Ca2+ Measurements of Recombinant Cell Lines

Cells were plated in 384-well plates. Cells were loaded with 1 μM Fluo-4 and 0.05% pluronic acid for 1 h at room temperature. Formalin-selectivity experiments were run with 0.003% formalin. Agonist EC50 curves used 0–25 μM AITC or 0–0.017% formalin. IC50 curves for TRPA1 antagonists were constructed by using 0.625–40 μM antagonist in the presence of 5 μM AITC or 0.001% formalin using data collected 3 min after agonist addition. Data were collected by using a Hamamatsu FDSS 6000 fluorescence-based plate reader and analyzed using IGOR Pro.

Cell lines

TRPA1-expressing HEK293 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

10 μM for 3 min

Applications

HC-030031 selectively inhibited TRPA1 activation by cinnamaldehyde and allyl isothiocyanate (AITC) in vitro [2]. HC-030031 inhibited the AITC- and formalin-induced Ca2+ increase in TRPA1-expressing cells [1].

Animal models

Male Sprague-Dawley rats model

Dosage form

100 or 300 mg/kg; p.o. for 60 min;

Applications

HC-030031 reduced inflammatory- and neuropathy-induced mechanical hypersensitivity. HC-030031 attenuated formalin- and allyl isothiocyanate-evoked pain behavior via inhibition of TRPA1 [2]. Moreover, HC-030031 alleviated behavioral mechanical hyperalgesia without affecting heat hyperalgesia in inflamed mice [3].

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

• Mol Cell Biochem (2020): 1-14. PMID:32627113

HC-030031 is a selective antagonist of transient receptor potential ankyrin subfamily, member 1(TRPA1) with IC50 value of 4.9μM [1].

TRP channels are found to take participate in the response of chemical stimuli and in mechanoreception. TRPA1 is the sole mammalian member of TRPA subfamily. As an inhibitor of TRPA1, HC-030031 can be used as a tool to study the role of this channel in pain perception. In the FLIPR calcium-influx assay, HC-030031 blocks cinnamaldehyde- and AITC- induced activation of TRPA1 with IC50 values of 4.9μM and 7.5μM, respectively. HC-030031 is selective against TRPA1. It shows no significant inhibitory activity against many other enzymes, receptors and transporters. It also has no effect on the activation of TRPV1, TRPV3 and TRPV4. In vivo, HC-030031 significantly reduces the hindpaw lifting induced by AITC as well as mechanical hypersensitivity induced by CFA in rats. Besides that, the administration of HC-030031(300mg/kg) can markedly attenuate the hypersensitivity (41%) induced by spinal nerve ligation [1].

References:
[1] Eid S R, Crown E D, Moore E L, et al. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory-and neuropathy-induced mechanical hypersensitivity. Mol Pain, 2008, 4(48): 1-10.