ML365 inhibits TWIK2 channel to block ATP-induced NLRP3 inflammasome

Xiao-yan Wu1, Jin-yan Lv1, Shi-qing Zhang2, Xin Yi1, Zi-wei Xu1, Yuan-xing Zhi1, Bo-xin Zhao3, Jian-xin Pang1, Ken Kin Lam Yung2, Shu-wen Liu1, Ping-zheng Zhou1
1 Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
2 Department of Biology, Faculty of Science, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
3 Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
Correspondence to: Ken Kin Lam Yung:, Shu-wen Liu:, Ping-zheng Zhou:,
DOI: 10.1038/s41401-021-00739-9
Received: 17 February 2021
Accepted: 30 June 2021
Advance online: 2 August 2021


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 (TWIK2I289A/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.

Keywords: TWIK2; K2P channels; NLRP3; inflammasome; innate immunity; inflammatory diseases

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