Structural and in Vitro Functional Characterization of a Menthyl TRPM8 Antagonist Indicates Species-Dependent Regulation

Abstract

TRPM8 antagonists derived from its cognate ligand, (−)-menthol, are underrepresented. We determine the absolute stereochemistry of a well-known TRPM8 antagonist, (−)-menthyl 1, using VCD and 2D NMR. We explore 1 for its antagonist effects of the human TRPM8 (hTRPM8) orthologue to uncover species-dependent inhibition versus rat channels. (−)-Menthyl 1 inhibits menthol-and icilin-evoked Ca 2+ responses at hTRPM8 with IC 50 values of 805 ± 200 nM and 1.8 ± 0.6 μM, respectively, while more potently inhibiting agonist responses at the rat orthologue (rTRPM8 IC 50 (menthol) = 117 ± 18 nM, IC 50 (icilin) = 521 ± 20 nM). Whole-cell patch-clamp recordings of hTRPM8 confirm the 1 inhibition of menthol-stimulated currents, with an IC 50 of 700 ± 200 nM. We demonstrate that 1 possesses ≥400-fold selectivity for hTRPM8 versus hTRPA1/hTRPV1. (−)-menthyl 1 can be used as a novel chemical tool to study hTRPM8 pharmacology and differences in species commonly used in drug discovery.

TRPM8 contributes to sex dimorphism by promoting recovery of normal sensitivity in a mouse model of chronic migraine

“…4e , p < 0.001 vs. wild-type), revealing a testosterone activity through TRPM8 in trigeminal neurons. Previous studies described structural and functional differences between murine and human TRPM8 35 . To evaluate this possibility, we assessed testosterone activity on HEK293 cells constitutively expressing murine or human TRPM8 (Fig.…”

“…IMR90 fibroblast-like cells (CCL-186 ATCC, Virgina, USA) were seeded in 12 mm coverslips at 50,000 cells/well and were maintained in MEM enriched with 10% FBS and 1% penicillin/streptomycin at 37 °C in 5% CO 2 atmosphere 88 . For the initial experiments assessing TRPM8 function, HEK293 cells constitutively expressing human or rat TRPM8 35 were obtained from Prof. Belmonte Laboratory (Instituto de Neurociencias, San Juan, Alicante, Spain). All calcium imaging and electrophysiological recordings took place when cells reached 60% of confluence and 72 h after transfection when applicable.…”

“…HEK293 cells expressing human TRPM8 35 ( Prof. Belmonte Laboratory, Instituto de Neurociencias, San Juan, Alicante, Spain) were cultured in EMEM (ATCC, 30-2003), 1% penicillin/streptomycin (Gibco, 15140-122), 1% geneticin (Gibco, 10131-019), 10% FBS (Gibco, 16000-044), 2 mM L-glutamine (Gibco, A2916801) and 2.5 mg/ml glucose (Sigma, G8270). T25 flasks at 80% confluency were transfected with DMEM plus Glutamax (Gibco, 61965-026), 1% Penicillin/Streptomycin (Gibco, 15140-122), 19.6 μl of Lipofectamine 3000 (Invitrogen, L3000-015), 1 μg of androgen receptor siRNA (Thermo Fisher, Ambion, s1538; sense GCCCAUUGACUAUUACUUUtt; antisense AAAGUAAUAGUCAAUGGGCaa) or negative control scramble siRNA (Thermo Fisher, Ambion, 4390843) and 6.5 μg of pcDNA3eGFP plasmid DNA ( Addgene plasmid #13031).…”

L-Menthol-Loadable Electrospun Fibers of PMVEMA Anhydride for Topical Administration

“…The menthol encapsulated into the experimental fibers after being dissolved in acetone, as for the cytotoxicity assays, induced intracellular Ca 2+ increases in a concentrationdependent fashion in treated HEK293-hTRPM8 cells (Figure 5). First, TRPM8 activation dynamics were determined for our experimental model using free menthol (Figure 5a,b), describing a dose-response curve as in previous works [49]. Follow-up studies showed that the menthol contained in both types of fibers was able to activate TRPM8 similarly, and also in a concentration-dependent manner (Figure 5c,d for F 119S and F 139S samples, respectively).…”

TRPM8 Channels: Advances in Structural Studies and Pharmacological Modulation

“…The work of different academic groups also contributed to the identification of novel chemotypes that inhibit TRPM8 receptor activation. In a very recent paper, Journigan et al described a (-)-menthyl derivative 29 (Figure 6, Table 2) as a potent antagonist of TRPM8 channels [163]. Calcium imaging assays were carried out to assess the (−)-menthyl 29 species-dependent inhibition of both menthol-and icilin-mediated responses, at either human or rat TRPM8, using strictly the same experimental conditions.…”