Aprepitant is a novel, selective activator of the K2P channel TRAAK

D Mccoull,E.L Veale,Y Walsh,L Byrom,T Avkiran,J.M Large,E Vaitone,F Gaffey,J Jerman,A Mathie,P.D Wright

doi:10.1016/j.bbrc.2021.12.031

Abstract

TRAAK (KCNK4, K2P4.1) is a mechanosensitive two-pore domain potassium (K2P) channel. Due to its expression within sensory neurons and genetic link to neuropathic pain it represents a promising potential target for novel analgesics. In common with many other channels in the wider K2P sub-family, there remains a paucity of small molecule pharmacological tools. Specifically, there is a lack of molecules selective for TRAAK over the other members of the TREK subfamily of K2P channels. We developed a thallium flux assay to allow high throughput screening of compounds and facilitate the identification of novel TRAAK activators. Using a library of ∼1200 drug like molecules we identified Aprepitant as a small molecule activator of TRAAK. Aprepitant is an NK-1 antagonist used to treat nausea and vomiting. Close structural analogues of Aprepitant and a range of NK-1 antagonists were also selected or designed for purchase or brief chemical synthesis and screened for their ability to activate TRAAK. Electrophysiology experiments confirmed that Aprepitant activates both the ‘long’ and ‘short’ transcript variants of TRAAK. We also demonstrated that Aprepitant is selective and does not activate other members of the K2P superfamily. This work describes the development of a high throughput assay to identify potential TRAAK activators and subsequent identification and confirmation of the novel TRAAK activator Aprepitant. This discovery identifies a useful tool compound which can be used to further probe the function of TRAAK K2P channels.

Highlights

There continues to be significant interest in examining and modulating the function of the 15 members of the two-pore domain potassium channel family (K2Ps) [1]
It has been shown that TRAAK is expressed in dorsal root ganglia (DRG) and trigeminal ganglia (TG) neurons by measuring K2P mRNA abundance using RT-qPCR [7]
In normal rodent DRG neurons [7] it was reported that TRAAK mRNA was the second highest in abundance of the K2P family

Summary

Introduction

There continues to be significant interest in examining and modulating the function of the 15 members of the two-pore domain potassium channel family (K2Ps) [1]. Activation of TRAAK by small molecules has been described, by non-selective activators of the closely related TREK-1 and TREK-2 channels. Compounds such as C3001a [14], BL-1249 [15], Flufenamic acid [16], ML67-33 [17], DCPIB [18], 2-APB [19], Arachidonic acid [5] and a group of phospholipid modulators [20] have been reported to activate the TRAAK channel at a number of distinct binding sites. We have confirmed and characterised this activity in more detail, in the context of other K2P channel selectivity and whole cell patch clamp electrophysiology experiments

Thallium flux assays

Whole-cell patch clamp electrophysiology

Compounds

Identification of novel TRAAK activators

Exploring compound SAR

Selectivity of Aprepitant