Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch- related behaviours

Shenbin Liu1,2,*, Jing Feng1,*, Jialie Luo1, Pu Yang1, Thomas J Brett3 and Hongzhen Hu1

BACKGROUND AND PURPOSE

TMEM16A, also known as anoctamin 1 channel, is a member of the Ca2+-activated chloride channels family and serves as a heat sensor in the primary nociceptors. Eact is a recently discovered small molecule activator of the TMEM16A channel. Here, we asked if Eact produces pain- and itch-related responses in vivo and investigated the cellular and molecular basis of Eact-elicited responses in dorsal root ganglia (DRG) neurons.

EXPERIMENTAL APPROACH
We employed behavioural testing combined with pharmacological inhibition and genetic ablation approaches to identify transient receptor potential vanilloid 1 (TRPV1) as the prominent mediator for Eact-evoked itch- or pain-related responses. We investigated the effects of Eact on TRPV1 and TMEM16A channels expressed in HEK293T cells and in DRG neurons isolated from wild type and Trpv1/ mice using Ca2+ imaging and patch-clamp recordings. We also used site-directed mutagenesis to determine the molecular basis of Eact activation of TRPV1.

KEY RESULTS
Administration of Eact elicited both itch- and pain-related behaviours. Unexpectedly, the Eact-elicited behavioural responses were dependent on the function of TRPV1, as shown by pharmacological inhibition and genetic ablation studies. Eact activated membrane currents and increased intracellular free Ca2+ in both TRPV1-expressing HEK293T cells and isolated DRG neurons in a TRPV1-dependent manner. Eact activation of the TRPV1 channel was severely attenuated by mutations disrupting the capsaicin binding sites.

CONCLUSIONS AND IMPLICATIONS
Our results suggest that Eact activates primary sensory nociceptors and produces both pain and itch responses mainly through direct activation of TRPV1 channels.

 

Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch- related behaviours

A Cell-Based Functional Assay Using a Green Fluorescent Protein-Based Calcium Indicator dCys-GCaMP
Bin Cai,1 Xia Chen,1 Fang Liu,1 Jun Li,1 Lijuan Gu,2 Jason R. Liu,3 and Jay Liu1
1Rugen Therapeutics Ltd., Suzhou Industrial Park, China. 2Biotech Development Co., Ltd., Suzhou Industrial Park, China. 3Summer Intern from Penn State University, University Park, Pennsylvania.

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Measurement of the changes in intracellular Ca2+ levels is an important assay for drug discovery. In this report, we describe a novel Ca2+ indicator, dCys-GCaMP, based on the green fluorescent protein and the development of a rapid and simple cell-based functional assay using this new Ca2+ indicator. We demonstrated the sensitivity and reliability of the assay by measuring the cellular responses to the agonists, antagonists, channel blockers, and modulators of the ionotropic N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. HEK293 cells coexpressing the NMDA receptor and dCys-GCaMP displayed a strong increase in fluorescence intensity when stimulated with the agonist glutamate. This increase in the fluorescence signal was agonist concentration dependent and could be blocked by NMDAR antagonists and channel blockers. The pharmacological parameters measured with the dCys-GCaMP assay are in close agreement with those derived from conventional assays with synthetic dye fluo-4 and literature values. In addition, we showed that this assay could be used on G protein-coupled receptors as well, as exemplified by studies on the a1A adrenergic receptor. A limited scale evaluation of the assay performance in a 96-well compound screening format suggests that the dCys-GCaMP assay could be easily adapted to a high-throughput screening environ- ment. The most important advantage of this new assay over the con- ventional fluo-4 and aequorin assays is the elimination of the dye-loading or substrate-loading process.

Plumbagin, Juglone, and Boropinal as Novel TRPA1 Agonists
Kerstin Hill,*,† Serena Fiorito,‡ Vito Alessandro Taddeo,‡ Anja Schulze,§ Marion Leonhardt,† Francesco Epifano,*,‡ and Salvatore Genovese‡
†Rudolf-Boehm-InstitutfürPharmakologieundToxikologie,UniversitaẗLeipzig,Har̈telstr.16-18,04107Leipzig,Germany ‡Department of Pharmacy, University “G. D’Annunzio” of Chieti-Pescara, Via dei Vestini 31, 66100 Chieti Scalo (CH), Italy §Fraunhofer-Institut für Zelltherapie und Immunologie IZI, Biozentrum, Weinbergweg 22, 06120 Halle, Germany

Plumbagin, Juglone, and Boropinal as Novel TRPA1 Agonists

ABSTRACT: A series of seven oxyprenylated phenylpropanoids and naphthoquinones were tested regarding their ability to activate transient receptor potential ankyrin subtype 1 channel (TRPA1). Three of the assayed compounds, namely, boropinal (3), juglone (5), and plumbagin (7), acted as strong modulators of TRPA1 channels with EC50 values of 9.8, 1.7,and 0.5 μM, respectively, as assessed by Ca2+ assays. Moreover, the compounds elicited TRPA1 currents in electrophysiological whole cell recordings. We additionally provide evidence that plumbagin activated TRPA1-positive neurons isolated from mouse dorsal root ganglion neurons but did not affect sensory neurons from TRPA1-deficient mice. The high potencies of plumbagin and juglone to activate TRPA1 channels may explain the molecular basis of the mucosal irritant properties of these compounds as well as of related naphthoquinones and phytopreparations, as widely reported in the literature.