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 Ca²⁺ responses at hTRPM8 with IC₅₀ values of 805 ± 200 nM and 1.8 ± 0.6 μM, respectively, while more potently inhibiting agonist responses at the rat orthologue (rTRPM8 IC₅₀ (menthol) = 117 ± 18 nM, IC₅₀ (icilin) = 521 ± 20 nM). Whole-cell patch-clamp recordings of hTRPM8 confirm the 1 inhibition of menthol-stimulated currents, with an IC₅₀ 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.

Figure 1

Monoterpene-derived TRPM8 antagonists. (a) Ref (11). (b) Ref (12), TRPM8 orthologue not specified. (c) Ref (13). (d) Ref (14).

Scheme 1

(a) NH₂OH, NaOH, EtOH/H₂O, rt, 95%; (b) Na⁺, EtOH/toluene, reflux, quantitative; (c) 1,1′-biphenyl carboxylic acid, EDCI, HOBt, Et₃N, THF, 40 °C, 76%.

Figure 2

VCD (upper frame) and IR (lower frame) spectra observed (blue) for compound 1 (left axes) compared with Boltzmann-averaged spectra of calculated (green) conformations for the (1R,2S,5R) configuration (right axes).

Figure 3

Characterization of hTRPM8 activity expressed in HEK293 cells by calcium imaging. (A) hTRPM8 activity evoked by icilin (black) or menthol (blue) (EC₅₀ icilin = 526 ± 24 nM, EC₅₀ menthol = 81 ± 17 μM). (B) hTRPM8 activity blocked by compound 1 using both agonists at its EC₅₀. (IC₅₀ = 1.8 ± 0.6 μM for icilin activation, IC₅₀ = 805 ± 200 nM for menthol activation). (C) Representative calcium traces for activity of hTRPM8 evoked by 500 nM icilin (control) in the presence of compound 1 from 0.1–10 μM. (D) Representative calcium traces for activity of hTRPM8 evoked by 100 μM menthol (control) in the presence of compound 1 from 0.1–10 μM. All data are expressed as mean ± SEM (n = 3, N = 3).

Figure 4

Characterization of rTRPM8 activity expressed in HEK293 cells by calcium imaging. (A) rTRPM8 activity evoked by icilin (black) or menthol (blue) (EC₅₀ icilin = 554 ± 12 nM, EC₅₀ menthol = 107 ± 8 μM). (B) rTRPM8 activity blocked by compound 1 using both agonists at its EC₅₀. (IC₅₀ = 521 ± 20 nM for icilin activation, IC₅₀ = 117 ± 18 nM for menthol activation). (C) Representative calcium traces for activity of rTRPM8 evoked by 500 nM icilin (control) in the presence of compound 1 from 0.1–10 μM. (D) Representative calcium traces for activity of rTRPM8 evoked by 100 μM menthol (control) in the presence of compound 1 from 0.1–10 μM. Data are expressed as mean ± SEM (n = 3, N = 3).

Figure 5

Comparative effect of compound 1 in hTRPM8 vs rTRPM8 activated by 100 μM menthol (A) or 500 nM icilin (B). Data are expressed as mean ± SEM (n = 3, N = 3). Data analyzed using two-way ANOVA followed by Bonferroni’s post hoc test. **P < 0.01, ***P < 0.001,**** P < 0.0001.

Figure 6

Convergence parameters of (−)-menthyl 1 and rTRPM8 during MD simulation. (a) Docking pose (pre-MD) of 1 in rTRPM8. (−)-Menthyl 1 is shown as green sticks. Important residues are shown as sticks. Individual helices are colored as follows: S1 (yellow), S2 (gold), S4 (orange), TRP helix (red). The Ca²⁺ ion (green) is shown in CPK representation. (b) Binding mode (post-MD) of 1 in rTRPM8 after 100 ns MD. (−)-Menthyl 1 is shown as magenta sticks. Important residues are shown as sticks. Individual helices are colored as follows: S1 (blue), S2 (light blue), S4 (cyan), TRP helix (green). The Ca²⁺ ion (magenta) is shown in CPK representation. (c) RMSD of 1 and rTRPM8 during MD simulation. (d) Energy decomposition of key residues in rTRPM8 that contributed to binding of 1. Our rTRPM8 homology model was constructed using the cryo-EM structure of TRPM8_FA (PDB 6BPQ) as a template.

Figure 7

Superimposition of the binding mode (post-MD) of (−)-menthyl 1 in rTRPM8 (this work) and hTRPM8. The MD pose of 1 and hTRPM8 is depicted in gray.

Figure 8

Whole-cell patch-clamp analysis of (−)-menthyl 1 inhibition of hTRPM8 validates species-dependent antagonism. (A) Normalized currents from menthol-evoked hTRPM8 show concentration-dependent inhibition. The IC₅₀ of 1 against 500 μM menthol is 700 ± 200 nM (n = 3 cells). Data were recorded in transiently transfected HEK293 cells at +80 mV over a range of 1 concentrations (1 nM – 0.1 mM). Error bars indicate standard error of the mean. (B) Representative inhibition data from a single cell stimulated by 500 μM menthol and exposed to 1 nM, 1 μM, and 100 μM of compound 1.