TRPV1 (EC50 = 11.6 ± 2.5 μM for activating outward currents at +60 mV in mTRPV1-expressing HEK293T cells) [1]
TMEM16A [1]

Eact Eact depends on TRPV1 to elicit itch and associated pain behaviors. With an EC50 of 11.6±2.5 μM, Eact stimulates membrane currents in HEK293T cells expressing mtrpv1 in a concentration-dependent manner. TRPV1 channels are damaged and rearranged when activated by the TMEM16A activator Eact [1].

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In HEK293T cells expressing mTRPV1, Eact (100 μM) evoked a substantial increase in intracellular free Ca2+ ([Ca2+]i), and cells also responded to capsaicin (500 nM). Eact had no effect on HEK293T cells expressing hTRPA1, hTRPM8, or rTRPV4, while respective agonists (AITC, menthol, GSK1016790A) activated them. [1]
Eact activated membrane currents in mTRPV1-expressing HEK293T cells in a concentration-dependent manner with an EC50 of 11.6 ± 2.5 μM (Hill coefficient 2.0 ± 0.4). The current-voltage relationship of Eact-activated currents was similar to that of capsaicin. Maximal current density evoked by 100 μM Eact was 404.0 ± 24.4 pA/pF, which was 69.4 ± 0.9% of the response evoked by 3 μM capsaicin (581.8 ± 33.9 pA/pF). [1]
In inside-out membrane patches excised from mTRPV1-expressing HEK293T cells, Eact significantly increased single-channel open probability (nPo) compared to baseline (P < 0.0001). [1]
Knockdown of endogenous TMEM16A by siRNA in HEK293T cells did not affect Eact activation of mTRPV1, as Eact (10 μM)-activated current densities were not significantly different between scrambled siRNA and TMEM16A siRNA groups. [1]
In cultured mouse DRG neurons, Eact (100 μM) activated a large outwardly rectifying current that was substantially reduced by co-application of TRPV1 inhibitors AMG9810 (0.1 μM) or capsazepine (10 μM), and was absent in Trpv1-/- DRG neurons. The selective TMEM16A inhibitor A01 (10 μM) did not significantly affect Eact-activated currents in WT DRG neurons. Eact-activated current persisted when recorded with a Cl−-free pipette solution, indicating it is not a chloride current. [1]
In DRG neurons, bath application of Eact (100 μM) alone elicited a [Ca2+]i response in 32.7 ± 2.2% of neurons tested, all of which responded to capsaicin (500 nM). Capsaicin activated a larger population (51.8 ± 5.1%). The Eact-evoked [Ca2+]i response was almost completely abolished by AMG9810 (0.1 μM) or genetic ablation of TRPV1. [1]

Intradermal (i.d.) injection of Eact (50 μL, 4.67 mM) into the rostral back of mice produced an intense scratching response (itch-related behaviour). This scratching was attenuated by pretreatment with the TRPV1 antagonist AMG9810 (50 mg/kg, i.p.) and was completely absent in Trpv1-/- mice. [1]
Intraplantar (i.pl.) injection of Eact (20 μL, 4.67 mM) into the hind paw immediately elicited a nocifensive response (flinching and licking) (pain-related behaviour). AMG9810 (50 mg/kg, i.p.) given 30 min before Eact significantly reduced this response, and genetic ablation of TRPV1 abolished it. [1]
Intraplantar injection of Eact (20 μL, 4.67 mM) also evoked a robust and sustained thermal hypersensitivity lasting at least 60 min, as measured by paw withdrawal latencies using the Hargreaves apparatus. AMG9810 (50 mg/kg, i.p.) significantly inhibited this effect, and Eact-elicited thermal hypersensitivity was absent in Trpv1-/- mice. [1]
Co-application of the selective TMEM16A inhibitor T16Ainh-A01 (A01) partially reversed Eact-elicited itch- and pain-related behaviours (Supporting Information Fig. S1). [1]

For live cell Ca2+ imaging, cultured DRG neurons or HEK293T cells expressing TRP channels were loaded with 4 μM Fura-2 AM in culture medium at 37°C for 60 min, then washed three times and incubated in HBSS at room temperature for 30 min. Fluorescence at 340 and 380 nm excitation wavelengths was recorded on an inverted microscope equipped with excitation filter wheels. Fura-2 ratios (F340/F380) reflect changes in [Ca2+]i. Threshold of activation was defined as 3 SDs above the average (~20% above baseline). [1]
For whole-cell patch-clamp recordings, an Axon 700B amplifier was used at room temperature (22-24°C). Pipettes (2-4 MΩ for whole-cell) were filled with solution containing 140 mM CsCl, 2 mM EGTA, and 10 mM HEPES (pH 7.3). Ca2+-free extracellular solution contained 140 mM NaCl, 5 mM KCl, 0.5 mM EGTA, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4). Whole-cell currents were recorded using voltage ramps from −100 to +100 mV for 500 ms at a holding potential of 0 mV. Data were filtered at 2 kHz and digitized at 10 kHz. [1]
For single-channel recordings, pipettes (8-10 MΩ) were filled with symmetrical solution (same as pipette solution). Inside-out patches were excised, and open probability was determined using half-amplitude threshold-crossing criteria. [1]
For Western blot, HEK293T cells were lysed in buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% SDS, 1 mM PMSF, NaF, NaVO3, leupeptin, pepstatin, aprotinin). Protein concentration was determined by BCA assay. 120 μg protein was boiled with SDS-PAGE sample loading buffer, separated on 12% SDS-PAGE, transferred to membrane, blocked with 5% BSA, incubated with rabbit anti-TMEM16A (1:1000) and HRP-anti-rabbit secondary (1:1000), and developed with ECL. Mouse HRP-anti-β-actin (1:5000) was used as loading control. Densitometry analysis was performed using Image J. [1]
For siRNA knockdown, HEK293T cells were transfected with 200 nM TMEM16A siRNA or scrambled siRNA control using Lipofectamine 2000 (20:2 ratio pmol siRNA:μL Lipofectamine 2000), co-transfected with mTRPV1. After 48 h, cells were plated onto coverslips and incubated for another 24 h before use. Knockdown was confirmed by Western blot. [1]

Wild-type (WT) and congenic Trpv1-/- mice on C57BL/6J background were used. All mice were housed under 12 h light/dark cycle with food and water ad libitum. Experiments were approved by the Animal Studies Committee at Washington University School of Medicine. [1]
For scratching behaviour, mice were placed individually in transparent cages for at least 30 min before assays. Eact (4.67 mM, 50 μL) or vehicle (0.9% saline + 1% DMSO + 0.1% Tween 80) was injected intradermally (i.d.) into the rostral back. The number of hind limb scratching bouts directed to the injection site over a 30 min period was counted. [1]
For nocifensive response, intraplantar (i.pl.) injection of Eact (4.67 mM, 20 μL) into the hind paw was used. Immediately after injection, mice were placed inside a Plexiglas chamber, and total time spent licking and lifting the injected paw was measured from video recordings over 5 min. AMG9810 (50 mg/kg) was administered intraperitoneally (i.p.) 30 min before Eact injection. Control mice received equal volume of vehicle. [1]
For thermal pain test, paw withdrawal latencies in response to radiant heat were measured using the Hargreaves apparatus. Mice were placed individually in a clear Plexiglas chamber (8×8×12 cm) and acclimated for at least 1 h before testing. The right hind paw was injected intraplantarly with 20 μL vehicle with or without Eact. Withdrawal latencies were measured before (0 min) and at 15, 30, 60, 90, and 120 min after injection. Infrared intensity was adjusted to obtain basal latencies of 10-15 s, with a 20 s cut-off to prevent tissue damage. [1]

[1]. Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch-related behaviours. Br J Pharmacol. 2016 Apr;173(7):1208-18.

Eact was originally discovered as a small molecule activator of TMEM16A (Ca2+-activated chloride channel) via cell-based high-throughput screening of ~110,000 compounds. It directly opens TMEM16A without increasing intracellular Ca2+ and stimulates chloride secretion. However, this study unexpectedly found that Eact also directly activates TRPV1 channels through interaction with the capsaicin-binding site. Mutations disrupting capsaicin binding (e.g., S513Y, Y512A/S513Y) severely attenuated Eact activation (EC50 increased by at least sixfold). Eact is therefore not an ideal tool to dissect TMEM16A function in primary sensory neurons because it directly activates TRPV1 in the same cells. The sensory hypersensitivity elicited by Eact might present an unwanted side effect for its potential applications to treat cystic fibrosis and related diseases. [1]

C22H24N2O5S

428.501364707947

428.141

461000-66-8

3173542

White to off-white solid powder

1.237±0.06 g/cm3(Predicted)

595.9±60.0 ℃(Predicted)

4.129

0

7

9

30

520

0

ZUXNHFFVQWADJL-UHFFFAOYSA-N

InChI=1S/C22H24N2O5S/c1-26-11-10-24(22-23-17(14-30-22)15-8-6-5-7-9-15)21(25)16-12-18(27-2)20(29-4)19(13-16)28-3/h5-9,12-14H,10-11H2,1-4H3

3,4,5-trimethoxy-N-(2-methoxyethyl)-N-(4-phenyl-1,3-thiazol-2-yl)benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~116.69 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)