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.

Transient receptor potential melastatin 8 (TRPM8) is a versatile target for chemotherapy-induced peripheral neuropathy,¹ neuropathic pain,² type 1 diabetes, and muscle wasting,³ likely due to its ability to integrate distinct stimuli as a polymodal sensor. Menthol is the cognate ligand for TRPM8.⁴ Its binding site has been explored in cryo-electron microscopy (cryo-EM) studies of collared flycatcher (avian) Ficedula albicollis TRPM8 (TRPM8_FA) bound to WS-12,⁵ backed by radioligand displacement and mutagenesis data.⁶ Many structurally disparate, non-menthol TRPM8 antagonist chemotypes have been reported, including several complexed with great tit (avian) Parus major TRPM8, implying a central binding cavity able to tolerate diverse scaffolds.⁷ These recent advances suggest a mature drug discovery landscape; however, few menthol analogues with an antagonist profile are known (Figure 1), representing a gap in the literature for this chemotype. Such antagonists along with agonists of the same chemotype, will provide tools to identify the molecular mechanisms of ligand-induced channel gating. The structure of TRPM8 with an open ion permeation pathway remains elusive⁸ despite known high-potency, menthol-derived agonists that could aid in its determination.⁹ The structural mechanism underlying the coupling of antagonist binding and channel gating was recently described using a potent benzimidazole analogue TC-I 2014.⁷ Complementary agonist derivatives within this series were not reported.¹⁰

Figure 1.

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

(−)-Menthyl 1 (Figure 1) is a potent antagonist of rat TRPM8 (rTRPM8) in spectrofluorometric calcium (Ca²⁺) flux assays using Fluo-4-AM as a fluorophore (IC₅₀ vs 20 μM menthol = 20 ± 2 nM; IC₅₀ vs 0.25 μM icilin = 50 ± 10 nM).¹¹ Its effects in the androgen-responsive prostate carcinoma cell line have also been explored.¹⁵ We recently reported its activity using Fura-2-based calcium imaging of HEK-293 cells stably expressing hTRPM8 at an IC₅₀ of 16 ± 1 μM, >300-fold lower than its reported IC₅₀ at the rat orthologue, using a similar concentration of the agonist icilin (IC₅₀ vs 0.5 μM icilin).¹⁶ Differences in observed IC₅₀ values for 1 may be dependent on TRPM8 orthologues (rat vs human) or different Ca²⁺-sensitive fluorescent indicators.¹⁷ The fluorescent Ca²⁺ assay is a standard functional assay for validating TRP-family ligands⁴ and amenable to high throughput; however, Ca²⁺ is an indirect signal indication of activity and does not offer real-time functional characterization of electrophysiology. Whole-cell patch-clamp measurements of 1 would validate both the nature of the functional effect and magnitude of antagonism.

In the same assay, benzoate 2 (Figure 1) has relatively low potency against menthol (100 μM) stimulated currents of TRPM8 (IC₅₀ = 2 ± 1 μM), although the orthologue is not specified.¹² The antagonist effect of D-1457 (Figure 1)¹³ is only described at a single high concentration of 10 μM in cell viability assays. Indeed, various functional assays are used to explore antagonist activity of these menthol-based compounds, at times with minimal or incomplete data. Conversely, cannabidivarin (Figure 1) bears a limonene group similar to menthol and has defined pharmacology. However, this compound is a nonselective TRPM8 antagonist, with bifunctional TRPA1 agonist activity.¹⁴ Well-characterized menthol-based antagonists are needed to serve as natural product TRPM8 probes and provide scaffolds as leads for drug discovery.

Results and Discussion

We first resynthesized and assigned absolute stereochemistry of the well-known TRPM8 antagonist (−)-menthyl 1 with VCD and 2D NMR, with special attention to the α-chiral amide, which was not described before. Our previous molecular dynamics (MD) studies suggest that configuration at this carbon positions the biphenyl moiety in the same region of the voltage-sensor-like domain (VSLD, transmembrane helices S1–S4) binding site as the methoxyphenyl moiety of agonist WS-12, with the 1′-phenyl ring extending into the floor of the pocket, toward the TRP helix.¹⁶ Comparison with the bound pose of TC-I 2014 shows that the 1′-phenyl ring of 1 occupies a similar space in the TRPM8 binding site as the 5-(2-trifluoromethyl-phenyl) of TC-I 2014. This suggests that a second phenyl ring could be partly responsible for functional profile differences of these two menthol analogues.

Ortar et al. described the preparation of 1(11) via (−)-menthylamine (1R,2S,5R)-2-isopropyl-5-methylcyclohexan-1-amine) using methodology by Schopohl and colleagues,¹⁸ followed by EDCI/HOBt-assisted amidation. However, the absolute stereochemistry of (−)-menthylamine was not confirmed to be consistent with the structure reported by the latter group, who reported its X-ray crystal structure via incorporation into a caffeine complex.¹⁹ We resynthesized (−)-menthylamine¹⁸ starting from l-menthone I-1 (Scheme 1). We prepared oxime I-2 using standard conditions from I-1 (95% yield), followed by Bouveault–Blanc reduction to I-3 in quantitative yield. Oxime I-2 and amine I-3 were not purified to prevent exclusion of any minor isomers. GC-MS traces and ¹H NMR spectra of I-2 and I-3 indicate that these reactions afford minimal side product formation (Supporting Information (SI), Figures S1–S7). EDCI/HOBt-assisted coupling of I-3 with 1,1′-biphenyl carboxylic acid afforded 1 in 76% yield, comparable to the reported yield (81%).¹¹

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%.

The absolute stereochemical configuration of 1 was established by VCD²⁰ and ¹H, ¹³C, gCOSY, NOESY, and gHSQCAD solution NMR experiments. The solvent-corrected IR and VCD spectra for (−)-menthyl 1 were obtained (SI, Figure S8). We performed a conformer search on the (1R,2S,5R) enantiomer of compound 1 using ComputeVOA from BioTools. We then optimized the 70 lowest energy conformers (8 kcal range from global minimum, SI, Figure S9) obtained with the MMFF94 force field and calculated IR and VCD frequencies using the B3LYP method and 6311Gdp basis set in Gaussian ’09. Convolved spectra from the 20 lowest energy unique conformers were averaged using Boltzmann weighting from the energies obtained from optimization and plotted using a scaling factor of 0.983. We compared their Boltzmann summation with the observed spectra of (−)-menthyl 1 (Figure 2). After X-axis scaling of the computed spectra, there was strong correlation to the experimental of both IR and VCD with a neighborhood similarity (Sfg)²¹ of 90.4 for IR and 78.6 for VCD and an enantiomeric similarity index (ESI)²¹ of 70.8. Given this agreement, the absolute configuration of (−)-menthyl 1 was assigned as 1R,2S,5R.

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).

Analysis of 2D NMR data including NOESY confirmed the correct relative stereochemistry (SI, Table S1, Figures S14–S17). Both data sets suggest chirality at the C1 position matching the reported structure,¹¹ for which stereochemical assignment is currently lacking in the literature. Our results also indicate that reduction favors formation of the desired amine, suggesting the isopropyl group provides sufficient steric bulk to confer stereoselectivity. Determining the stereochemistry at this position is crucial to explore agonist vs antagonist binding epitopes that could explain differences in functional activity because this chiral center determines orientation of the biphenyl moiety in the TRPM8 binding site.

To assess (−)-menthyl 1 for species-dependent inhibition of both menthol and icilin-mediated responses, we used calcium imaging at both human and rat TRPM8 carried out under the same conditions. We used the Fluo-4 NW probe that allows us to measure maximum responses after addition of the agonist, without saturating the signal, contrary to the Fura-2 probe. We first determined whether rat and human TRPM8 elicit different sensitivities to menthol and icilin in this assay. To this end, we obtained EC₅₀ values of 81 ± 17 and 107 ± 8 μM for menthol stimulation of human and rat TRPM8 (Figures 3a and 4a), respectively. For icilin, we obtained EC₅₀ values of 526 ± 24 nM and 554 ± 12 nM at human and rat TRPM8 (Figures 3a and 4a), respectively. These potencies are similar to those determined in CHO cells transfected with mouse TRPM8 using calcium imaging²² and trigeminal neurons using patch-clamp electrophysiology.⁴ Importantly, our measured EC₅₀ values demonstrate equivalency for these agonists at both orthologues. In contrast, at hTRPM8 channels (−)-menthyl 1 inhibits both menthol and icilin responses at higher concentrations than the rat orthologue, with IC₅₀ values of 805 ± 200 nM and 1.8 ± 0.6 μM, respectively (Figure 3b). At rTRPM8, 1 inhibits responses by both agonists more potently, with IC₅₀ values of 117 ± 18 nM and 521 ± 20 nM against menthol and icilin stimulation, respectively (Figure 4b).

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).

The antagonist effects of 1 (0.1, 1, 10 μM) against menthol stimulation at both human and rat TRPM8 are significantly different (P < 0.001 and P < 0.0001, two-way ANOVA followed by Bonferroni’s test) (Figure 5). (−)-Menthyl 1 also exerts significantly different antagonist effects against icilin at both orthologues, at concentrations up to 10 μM (P < 0.01, two-way ANOVA followed by Bonferroni’s test). Taken together, our data demonstrates that the potent antagonist effects of 1 observed at the rat orthologue result from species differences between rat and human TRPM8 channels. We propose that (−)-menthyl 1 can be used as a chemical tool to study species differences between rat and human TRPM8.

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.

Other ligands with appreciable TRPM8 activity show species-dependent effects in vitro. The monoterpene eucalyptol activates hTRPM8 at an EC₅₀ of 145.6 μM, although rodent orthologues (rat and mouse) respond to eucalyptol only at higher EC₅₀ values of 1.21 mM and 924.5 μM, respectively.²³ Menthol also displays species-dependent effects, activating chicken TRPM8 more potently than the mouse orthologue in both Ca²⁺ imaging and patch clamp assays.²⁴ On the other hand, icilin does not activate cTRPM8-expressing oocytes or HEK293 cells at a 10-fold higher concentration than required for a peak agonist response at rTRPM8.

Differential sensitivity among TRPM8 orthologues to icilin stimulation is attributed to Ala796 in the VSLD S3 helix, as Ala → Gly exchange confers avian sensitivity.²⁵ Yin et al. introduced the same mutation at the equivalent position (805) in TRPM8_FA to confer icilin sensitivity and elucidated the icilin-bound structure via cryo-EM (PDB 6NR3).⁵ Our previous MD studies of 1 carried out in the VSLD of our hTRPM8 homology model suggest that 1 binds in a pocket defined partially by Ala805.¹⁶ This suggests residues responsible for differential sensitivities to compounds such as 1 may reside in the VSLD. On the other hand, sequence alignment of TRPM8 orthologues within the transmembrane domain, containing the VSLD, S5–S6 helices within the pore domain (PD), as well as the pore helix (PH) and TRP helix,²⁶ show that hTRPM8 differs from the rat orthologue at 10 unique positions. Eight of these residues are outside the VSLD binding site, in the upper S2 helix (Pro766, Val770, Ser773) and lower S2 helix (Val788). Remaining residues are distant to the binding cavity, in the loop connecting the PH and S6 helices (Gly921, Ala927, Thr932, His946) and the loop connecting the S6 and TRP helices (Thr985). Ser1007 (TRP helix) is the only point of differentiation close to the binding site for the two orthologues (Asn1007 in rTRPM8).

We previously reported MD simulations of 1 in our hTRPM8 model.¹⁶ To explore the potential binding mode of 1 at rTRPM8 and provide structural context for our experimental data, we built a model of the rat orthologue using similar methodology as we reported previously.¹⁶ Briefly, we built the rTRPM8 homology model based on the cryo-EM structure of TRPM8_FA (PDB 6BPQ), which shares 80% sequence identity to rTRPM8. A calcium ion was placed in the VLSD, coordinating to Glu782, Gln785, Asn799, and Asp802, in agreement with our previous human model and calcium-bound structure of great tit Parus major TRPM8.⁷ We then performed 100 ns MD simulations for 1 in rTRPM8 using our reported protocol.¹⁶ Our results show that both root-mean-square deviation (RMSD) of rTRPM8 (∼2.0 Å) and (−)-menthyl 1 (∼4.0 Å) kept stable after 60 ns during the simulation, indicating that the time scale of 100 ns is reasonable (Figure 6c). Interactions between 1 and some residues can be observed during the MD. For example, the carbonyl of 1 interacts with the guanidine moiety of a conserved Arg1008 (TRP helix, 3.1 Å) (Figure 6b). The isopropyl group of the (−)-menthyl core scaffold forms hydrophobic contacts with the backbone of Arg842 (S4, 3.7 Å) and Leu778 (S2, 3.9 Å). To explore the contribution of these residues to the binding of 1, we decomposed the free energy using the MM/GBSA method (Figure 6d). From the energy decomposition, we found that Arg1008 contributed greatly to binding of 1.

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.

We then compared the predicted binding mode of 1 at rTRPM8 to our previous MD studies of this antagonist in our hTRPM8 homology model¹⁶ via superimposition. As shown in Figure 7, 1 binds to rTRMP8 and hTRPM8 (gray) in a similar conformation, and key residues are oriented identically, with the exception of Phe779 and Ser/Asn1007. Arg1008 forms key interactions to 1 in both models, although at rTRPM8 this residue hydrogen-bonds to the 1 carbonyl via its guanidine moiety (Figure 6b), versus forming only hydrophobic contacts with the (−)-menthyl group via the three-carbon backbone at hTRPM8. We posit that at rTRPM8, Arg1008 is free to sample its conformational space due in part to the neighboring Asn1007 residue located 90° away, which forms hydrogen bonds to Glu1004 (2.9 Å). In contrast, at hTRPM8, the guanidine of Arg1008 is situated near Ser1007 (Figure 7), within 2.85 Å, and may anchor Arg1008 in position to form only hydrophobic interactions with 1. These studies suggest that Ser/Asn1007 influences the binding pockets of both orthologues via spatial control of a conserved Arg1008 residue that forms key interactions with 1.

Figure 7.

Superimposition of the binding mode (post-MD) of (−)-menthyl 1 in rTRPM8 (this work) and hTRPM8.¹⁶ The MD pose of 1 and hTRPM8 is depicted in gray.

Rodent and human TRPM8 also exhibit species differences in modulation by the accessory protein PIRT (phosphoinositide-interacting regulator of TRP). hPIRT attenuates menthol and cold-evoked hTRPM8 currents, whereas mPIRT increases the equivalent responses.²⁷ Innate differences in voltage-stimulated current densities have also been identified between orthologues.²⁷ Taken together, compounds such as 1 will be useful to identify mechanisms that underlie TRPM8 species-dependent effects and are anticipated to streamline drug discovery.

Whole-cell patch-clamp is the gold standard for deciphering ion channel molecular pharmacology, as it is a direct measurement of current flux through the channel, whereas Ca²⁺ flux assays are indirectly coupled to channel function, typically through a fluorophore. To validate our calcium fluorescent-based assays at hTRPM8, we performed electrophysiology of variable concentrations of 1 against 500 μM menthol (Figure 8) using our reported protocol.¹⁶ In correlation with calcium imaging, whole-cell electrophysiological recordings showed that the antagonist activity of 1 at hTRPM8 is in the high nM range, with an IC₅₀ value of 700 ± 200 nM. Notably, the IC₅₀ value of 1 at hTRPM8 surpasses that of benzoate 2 (IC₅₀: 2 ± 1 μM)¹² in the same assay against the effect of 100 μM menthol, although the TRPM8 orthologue used was not reported. To our knowledge, 1 represents one of the most potent natural product-derived antagonists at hTRPM8 that possesses a strictly menthol chemotype.

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.

Limited selectivity data are available for 1. This antagonist is described as 200-fold selective for rTRPM8 versus rTRPA1 based on its measurable TRPA1 agonist activity (EC₅₀: 4.1 ± 0.01 μM). This may arise from the (−)-menthyl portion of 1 because menthol acts as an agonist at mouse TRPA1 (EC₅₀: 95 ± 15 μM)²⁸ and exerts significant TRPA1-mediated sensory effects in vivo.²⁹ Potential antagonist effects of 1 at this subtype were not determined.¹¹ In addition, 1 does not stimulate hTRPV1, although the concentration used in this assay was not disclosed, further arguing for antagonist studies at this subtype. Use of different orthologues (rTRPA1 and hTRPV1) for selectivity profiling of 1 may also pose further limitations. In light of sparse available selectivity data, the intrinsic (agonist) and antagonist activity of 1 at hTRPA1 and hTRPV1 was determined by Ca²⁺ flux experiments, using the standard agonists allyl isothiocyanate (AITC) and capsaicin and antagonists ruthenium red and capsazepine for each subtype, respectively. Experiments were conducted in HEK293 cells stably expressing each subtype, as we previously described.¹⁶ We found 1 has negligible intrinsic (agonist) and antagonist activity in Ca²⁺ flux assays at both subtypes, at concentrations ≥400-fold higher than its hTRPM8 IC₅₀ value (Table 1). Specifically, 1 did not activate either subtype >25% when normalized to the E_max concentration of AITC (300 μM) and capsaicin (3 μM) at all concentrations tested. Similarly, 1 did not inhibit either subtype >25% when challenged with the EC₈₀ concentrations of AITC and capsaicin (10 and 0.1 μM, respectively). Thus, this antagonist demonstrates at least 400-fold selectivity for hTRPM8 versus other TRP subtypes when compared across uniform (human) orthologues. Although 1 does not achieve high potency at hTRPM8, it is well-characterized for its antagonist activity versus other menthol-based antagonists (Figure 1) and is ≥400-fold selective. These data suggest that 1 has worthy attributes to serve as a menthol-based antagonist probe to investigate TRPM8-mediated phenomena.

Table 1. Intrinsic and Antagonist Functional Activities of (−)-Menthyl 1 at hTRPA1 and hTRPV1 Receptors, Determined in Ca²⁺ Flux Assays.

compd	concentration tested (×TRPM8 IC50)	hTRPA1% activation (n = 3)a	hTRPA1% inhibition (n = 3)b	hTRPV1% activation (n = 3)a	hTRPV1% inhibition (n = 3)b
1,	66.7 μM (83-fold)	–0.83 ± 0.78	18.3 ± 2.66	1.3 ± 0.22	–3.6 ± 0.77
hTRPM8 IC50 (menthol): 805 ± 200 nM	133 μM (165-fold)	–0.20 ± 0.49	24.9 ± 1.67	3.6 ± 0.40	–7.44 ± 5.31
	200 μM (248-fold)	0.95 ± 0.80	18.7 ± 3.78	4.8 ± 0.20	–5.89 ± 2.96
	267 μM (332-fold)	2.1 ± 0.43	16.8 ± 3.20	4.57 ± 2.06	6.79 ± 6.71
	333 μM (414-fold)	3.4 ± 0.40	11.9 ± 3.10	7.9 ± 0.15	7.64 ± 4.47

^aData normalized to concentration corresponding to E_max value of TRPA1 and TRPV1 agonists AITC (300 μM) and capsaicin (3 μM), respectively.

^bData normalized to EC₈₀ of AITC (10 μM) and capsaicin (0.1 μM).

We present (−)-menthyl 1 as a well-characterized and selective menthol-based TRPM8 antagonist at the human orthologue for use as a chemical tool based on a menthol chemotype. Notably, (−)-menthyl 1 demonstrates a greater degree of TRPM8 inhibition in whole-cell patch-clamp electrophysiology assays measuring direct blockade of the channel than another reported menthol-based antagonist, benzoate 2.¹² Previous studies describe 1 as a highly potent TRPM8 antagonist at the rat orthologue; however, we show species-dependent chemical regulation of rat versus human TRPM8 using this antagonist. (−)-menthyl 1 can be used as a tool to further study TRPM8 species differences and strongly supports that in vitro screening of ligands for TRPM8 activity should be performed using the human orthologue before conducting preclinical studies in rodents (rats). Initial in vitro calcium flux studies conducted only at rTRPM8 and subsequent in vivo studies in rats or mice may be misleading when considering the clinical translational potential of a chemical series. Similar cautions have been put forth for TRPA1³⁰ and TRPV1.³¹ These studies should inform early drug discovery efforts to discover novel menthol-based TRPM8 antagonists as pharmacotherapies.

Glossary

Abbreviations

EDCI

N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide

HOBt

1-hydroxybenzotriazole hydrate

gHSQCAD

gradient-enhanced heteronuclear single quantum coherence with adiabatic pulses

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00001.

• Compound synthesis, functional assays, molecular modeling, GC-MS traces, NMR spectra, and VCD spectra (PDF)

• Lay summary (PDF)

Author Contributions

V.B.J. and D.A.A. contributed equally. The manuscript was written through contributions of all authors, who have approved the final version of the manuscript.

The authors declare no competing financial interest.