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

Article abstract of DOI:10.1021/acsmedchemlett.1c00001

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.

Full text of DOI:10.1021/acsmedchemlett.1c00001

pubs.acs.org/acsmedchemlett
Letter
Structural and in Vitro Functional Characterization of a Menthyl
TRPM8 Antagonist Indicates Species-Dependent Regulation
V. Blair Journigan,* David Alarcón-Alarcón, Zhiwei Feng, Yuanqiang Wang, Tianjian Liang,
Denise C. Dawley, A. R. M. Ruhul Amin, Camila Montano, Wade D. Van Horn, Xiang-Qun Xie,
Antonio Ferrer-Montiel, and Asia Fernández-Carvajal
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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
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
0.6 μM, respectively, while more potently inhibiting
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.
KEYWORDS: TRPM8, menthol, antagonist, species, chemical regulation
Complementary agonist derivatives within this series were not
reported.10
ransient receptor potential melastatin 8 (TRPM8) is a
Tversatile 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 (TRPM8FA) bound to WS-12,5 backed by
radioligand displacement and mutagenesis data.6 Many
structurally disparate, non-menthol TRPM8 antagonist chemo-
types 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
(−)-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
Received: January 11, 2021
Accepted: March 26, 2021
Published: March 31, 2021
https://doi.org/10.1021/acsmedchemlett.1c00001
© 2021 American Chemical Society
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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 111 via (−)-menthyl-
amine (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 stereo-
chemistry 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
Figure 1. Monoterpene-derived TRPM8 antagonists. (a) Ref 11. (b)
Ref 12, TRPM8 orthologue not specified. (c) Ref 13. (d) Ref 14.
The absolute stereochemical configuration of 1 was
established by VCD20 and H, 13C, gCOSY, NOESY, and
1
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.
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 (−)-menth-
yl 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.
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 non-
selective 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, trans-
membrane helices S1−S4) binding site as the methoxyphenyl
moiety of agonist WS-12, with the 1′-phenyl ring extending into
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
a
Scheme 1
a
(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%.
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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).
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.
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
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 electro-
physiology.4 Importantly, our measured EC50 values demon-
strate equivalency for these agonists at both orthologues. In
and 521
20 nM against menthol and icilin stimulation,
respectively (Figure 4b).
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.
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
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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).
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 TRPM8FA
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 TRPM8FA
(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
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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.
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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 TRPM8FA (PDB 6BPQ) as a template.
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.
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
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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.
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-depend-
ent 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.
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.
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 ortho-
logues.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 electro-
physiology 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
derived antagonists at hTRPM8 that possesses a strictly menthol
chemotype.
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 Emax
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
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-
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Table 1. Intrinsic and Antagonist Functional Activities of (−)-Menthyl 1 at hTRPA1 and hTRPV1 Receptors, Determined in Ca2+
Flux Assays
concentration tested
(×TRPM8 IC50)
hTRPA1% activation
hTRPA1% inhibition
hTRPV1% activation
hTRPV1% inhibition
a
b
a
b
compd
(n = 3)
(n = 3)
(n = 3)
(n = 3)
1,
66.7 μM (83-fold)
133 μM (165-fold)
200 μM (248-fold)
267 μM (332-fold)
333 μM (414-fold)
−0.83 0.78
−0.20 0.49
0.95 0.80
2.1 0.43
18.3 2.66
24.9 1.67
18.7 3.78
16.8 3.20
11.9 3.10
1.3 0.22
3.6 0.40
4.8 0.20
4.57 2.06
7.9 0.15
−3.6 0.77
−7.44 5.31
−5.89 2.96
6.79 6.71
7.64 4.47
hTRPM8 IC50 (menthol):
805 200 nM
3.4 0.40
a
Data normalized to concentration corresponding to Emax value of TRPA1 and TRPV1 agonists AITC (300 μM) and capsaicin (3 μM),
b
respectively. Data normalized to EC80 of AITC (10 μM) and capsaicin (0.1 μM).
Authors
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.
David Alarcón-Alarcón − IDiBE: Instituto de Investigación,
Desarrollo e innovación en Biotecnología Sanitaria de Elche,
Universitas Miguel Hernández, 03202 Elche, Spain
Zhiwei Feng − Department of Pharmaceutical Sciences and
Computational Chemical Genomics Screening Center, School
of Pharmacy, NIDA National Center of Excellence for
Computational Drug Abuse Research, and Drug Discovery
Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
15261, United States; orcid.org/0000-0001-6533-8932
Yuanqiang Wang − Department of Pharmaceutical Sciences
and Computational Chemical Genomics Screening Center,
School of Pharmacy, NIDA National Center of Excellence for
Computational Drug Abuse Research, and Drug Discovery
Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
15261, United States
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.
Tianjian Liang − Department of Pharmaceutical Sciences and
Computational Chemical Genomics Screening Center, School
of Pharmacy, NIDA National Center of Excellence for
Computational Drug Abuse Research, and Drug Discovery
Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
15261, United States
Denise C. Dawley − Department of Pharmaceutical Sciences,
School of Pharmacy, Marshall University, Huntington, West
Virginia 25755, United States
A. R. M. Ruhul Amin − Department of Pharmaceutical Sciences,
School of Pharmacy and Department of Biomedical Sciences,
Joan C. Edwards School of Medicine, Marshall University,
Huntington, West Virginia 25755, United States
Camila Montano − The School of Molecular Sciences, The
Virginia G. Piper Center for Personalized Diagnostics,
Biodesign Institute, and The Magnetic Resonance Research
Center, Arizona State University, Tempe, Arizona 85287,
United States
Wade D. Van Horn − The School of Molecular Sciences, The
Virginia G. Piper Center for Personalized Diagnostics,
Biodesign Institute, and The Magnetic Resonance Research
Center, Arizona State University, Tempe, Arizona 85287,
United States; orcid.org/0000-0002-2493-5578
Xiang-Qun Xie − Department of Pharmaceutical Sciences and
Computational Chemical Genomics Screening Center, School
of Pharmacy, NIDA National Center of Excellence for
Computational Drug Abuse Research, and Drug Discovery
Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
15261, United States
ASSOCIATED CONTENT
■
sı
* Supporting Information
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 INFORMATION
■
Corresponding Author
V. Blair Journigan − Department of Pharmaceutical Sciences,
School of Pharmacy and Department of Biomedical Sciences,
Joan C. Edwards School of Medicine, Marshall University,
Huntington, West Virginia 25755, United States;
orcid.org/0000-0002-4582-5095; Email: journigan@
marshall.edu
Antonio Ferrer-Montiel − IDiBE: Instituto de Investigación,
Desarrollo e innovación en Biotecnología Sanitaria de Elche,
Universitas Miguel Hernández, 03202 Elche, Spain
765
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Letter
benzimidazole-containing transient receptor potential melastatin 8
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Asia Fernández-Carvajal − IDiBE: Instituto de Investigación,
Desarrollo e innovación en Biotecnología Sanitaria de Elche,
Universitas Miguel Hernández, 03202 Elche, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.1c00001
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.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank T. Voets (KU Leuven) for supplying the HEK293-
hTRPM8 stable cell line. We thank T. Trejos and W. Feeney
(West Virginia University) for GC-MS data and G. Rankin
(Marshall University) for critical reading of the manuscript. This
work was supported by the National Institutes of Health
NIGMS U54GM104942−04 (V.B.J.) and R01GM112077
(W.V.H.), NIDA P30DA035778 (X-Q.X,); and by the
Ministerio de Ciencia, Innovación y Universidades (MICIU)
RTI2018-097189-B-C21 (A.F.M., A.F.C.).
ABBREVIATIONS
■
EDCI, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide;
HOBt, 1-hydroxybenzotriazole hydrate; gHSQCAD, gradient-
enhanced heteronuclear single quantum coherence with
adiabatic pulses
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