin Lam Yung;Shu-wen Liu;Ping-zheng Zhou

Guangdong Provincial Key Laboratory of New Drug Screening,School of Pharmaceutical Sciences,Southern Medical University,Guangzhou 510515,China;Department of Biology,Faculty of Science,Hong Kong Baptist University,Kowloon Tong,Hong Kong,China;Department of Pharmacy,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China

中国药理学报（英文版）

[1]Xiao-yan Wu;Jin-yan Lv;Shi-qing Zhang;Xin Yi;Zi-wei Xu;Yuan-xing Zhi;Bo-xin Zhao;Jian-xin Pang;Ken Kin Lam Yung;Shu-wen Liu;Ping-zheng Zhou-.ML365 inhibits TWIK2 channel to block ATP-induced NLRP3 inflammasome)[J].中国药理学报（英文版）,2022(04):992-1000

ML365,TWIK2,TWIK21289A,L290A,Y308A,DCPIB,ML335,TKDC,TWIK1,THIK1,endotoxic

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