Structure-Based Design of Novel Biphenyl Amide Antagonists of Human Transient Receptor Potential Cation Channel Subfamily M Member 8 Channels with Potential Implications in the Treatment of Sensory Neuropathies

Article abstract of DOI:10.1021/acschemneuro.9b00404

Structure-activity relationship studies of a reported menthol-based transient receptor potential cation channel subfamily M member 8 channel (TRPM8) antagonist, guided by computational simulations and structure-based design, uncovers a novel series of TRP

Full text of DOI:10.1021/acschemneuro.9b00404

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Article
Structure-based design of novel biphenyl amide antagonists of human
Transient Receptor Potential Cation Channel Subfamily M Member 8 channels
(TRPM8) with potential implications in the treatment of sensory neuropathies
V. Blair Journigan, Zhiwei Feng, Saifur Rahman, Yuanqiang Wang, A.R.M. Ruhul Amin, Colleen E. Heffner,
Nicholas Bachtel, Siyi Wang, Sara Gonzalez-Rodriguez, Asia Fernández-Carvajal, Gregorio Fernández-
Ballester, Jacob K. Hilton, Wade D. Van Horn, Antonio Ferrer-Montiel, Xiang-Qun Xie, and Taufiq Rahman
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00404 • Publication Date (Web): 18 Dec 2019
Downloaded from pubs.acs.org on December 18, 2019
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TITLE PAGE:
Structure-based design of novel biphenyl amide antagonists of human Transient Receptor
Potential Cation Channel Subfamily M Member 8 channels (TRPM8) with potential
implications in the treatment of sensory neuropathies
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V. Blair Journigan *,†,‡,§, Zhiwei Feng §, ∫, ∆, ┴, Saifur Rahman §, ‖, Yuanqiang Wang §, ∫, ∆, ┴, A.R.M.
Ruhul Amin †, ‡, Colleen E. Heffner , Nicholas Bachtel , Siyi Wang ∫, ∆, ┴, Sara Gonzalez-Rodriguez
†
†
⸠
Asia Fernández-Carvajal , Gregorio Fernández-Ballester , Jacob K. Hilton ⸠ , ⸠ , ⌡, Wade D.
⸠
⸠
,
Van Horn ⸠ , ⸠ , ⌡, Antonio Ferrer-Montiel , Xiang-Qun Xie
⸠
*, ∫, ∆, ┴
, Taufiq Rahman *, ‖
†
Department of Pharmaceutical Sciences, School of Pharmacy, Marshall University, Huntington,
West Virginia 25755, United States
‡
Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University,
Huntington, West Virginia 25755, United States
‖
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, United
Kingdom
∫
Department of Pharmaceutical Sciences and Computational Chemical Genomics Screening Center,
School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States
∆
NIDA National Center of Excellence for Computational Drug Abuse Research, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261, United States
┴
Drug Discovery Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States
⸠
The School of Molecular Sciences, Arizona State University, Tempe, Arizona, 85287, United
States
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⸠
the Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State
University, Tempe, Arizona 85281
⌡The Magnetic Resonance Research Center, Arizona State University, Tempe, Arizona 85287
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⸠
IDiBE: Instituto de Investigación, Desarrollo e innovación en Biotecnología sanitaria de Elche,
Universitas Miguel Hernández, 03202 Elche, Spain
§
These authors contributed equally to this work
*
Correspondence and requests for materials should be addressed to V.B.J. (email:
journigan@marshall.edu) or X.-Q.X. (email: xix15@pitt.edu) or to T.R. (email: mtur2@cam.ac.uk)
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ABSTRACT
SAR studies of a reported menthol-based TRPM8 antagonist, guided by computational simulations
and structure-based design, uncovers a novel series of TRPM8 antagonists with >10-fold selectivity
versus related TRP subtypes. Spiro[4.5]decan-8-yl analog 14 inhibits icilin-evoked Ca2+ entry in
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HEK-293 cells stably expressing human TRPM8 (hTRPM8) with an IC50: 2.4 ± 1.0 nM, while in
whole-cell patch-clamp recordings, this analog inhibits menthol-evoked currents with an hTRPM8
IC50: 64 ± 2 nM. Molecular dynamics (MD) simulations of compound 14 in our homology model of
hTRPM8 suggests that this antagonist forms extensive hydrophobic contacts within the orthosteric
site. In the wet dog shakes (WDS) assay, compound 14 dose-dependently blocks icilin-triggered
shaking behaviors in mice. Upon local administration, compound 14 dose dependently inhibits cold
allodynia evoked by the chemotherapy oxaliplatin in a murine model of peripheral neuropathy at
microgram doses. Our findings suggest that 14 and other biphenyl amide analogs within our series
can find utility as potent antagonist chemical probes derived from (−)-menthol, as well as small
molecule therapeutic scaffolds for chemotherapy-induced peripheral neuropathy (CIPN) and other
sensory neuropathies.
KEYWORDS
Menthol, molecular dynamics simulations, chemical probe, TRPM8, sensory neuropathy, TRP
channel
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INTRODUCTION
TRPM8, often referred to as the ‘cold receptor’, is a polymodal nociceptor of the somatosensory
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nervous system and a major sensor of cold nociception in humans. TRPM8 is expressed, albeit
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differentially compared to other temperature sensitive TRP channels TRPA1 and TRPV1, in
primary sensory neurons (Aδ and C-fibers) of the dorsal root ganglia (DRG) and trigeminal ganglia
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(TG), as well as in some other non-neuronal tissues. Within primary sensory neurons, TRPM8 is
present on both the peripheral and central nerve terminals, supporting a role in the somatosensory
3
and central nervous systems. This cold-sensing nociceptor initiates sensory nerve impulses after
o
activation by mildly-cool temperatures (15-25 C), chemical agents such as (−)-menthol (1R,2S,5R)-
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-methyl-2-(1-methylethyl)-cyclohexanol), the ‘super-agonist’ icilin (3-(2-hydroxyphenyl)-6-(3-
nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one, also known as AG 3-5), and the endogenous lipid
.4 Maximal stimulation of TRPM8 by menthol occurs via a ligand stoichiometry of four
PIP
2
menthol molecules each occupying a monomer of one functional TRPM8 tetramer.5
TRPM8 appears to be a potential target for multiple therapeutic interventions. TRPM8 agonists
have been explored for benign prostatic hyperplasia and prostate cancer, while antagonists have
spurred more interest, for cold hypersensitivity in neuropathic pain, and migraine, among others.6
A number of proprietary TRPM8 antagonists, discovered largely through high throughput
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screening of in-house chemical libraries, are known. However, the precise molecular details of
how these antagonists bind to human TRPM8 and subsequently influence its gating mechanism, as
well as their underlying pharmacophores, remains to be elucidated, especially in the wake of recent
success with TRPM8 and TRP channel structural biology revealed through cryo-electron
microscopy (cryo-EM). 7-8
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In the TRPM8-mediated Ca2+ flux assay, well-known TRPM8 antagonists appear to manifest
either modest or variable potencies. For example, quinoline-based PF-05105679 (Pfizer, Phase 1,
NCT01393652) inhibits the effects of voltage- and WS-12-mediated activation at human TRPM8
with IC50 values of 103 ± 29.4 nM and 181 ± 7.21 nM, 1, 9 while 5,6,7,8-tetrahydro-1,7-
naphthyridine analog AMG2850 (Amgen) exhibits IC50 values ranging from 7 -156 nM, against
menthol, icilin and cold activation at rat and human TRPM8.10-11 2-benzyloxy-benzoic acid amide
derivative AMTB (Bayer) affords weaker inhibition against icilin-mediated responses at human
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TRPM8, with a pIC50: 6.23 ± 0.02. To date, no TRPM8 antagonists have advanced to clinical use;
for example, PF-05105679 does not possess a measurable therapeutic index at high doses (600 mg
and 900 mg) required to achieve unbound plasma concentrations greater than its IC50 value.1
Likewise, Phase 1 studies of nicotinic acid analog AMG 33313 (Amgen, NCT02132429) were
terminated for undisclosed reasons. There remains a need for introducing more TRPM8 antagonists
that are potent, reasonably specific and thereby can sustain the possibility of successfully passing
through clinical trials after successful preclinical development.
Moreover, a variety of both agonist and antagonist scaffolds with wide chemical diversity have
been used to decipher the role of TRPM8 in various disease states, such as orofacial and
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neuropathic pain, overactive bladder (OAB) and painful bladder syndrome, diseases involving
thermoregulation,15 oral squamous cell carcinoma,16 nicotine addictive behaviors,17 and chronic
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obstructive pulmonary disease. In some cases, use of these ligands as tool molecules to probe the
pharmacology of TRPM8 have led to conflicting data, particularly in determining its role in chronic
neuropathic pain.3, 14, 19 Highly potent chemical probes based on the cognate ligand (−)-menthol
with an antagonist profile, are needed to revisit the TRPM8 field from an early drug discovery
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perspective, which currently lacks such small molecules of this chemotype. De novo design of these
scaffolds is enabled by contemporary knowledge of the TRPM8 small molecule binding site.
Recent cryo-EM structures of both apo- and agonist-bound avian TRPM8 (TRPM8FA, PDB
codes 6BPQ (4.1 Å), 6NR2 (4 Å) and 6NR3 (3.4 Å)), 7-8 coupled with supporting in vitro and in
vivo mutagenesis studies at mouse, rat, squirrel and human orthologs, point to a definitive binding
site for the prototypical ligands menthol and icilin that is likely decoupled from cold sensing.
Mutagenesis studies have suggested a conserved Tyr745, located in the middle of the
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transmembrane S1 helix of the voltage-sensor-like domain (VSLD), is crucial for both menthol
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binding, as determined by radioligand displacement studies using [ H]-menthol,
as well as
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efficacy. These studies are consistent with the structure of avian TRPM8 complexed with menthol
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analog WS-12 (PDB 6NR2). Constructs of the isolated S1-S4 sensing domain also suggest this
region to be crucial for menthol recognition, as determined by microscale thermophoresis and
NMR.22 Other menthol-sensitive residues include Tyr1005 and Leu1009, both of which are
conserved21 and located in the TRP helix transmembrane domain, near the cytosolic domain
interface. The positional equivalent of these two residues can be seen in the WS-12 bound structure
(
Tyr1004 and Leu1008 in TRPM8FA). This pocket also binds icilin with similar residues, including
Tyr745 8, 21 and Leu1009; 21 as well as Gly805, Asn799 and Asp802 in the S3 helix.8, 23 The cryo-
EM structure of icilin-bound avian TRPM8 reinforces the notion that Tyr745, a residue conserved
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across all TRPM subtypes, is a common TRPM8 residue for both menthol and icilin recognition,
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despite their different chemotypes, while Asn799 and Asp802 are necessary for Ca -dependent
icilin binding, along with Gln782 and Glu785. Additional icilin-binding residues line the orthosteric
site within the VSLD and TRP helix, including Phe838, Arg841, His844 and Tyr1004 (Phe839,
8
Arg842, His845 and Tyr1005 at hTRPM8, respectively). This ligand-binding site also overlaps
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with a crucial voltage- and cold-sensitive residue: a conserved Arg841 in the S4 helix (Arg842 at
hTRPM8), 20 indicating that the ability of TRPM8 to function as a polysensor of ligand and voltage
stimuli is derived in part from this common site, comprised of S1-S4 helices within the VSLD.
Transgenic models of hibernating ground squirrels and rats implicate residues responsible for cold
vs. icilin activation, scattered throughout the VSLD in the loops linking the TM helices, at position
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26 (pre-S1 domain), 762 (S1-S2 loop), 819 (S3-S4 loop), and 927, 946, 947 in the loop connecting
the pore helix (PH) and S6 helix.25 From the sequence alignments of rat, squirrel and human
TRPM8 orthologs,7, 25 four of the six cold-conferring residues in rats are conserved in the human
ortholog (Tyr726, Ser762, Ser819 and Asn947), raising the possibility that ligand vs. cold
activation in humans occurs at topographically distinct regions. On the other hand, the remaining
two mismatched residues are conserved in humans and cold-tolerant hibernating ground squirrels
(
Ala927, His946), suggesting either that these residues do not support a cold phenotype, or
contribute to a cold response via allosteric regulation.
In light of these recent developments in the TRPM8 field, we hypothesize that reported small
molecule TRPM8 antagonists with both structural similarities and conformational rigidity can
reveal similar molecular determinants for ligand recognition, via their poses from molecular
dynamics (MD) simulations in our human TRPM8 homology model based on the avian cryo-EM
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structure TRPM8FA (PDB 6BPQ, ~4.1 Å). In turn, these insights can allow for the design of novel
TRPM8 chemical probes and therapeutic scaffolds for TRPM8-mediated sensory neuropathies. We
employed this structure-based approach using reported, selective TRPM8 antagonists such as (−)-
menthyl 126 (rTRPM8 IC50: 20 ± 2 nM; 50 ± 10 nM) and 5,6,7,8-tetrahydro-1,7-naphthyridine
AMG2850,10, 11 (Figure 1) containing similar hydrophobic moieties such as (−)-menthyl and
trifluoropropanyl groups, as well as amides/ureas, and cycloalkyl and aromatic 6-membered rings.
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These moieties are also present in other well-known TRPM8 antagonists such as 2-benzyloxy-
benzoic acid amide derivative AMTB, as well as TRPV1 antagonists with bifunctional TRPM8
antagonist activity, such as pyridylpiperazine carboxamide BCTC and urea SB-452533 (Figure 1).
Using this approach, we herein report the discovery of a novel biphenyl amide series with potent
TRPM8 antagonist activity at hTRPM8, and tractable SAR to inform our drug discovery efforts.
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Figure 1. Reported TRPM8 antagonists. IC50 values (antagonists) determined in the Ca flux assay
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against the effect of menthol and/or icilin. Ref. , Ref. , Ref. , Ref , Ref , Ref , Ref.
29, h
Ref , Ref. . icilin: 3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-3,4-dihydropyrimidin-2(1H)-one.
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i
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PHARMACOLOGICAL CHARACTERIZATION
Evaluation of TRPM8 agonist and antagonist activity using Ca imaging. Analogs 2-15 were
2
+
2
+
evaluated for their ability to modulate TRPM8 functions with Fura-2 based Ca imaging in HEK-
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93 cells stably expressing hTRPM8. The latter were a kind gift from Thomas Voets (Laboratory of
Ion Channels Research, KU Leuven, Belgium). The concentration of intracellular free Ca2+
2
+
) was monitored as a Fura-2 fluorescence ratio (F355/F380). 32-34
(
[Ca ]
i
2
+
2+
33
The analogs were assessed against Ca influx ([Ca ] ) evoked by icilin (AG-3-5). The latter
i
2+
was used at 500 nM, as maximum Ca entry was found to occur at this concentration in our dose-
response study. We chose icilin to stimulate TRPM8 in our assay since it was markedly more potent
(
EC50: ~70 nM vs 6 µM) than (−)-menthol in our initial concentration-response studies (Figures
S2a-b) and this broadly agrees with previous reports.33-34
2
+
TRPV1 and TRPA1 agonist and antagonist Ca flux assay (selectivity profiling). Compounds
with hTRPM8 IC50 values ≤ 100 nM were evaluated for off-target effects at two related
temperature-sensitive subtypes hTRPA1 and hTRPV1, using high-throughput FLIPR-based Ca2+
flux assays at Eurofins Pharma Discovery Services (St. Charles, MO). Compounds were evaluated
at single concentrations corresponding to approximately 90% target coverage at TRPM8, followed
by single point assays at 10-fold higher concentrations to demonstrate >10-fold selectivity. Agonist
(
intrinsic) activity was evaluated in recombinant HEK293 cell lines stably expressing either human
TRPA1 or TRPV1, and normalized to the maximum effect (Emax) of the agonists allyl
isothiocyanate or capsaicin, respectively. Antagonist activity was evaluated by measuring the
2
+
ability of the analogs to inhibit AITC- or capsaicin-induced increases in cytosolic [Ca ] by
2
+
inhibiting Ca influx in these cell lines, and compared to the effect of the ruthenium red (TRPA1)
and capsazepine (TRPV1) used as reference antagonists.
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Whole-cell patch clamp electrophysiology of 14. The functional activity of 14 in calcium flux
assays was confirmed with whole-cell patch-clamp electrophysiology. Human TRPM8 whole-cell
patch-clamp electrophysiology was done at 23 ± 1 °C on human embryonic kidney (HEK) 293 cells
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(
ATCC CRL-1573) authenticated by polymorphic genetic marker testing (DNA Diagnostics Center
Medical) as described previously.22, 35 A series of 500 µM menthol and variable concentration (from
0
to 1 µM) 14 solutions were used. Cellular current response was measured at each concentration of
1
4. To determine the IC50, normalized currents from six cells were averaged at each concentration
of antagonist, and the standard error of the mean was calculated at each point. The data were fit to a
single site competition model to obtain IC50. The IC50 whole-cell patch-clamp experiments were
deliberately performed against saturating (500 µM) menthol concentrations, because menthol
2+
currents are not Ca dependent and give reproducible and robust currents. Similarly, studying the
inhibitory effects of 14 against saturating menthol, allows direct comparison to other published
TRPM8 antagonism studies that used these conditions in whole-cell patch-clamp measurements.32,
36-38
In vitro metabolic stability. 14 was evaluated for metabolic stability in mouse liver microsomes by
Absorption Systems (Exton, PA), by determining the percent remaining at 0, 10, 20, 30 and 60
minutes when incubated with mouse liver microsomes in the presence of NADPH. The half-life
(
(
t
1/2) and in vitro intrinsic clearance (CLint) of 14 was calculated, in addition to the control
testosterone).
RESULTS AND DISCUSSION
The available functional data on known TRPM8 antagonists including (−)-menthyl 1 and
AMG2850 are from rat and human TRPM8, for which we are yet to have solved structures, while
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the structure of the avian ortholog has recently been elucidated using cryo-EM. To first evaluate
the degree of sequence conservation across these species, we carried out sequence alignment of the
transmembrane S1-S4 helices (VSLD), S5-S6 helices (pore domain), pore helix (PH) and TRP helix
of avian, human and rat TRPM8 (Figure S1). As detailed above, residues within the VSLD and
TRP helix are reported to be crucial for agonist (menthol) binding, agonist (menthol, icilin)
response or efficacy and voltage sensitivity. Interestingly, the high degree of homology between
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full-length avian and human TRPM8 (83%) is retained within this transmembrane region
containing the ligand binding site (86%); additionally there is a comparatively high sequence
identity (87%) between the avian and rat orthologs in this same region (Figure S1). We therefore
considered the avian TRPM8 structure to be a reliable template for building a homology model of
hTRPM8 with a view to first predicting plausible binding modes of known TRPM8 antagonists (−)-
menthyl 1 and AMG2850 and establishing a relevant structure-function correlation. The latter in
turn, could inform our structure-based strategy for designing novel TRPM8 antagonist probes.
MD simulations. To date, no structure of hTRPM8 is available. We first built the homology model
of hTRPM8 based on the cryo-EM structure of the collared flycatcher Ficedula albicollis
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(
TRPM8FA, PDB: 6BPQ), with which hTRPM8 shares 83% sequence identity to full length
TRPM8FA and 86% in region containing the ligand binding site. We mainly focused on the
transmembrane region of TRPM8, comprised of the VSLD (S1-S4 helices, also termed the sensing
domain, SD) and pore domains, as well as the TRP helix, and built our homology model of
hTRPM8 with a sequence from Gln671 to Asn1010 (340 residues). The VSLD and TRP helix binds
8
small molecule agonists, as discussed above, while the isolated VSLD (including the pre-S1
domain) is reported to recapitulate binding phenotypes that agree with functional activation of full-
length hTRPM8 (1104 residues).22
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2+
A Ca ion was placed in the VLSD, coordinating to Glu782, Gln785, Asn799 and Asp802, to
allow our human model to more closely resemble the cryo-EM structures of related human
melastatin subtypes M2 (PDB 6MJ2) 39 and M4 (PDB 6BQV) , and provide structural context for
our Ca2+ flux studies using the TRPM8 agonist icilin as an activator (Table 1). These structures
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capture a bound Ca ion coordinating to four side chains oriented similarly in the VLSD, three of
which are conserved among TRPM8, TRPM2 and TRPM4: Glu782 (TRPM8, S2 helix)/Glu843
(
TRPM2, S2)/Glu828 (TRPM4, S2); Gln785 (TRPM8, S2)/Gln846 (TRPM2, S2)/Gln831 (TRPM4,
S2); and Asn799 (TRPM8, S3)/Asn869 (TRPM2, S3)/Asn865 (TRPM4, S3). Taking into
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consideration the remaining non-conserved Ca binding residue, we chose Asp802 (S3) as the final
chelating amino acid to satisfy the tetrahedral complex, corresponding to Asp868 in the M4 subtype
S3 helix). Notably, both Asn799 and Asp802 are well-known icilin sensitive residues.23 The
(
2
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recently reported icilin-bound structure of TRPM8 (PDB 6NR3, 3.4 Å) reveals a Ca ion bound to
2
+
these four residues within the now well-defined Ca binding site, in agreement with our homology
model.
Sequentially, we docked (−)-menthyl 1, AMG2850 and 14 into the ligand binding site of
hTRPM8, carried out 100 ns molecular dynamics (MD) simulations for each compound in
hTRPM8, and analyzed the results as detailed below. (−)-menthyl 1, described as a potent and
26
2+
selective TRPM8 antagonist at rat TRPM8 in Ca flux assays (IC50 vs. 20 µM menthol, 20 ± 2
nM; IC50 vs. 0.25 µM icilin, 50 ± 10 nM), is currently used in the literature as a TRPM8 tool
40
41
molecule (also described as OMDM233), as well as AMG2850.
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(
−)-menthyl 1 and hTRPM8. As shown in Figure 2c, our results show that both the root mean
square deviation (RMSD) of hTRPM8 (about 3.9 Å) and (−)-menthyl 1 (1.8 Å) kept stable after 50
ns during the simulation, indicating that the time scale of 100 ns is reasonable.
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The biphenyl of 1 kept stable during the MD simulation, projecting into the base of the binding
cavity in an extended conformation, towards the TRP helix, which forms strong hydrophobic
interactions with Asn741 (S1, ~4.0 Å, not shown) and a conserved Val849 (S4, 4.4 Å), as shown in
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Figure 2b, occupying a similar space as the corresponding methoxyphenyl of WS-12. This pose is
consistent with SAR studies on this scaffold, in which a biphenyl substitution affords the highest
TRPM8 potency among all analogs studied.26
In contrast to the biphenyl moiety, we found that the flexible part of 1, the (−)-menthyl moiety,
endures a change in conformation during the MD simulation, when comparing its binding mode
between pre-MD (Figure 2a) and post-MD (Figure 2b). However, interactions between 1 and some
residues can be observed during the MD. For example, the (−)-menthyl of 1 interacts with a
conserved Arg1008 (TRP helix, 3.5 Å, not shown) (Figure 2b), which assumes a folded
conformation similar to that seen in the WS-12 bound structure, and may represent a common
TRPM8 binding residue for small molecule ligands. To explore the contribution of these residues to
the binding of 1, we decomposed the free energy using the MM/GBSA method, as shown in Figure
2
d. From the energy decomposition, we found that Arg1008 contributed greatly to the binding of 1.
Although the distance between 1 and Arg842 (S4) was about 3.9 Å, the (−)-menthyl moiety
approaches a conserved Leu778 (S2, ~3.8 Å, not shown) during the MD simulation (Figure 2b),
overlapping with the (−)-menthyl and nitrophenyl binding regions of WS-12 and icilin,
8
respectively, making Arg842 contribute less to the binding free energy than Leu778. The results
are consistent with that of the free energy decomposition using the MM/GBSA method (Figure 2d).
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Interestingly, the (−)-menthyl group does not contact Tyr745 (S1), as suggested for (−)-menthol by
radioligand displacement studies, as well as the WS-12-bound structure, likely due to the anchoring
nature of the biphenyl, which forms extensive hydrophobic interactions with the base of the
orthosteric site. Our studies here provide the first forays into investigating menthol-based antagonist
mediated molecular events using the TRPM8 structural biology described to date.
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(−)-menthyl 1
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Figure 2. Convergence parameters of (−)-menthyl 1 and hTRPM8 during MD simulation. (a)
The docking pose (pre-MD) of 1 in hTRPM8. (−)-menthyl 1 is shown as magenta sticks. Important
residues are shown as sticks. Individual helices are colored as follows: S1(blue), S2 (faded green),
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S4 (yellow green), TRP helix (red). The Ca ion (green) is shown in CPK representation. (b) The
binding mode (post-MD) of 1 in hTRPM8 after 100 ns MD. (−)-menthyl 1 is shown as gray sticks.
(
c) The RMSD of 1 and hTRPM8 during MD simulation. (d) Energy decomposition of key residues
in hTRPM8 that contributed to the binding of 1. Our hTRPM8 homology model was constructed
using the cryo-EM structure of TRPM8FA (PDB 6BPQ) as a template.
AMG2850 and hTRPM8. We also performed 100 ns MD simulation for AMG2850 with
hTRPM8. We found that the RMSD of hTRPM8 equilibrates (about 4.4 Å) after 60 ns, as shown in
Figure 3c. The RMSD of AMG2850 kept stable at 1.6 Å (Figure 3c). Due to the stability of
AMG2850, the time scale of 100 ns is reasonable to explore its potential binding mode. Comparing
the binding mode of AMG2850 between pre-MD (Figure 3a) and post-MD (Figure 3b), we found
that several important interactions between AMG2850 and hTRPM8 kept stable. For example, a
conserved Arg842 (S4) forms a strong hydrogen bond with the urea carbonyl (3.1/3.0 Å) of
AMG2850, similar to the WS-12 amide carbonyl and icilin dihydropyrimidinone carbonyl,8
suggesting a common H-bond acceptor feature for TRPM8 ligand recognition via this common
residue. Consistent with this predicted interaction, SAR studies of the AMG2850 precursor series,
containing a tetrahydrothienopyridine core, highlight the importance of the urea moiety, where
42
deletion of this group negates TRPM8 activity (derivatives 24-26 reported by Tamayo et al ). Our
MD results also show that a conserved Trp798 (S3) forms a hydrogen bond with the trifluorophenyl
CF
3
group of AMG2850, with a distance of 3.2 Å, in agreement with SAR studies of the precursor
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tetrahydrothienopyridine series, wherein a 4-CF affords optimal functional and pharmacokinetic
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2
properties. Interestingly, the inactive S-enantiomer of a related tetrahydroisoquinoline analog of
AMG2850 positions this trifluorophenyl in the opposite direction, away from Trp798, while the
corresponding R-enantiomer has a reported TRPM8 IC50: 56 ± 24 nM (derivatives 87-88 reported
by Tamayo et al 42). Moreover, Leu841 (S4) (distance: 4.2 Å, not shown) and the backbone of
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Arg842 (distance: 3.3 Å, not shown) interact with the trifluorophenyl CF group of AMG2850 via a
3
hydrophobic interaction. Tyr1005 (conserved residue, TRP helix) forms strong hydrophobic
interactions with the 5,6,7,8-tetrahydro-1,7-naphthyridine, with a distance of 4.4 Å. This residue
(
Tyr1004 in TRPM8FA) hydrogen bonds to the icilin nitro group and WS-12 amide nitrogen,
suggesting another common TRPM8 binding residue, in agreement with functional studies of
menthol.21 Again, this interaction is consistent with the SAR data for this series, suggesting the
importance of hydrophobic interactions between the ion channel and the azatetrahydroquinoline.
Modifications including removal of the aromatic ring, expansion of the piperidine by one carbon (to
an azepine), and conformational restriction via methyl substitution at the 8-position, results in
significant loss or negates TRPM8 activity (derivatives 32, 72 and 86 reported by Horne et al 10 and
4
2
Tamayo et al ), which may disturb this hydrophobic interaction. Alternatively, these analogs may
disrupt the hydrogen bond between Arg842 and the urea carbonyl of AMG2850, resulting in a
dramatic loss of TRPM8 activity.
On the opposite side of the molecule, we observe that Asp781 (S2) interacts with the
trifluoropropanyl CF via hydrogen bonding with a distance of 3.4 Å. Similar to the previously
3
discussed interactions, this interaction is consistent with SAR studies of this region on the
azatetrahydroquinolinone and tetrahydroisoquinoline scaffolds, where the trifluoropropanyl group
affords high TRPM8 potency.10 Together with hydrogen bonding interactions with Arg842 and
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Trp798, these orient AMG2850 in a T-shape conformation, with either ends of the molecule
spanning the upper portion of the VSLD pocket, vs. the extended conformation seen with (−)-
menthyl 1. On the other hand, the azatetrahydroquinoline ring of AMG2850 penetrates deeply into
the cavity, towards the TRP helix, similar to the biphenyl of 1. Moreover, we observe that Leu778
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forms strong hydrophobic interactions with trifluoropropanyl CF with a distance of 3.6 Å (not
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shown), similar to the (−)-menthyl of 1. These results are consistent with that of the free energy
decomposition using the MM/GBSA method (Figure 3d).
Notably, from our MD studies we did not find any direct interactions between the two
2
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antagonists studied and the Ca binding residues, which could point to a possible structure-based
explanation of their ability to function as antagonists. We did observe interactions between
AMG2850 and Arg842, which could in theory reinforce the tetrahedral Ca2+ complex in human
TRPM8 via Asp802, similar to that seen in the human TRPM4 structure (corresponding TRPM4
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residues are Arg905 and Asp868). Such interactions with Arg842 could work to destabilize the
2+
bound Ca , resulting in channel inhibition. On the other hand, the TRPM8FA (avian) structure does
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not depict the corresponding residue Arg841 as interacting with Ca binding residues, but instead
makes a direct interaction with the icilin carbonyl.8
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Figure 3. Convergence parameters of AMG2850 and hTRPM8 during MD simulation. (a) The
docking pose (pre-MD) of AMG2850 in hTRPM8. AMG2850 is shown as magenta sticks.
Important residues are shown as sticks. Individual helices are colored as follows: S2 (faded green),
S3 (green), S4 (yellow-green), TRP helix (red). The Ca2+ ion (green) is shown in CPK
representation. (b) The binding mode (post-MD) of AMG2850 in hTRPM8 after 100 ns MD.
AMG2850 is shown as gray sticks. (c) The RMSD of AMG2850 and hTRPM8 during MD
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simulation. (d) Energy decomposition of key residues in hTRPM8 that contributed to the binding of
AMG2850. Our hTRPM8 homology model was constructed using the cryo-EM structure of
TRPM8FA (PDB 6BPQ) as a template.
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Structure-based design strategy. By superimposing the predicted binding modes of AMG2850
and (−)-menthyl 1 from MD simulations, we observe several important residues that form a
hydrophobic pocket within the VSLD, in the S2 and TRP helix (Figure 4). Leu778 (S2) interacts
with both the (−)-menthyl and trifluoropropanyl groups of 1 and AMG2850. In the same vicinity,
Arg1008 (TRP helix) form a key hydrophobic interaction with the (−)-menthyl group. These
interactions allow the aforementioned groups to superimpose in the VSLD, and suggests that the 1
scaffold can be substituted with other lipophilic groups such as ring-expanded analogs of the core
cyclohexyl ring, bi- and tricyclic rings, aromatic-containing groups and spiro-substituted bicyclics.
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Figure 4. Superimposition of AMG2850 and (−)-menthyl 1 from their MD poses. (Green and
magentas: MD pose of TRPM8 and (−)-menthyl 1, Blue and gray: MD pose of TRPM8 and
AMG2850)
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
+
We explored these substitutions on the 1 scaffold at human TRPM8 via ratiometric Ca imaging
(
representative experiments are shown in Figure 5, corresponding histograms are shown in Figure
S3), while retaining the biphenyl, as this was the optimal substitution for potent TRPM8 antagonist
activity, from previous SAR studies.26 To this end, various lipophilic substitutions of the (−)-
menthyl moiety of 1 were prepared as shown in Scheme 1, via standard EDCI/HOBt-mediated
amidations of commercially available substituted amines and [1,1ʹ-biphenyl]-4-carboxylic acid I-1.
O
CO H
R1
2
a
N
H
Ph
Ph
I-1
2-15
Scheme 1. Synthesis of 2-15. Reagents and conditions: (a) R
1
-NH
2
, EDCI, HOBt, Et N.
3
Structure-activity relationship (SAR) analysis of (−)-menthyl 1. Dose-response studies of the
prototypical icilin in this assay induces Ca2+ influx at an EC50 of 74 ± 3 nM (Figure S2a) and
appears to be more potent than the previously-reported values lying within 120-200 nM range,29, 33
3
3
but this could very well be due difference in cell type (CHO vs HEK cells) or species-dependence
variation in icilin sensitivity (human versus mouse).29 In our hands, icilin-evoked Ca2+ signals
began to plateau at 500 nM. We used RQ-00203078 as a positive control as well as a reference
43
standard for our SAR studies, due to its potent TRPM8 antagonist activity. In our assays, RQ-
0
0203078 inhibits icilin-evoked Ca2+ signals at an IC50 of 2.96 ± 0.99 nM (Figure S4), which is
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lower than the value (~8 nM) previously reported against 30 µM menthol-evoked Ca influx in
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
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5
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5
5
5
6
HEK cells expressing hTRPM8.43
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Evaluation of test compounds for any effect on icilin-evoked Ca2+ entry signals using
2
+
fura-2 based Ca imaging of HEK-293 cells stably expressing human TRPM8. The fura-2 loaded
2+
cells bathed in Ca -free extracellular solution were exposed to icilin (500 nM) with or without the
synthesized compounds or the known TRPM8 antagonist RQ-00203078 at various concentrations
(For this figure, all were at 3 nM. The corresponding histogram is provided in Figure S3). After ∼ 4
2+
2+
min of compound addition, Ca free bath solution was replaced by the one containing 2 mM Ca .
2
+
Ca influx was monitored as fura-2 fluorescence ratio (F355/F380). Each data point represents mean
± SEM (n ≥ 35 cells from 3 independent experiments done in three different days).
For our synthesized analogs, IC50 values are shown in Table 1, and expressed as the
2
+
concentration required to produce half-maximal inhibition of icilin-stimulated Ca influx. We also
resynthesized (−)-menthyl 1 using methodology reported by Ortar and colleagues 26 to serve as an
additional standard for our biochemical assays at hTRPM8, due to its potent antagonist activity at
rTRPM8 in calcium flux assays (IC50 vs. 20 µM menthol, 20 ± 2 nM; IC50 vs. 0.25 µM menthol,
5
0 ± 10 nM) and weak intrinsic (agonist) activity at hTRPV1 and rTRPA1. Interestingly, 1 shows
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1
1
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2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
4
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5
5
5
5
6
dramatically reduced potency (IC50 of 16 ± 1 µM) in our assays in HEK-293 cells transfected with
human TRPM8 (Table 1). Differences in observed IC50 values in the calcium flux assay at human
vs. rat TRPM8 suggests species differences among TRPM8 orthologs for this compound. Species
differences are known to exist between different TRPM8 orthologs despite high sequence
conservation.35, 44 Additional studies at rTRPM8 could be performed on all analogs of this series, to
determine whether similar potency shifts are observed. Although human TRPM8 is highly
homologous to the rat ortholog, with 96% sequence identity in the VSLD domain, pore domain and
TRP helix (residues 734-1009, calculated by Clustal Omega, data not shown), there are a small
number of different amino acid residues, possibly accounting for the discrepancy seen between
these orthologs in the calcium flux assay. One such residue at position 1007, in the TRP helix, is
situated in the binding pocket (serine at hTRPM8, asparagine at rTRPM8). Studies are ongoing to
further characterize 1 and will be reported in due course.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Removing the chirality of lead 1 to give isomeric mixture 2 increases hTRPM8 activity by 30-
fold (Table 1) in our assays (hTRPM8 IC50: 468 ± 1 nM) relative to 1, indicating the mixture
contains isomers with a more optimal stereochemical arrangement at the C1, C2 and C5 positions
than 1. Relative to menthol isomeric mixture 2, removal of the C5 methyl and C2 isopropyl groups
(
as in 3) and conformational restriction of the core cyclohexyl ring (as in 4) affords reduced
TRPM8 potency, in the µM range, highlighting the importance of the entire (−)-menthyl scaffold as
well as the conformational flexibility of the cyclohexyl ring, and possibly displacing hydrophobic
interactions with Leu778 and Arg1008, as suggested by our MD studies.
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Table 1. IC50 values of 1 analogs in nM, determined by cellular Ca flux assays in HEK-293 cells
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
stably expressing hTRPM8 and represented as mean ± SEM from three independent experiments.
2
+
IC50 is defined by concentration of the compounds that inhibit 50% of icilin-evoked Ca entry.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
R1
Functional Activity
N
H
Ph
hTRPM8 potency
Cmpd
R
1
IC50 ± SEM
1
2
16 ± 1 µM
468 ± 1 nM
3
4
5
49 ± 1 µM
7.1 ± 0.9 µM
52 ± 1 nM
6
7
692 ± 1 nM
62 ± 1 nM
8
9
21 ± 1 µM
55 ± 1 µM
1
0
6
± 1 nM
(diastereomeric
mixture)
11
52 ± 1 nM
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1
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1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
H 1
8
5
1
2
2
7
6
9
1.4 ± 1.0 nM
40 ± 1 nM
10
3
(isolated
H 4
diastereomer)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
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7
8
9
0
1
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
14
2.4 ± 1.0 nM
16 ± 1 nM
1
5
RQ-00203078
icilin
3 ± 1 nM
74 ± 3 nM
EC50
EC50
(−)-menthol
5.7 ± 0.72 µM
On the other hand, ring expanded analogs (cycloheptyl 5 and cyclooctyl 7) improve hTRPM8
potency by 8-9 fold relative to 2, with IC50 values of 52 ± 1 nM and 62 ± 1 nM, respectively,
suggesting the ability to capture additional interactions in the pocket. In order to rationalize these
results, we docked analogs 5 and 7 into our hTRPM8 homology model (Figure 6). For both analogs,
the biphenyl ring is positioned between the S1 and S4 helices and TRP helix, similar to 1. The
biphenyl ring of both analogs forms strong hydrophobic interactions with Val849 (conserved, S4,
3
3
.8/3.8 Å), the same interaction seen for the 1 biphenyl, in addition to Phe738 (conserved, S1,
.5/3.7 Å), Leu1001 (conserved, TRP helix, 3.7/3.7 Å) and menthol- and icilin-sensitive Tyr1005
(conserved, TRP helix, 4.0/4.0 Å). The 1-phenyl group forms edge-to-face π-π stacking with both
Phe738 and Tyr1005. These additional interactions may contribute to the enhanced potency of these
analogs in the Ca2+ flux assay, versus 1. Interestingly, the 5,6,7,8-tetrahydro-1,7-naphthyridine of
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AMG2850 forms hydrophobic contacts with Tyr1005 (Figure 3), in the same vicinity as the 1-
phenyl of 1 (Figure 4). This reinforces the notion of Tyr1005 as a common small molecule binding
residue. Both the amide NH and carbonyl O of 5 and 7 interact with Arg1008 (conserved, TRP
helix) with strong hydrogen-bond interactions, possibly driving its potent functional activity; vs.
that seen in 1 where this residue only forms hydrophobic contacts with the (−)-menthyl moiety.
These results suggest that Arg1008 is a key residue for the recognition of both 1 and its analogs. In
addition, the cycloheptyl and cyclooctyl moieties of both ligands approached three residues with
similar hydrophobic interactions, including voltage- and icilin-sensitive Arg842 (conserved, S4,
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
4
4
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4
4
4
5
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5
5
5
5
5
5
5
6
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
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5
6
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1
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3
4
5
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7
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2
3
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5
6
7
8
9
0
4
.2/4.0 Å), icilin-sensitive His845 (conserved, S4, 3.7/4.0 Å), and Ile846 (conserved, S4, 3.6/3.5
Å), located on the opposite side of the VSLD as Leu778 and Arg1008. Together, these results
suggest that the orthosteric site is able to accommodate larger hydrophobic substitutions than that
seen in 1, with a number of other lipophilic residues as mentioned above, while the biphenyl
provides an anchoring interaction towards the floor of the cavity.
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1
1
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2
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2
3
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3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 6. Binding mode of 5 and 7 in hTRPM8. Analogs 5 and 7 are shown as yellow and orange
sticks, respectively. Important residues are shown as sticks. Individual helices are colored as
2+
follows: S1(blue), S2 (faded green), S4 (yellow green) and TRP helix (red). The Ca ion (green) is
shown in CPK representation. Our hTRPM8 homology model was constructed using the cryo-
electron microscopy (cryo-EM) structure of TRPM8FA (PDB 6BPQ) as a template.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
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9
0
1
2
3
4
5
6
7
8
9
0
Similar to 3 and 4, conformational restriction of 5 (to bicyclo[3.2.1]octan-3-yl 6) results in a
sharp decrease in potency, possibly due to the loss of multiple H-bond contacts to Arg1008.
Exploration with bicyclic and tricyclic ring replacements were carried out to further probe the
lipophilic space around our series.
Decahydronapthalen-1-yl 8 results in a significant drop in hTRPM8 potency, relative to 5 and 7,
in the µM range, indicating that the pocket is of a discriminate volume; similarly,
tetrahydronaphthalen-1-yl 9 suffers a 3-fold loss of potency vs. 8, again suggesting the need for
flexibility at this region of the scaffold. In striking contrast, decahydronaphthalen-2-yl analog 10, a
mixture of two isomers, enhances TRPM8 potency by 3,500-fold relative to 8 (IC50: 6 ± 1 nM), and
has 10-fold higher potency than both the cycloheptyl 5 and cyclooctyl 7 analogs, suggesting the
decahydronapthalen-2-yl ring occupies the lipophilic pocket to maximize available hydrophobic
interactions. Consistent with the SAR trends seen with 8 and 9, substitution with a
tetrahydronaphthalen-2-yl group (analog 11) decreases hTRPM8 potency by 9-fold, though retains
nM potency at hTRPM8 with an IC50: 52 ± 1 nM. Separating out one of the isomers of analog 10
via chromatography to give 12, gratifyingly affords potency in the single digit nM range, with an
IC50: 1.4 ± 1.0 nM, 4-fold higher than the diastereomeric mixture. It is possible that 10 forms
multiple contacts with residues within the VSLD to give high hTRPM8 potency, similar to the
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single isomer 12. LC-MS analysis of 10 and 12 (Figures S20 and S22, respectively) indicates that
0, a mixture of two diastereomers, is comprised of >85% of the 12 isomer, thus accounting for its
potent IC50 value.
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
The relative stereochemical configuration of 12 was established by H, C, gCOSY, NOESY,
gHSQCAD, gHMBCAD and gH2BC (Table S6, Figures S24-S30). Analysis of NMR data
including comparison of decalin ring proton and carbon chemical shifts to literature values indicates
that the compound is a cis-decalin with the biphenyl amide moiety in the 2-position. Analysis of
NOESY data indicates that the amide group is trans to the decalin bridge protons (see structure in
Table S6, absolute stereochemistry cannot be established).
The structure was determined to be cis-decalin because the carbon and proton chemical shifts are
45
more consistent with that configuration (see, Dodziuk et al. ). Cis-decalin bridge carbons (C9,10)
resonate at 36.1 ppm, whereas those in trans-decalin resonate at 42.5 ppm. Furthermore, the
bridgehead protons resonate at lower ppm in the trans-decalin than that observed for this structure
compare 1.91, 1.68 ppm vs. 0.87 ppm for trans-decalin). Also see, Browne et al.46
(
The relative stereochemistry of the amide moiety is a bit more challenging to ascertain. NOESY
data is consistent with the proposed structure; however, the severe chemical shift overlap,
particularly with H10 and H1, renders unambiguous assignment impossible. NOESY data does
establish that H9 (1.91 ppm) and H2 (4.01 ppm) are on the same face. This result coupled with the
chemical shift data is consistent with a cis-decalin substituted in the trans-2-position with a
biphenyl carboxamide.
We then probed spiro-substituted bicyclic replacement of the (−)-menthyl group of lead 1. Spiro
[
5.5]-undecan-3-yl analog 13 retains similar potency as 5 and 7 with an IC50: 40 ± 1 nM, though
not in the high nM range as decahydronapthalen-1-yl analogs 10 and 12, suggesting the
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
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4
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4
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5
5
5
5
5
5
5
5
5
5
6
complementary region of the receptor cavity is of a fixed volume and cannot tolerate a spiro-
arrangement of the two cyclohexyl rings.
Ring contraction of 13 by one carbon (to spiro[4.5]decan-8-yl analog 14) recapitulates the potent
activity of decahydronapthen-1-yl isomer 12 (IC50: 2.4 ± 1.0 nM), with comparable activity as the
highly potent TRPM8 antagonist RQ-00203078 in our assays.43 Spiro[4.5]decan-8-yl 14 is of
similar potency as 12, further suggesting the constrained or discerning nature of the pocket. The
binding site for this series could be determined unequivocally using structural biology techniques
such as cryo-EM or X-ray crystallography to produce a ligand bound structure of human TRPM8,
coupled with mutagenesis studies. Currently, the structure of human TRPM8 is not known.
Notably, analog 14 shares several common structural features with other potent TRPM8 antagonists
reported in the scientific and patent literature. Both a spiro group (spiro-isoxazoline) and biphenyl
aromatic arrangement are also seen in highly potent benzimidazole-based TRPM8 antagonist
JNJ41876666/ TC-I 2014 47 (cTRPM8 IC50 (icilin): 0.8 nM). Another TRPM8 antagonist reported
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
45
2+
by Glenmark Pharmaceuticals, (R)-(─)-10e (hTRPM8 IC50 ( Ca uptake): 8.9 nM), contains a 3,4-
4
8
dihydrospiro[chromene-2,4ʹ-piperidine. Similarly, Raqualia Pharma reported a series of azaspiro
TRPM8 antagonists containing ring systems such as 1-oxa-3-azaspiro[4.5]decan-2-ones, 1,3-
diazaspiro[4.5]decane-2,4-diones and 1-oxa-3-azaspiro[4.5]decane-2,4-diones, linked via a ketone,
to phenyl rings para substituted with aromatic and heterocyclic rings. 49
We then constructed a dose-response curve of 14 from the data shown in Figure S3. We selected
14 for further characterization, given its lack of chiral centers unlike 12. As shown in Figure S5,
2+
compound 14 dose-dependently inhibits icilin-induced Ca influx with an IC50 = 2.4 ± 1.0 nM and
2
+
at higher concentrations, nearly abolishes the Ca influx (Figure S3, S5).
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5
To determine the ability of 14 to inhibit menthol in the Ca2+ flux assay, we first evaluated the
menthol response in our HEK-293 cells stably expressing hTRPM8 and found an EC50 ~ 6 µM
(Figure S2b). The observed menthol potency was comparable to the values previously reported for
hTRPM8 in similar calcium imaging of HEK293 cells.50-51 The lowest concentration at which
compound 14 was still able to cause significant reduction in icilin-evoked TRPM8-mediated Ca2+
entry was 3 nM (Figure S5). We tested compound 14 at the same lowest (i.e. 3 nM) concentration
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2+
51
against the maximal (100 µM) menthol -evoked TRPM8-mediated Ca entry (Figure S6a). As
can be seen in Figure S6b, compound 14 at 3 nM was also able to significantly (P<0.01) reduce
TRPM8-mediated Ca2+ entry triggered by menthol. These studies provide additional support for
exploring the effects of 14 in this assay using menthol as an agonist, in addition to our SAR studies
carried out with icilin.
Adamantyl 15 affords slightly lower potency than 14, with an IC50: 16 ± 1 nM, indicating that
tricyclic rings are tolerated, as well as bicyclics 10-14. Altogether, the antagonist activity of 10, 12,
1
4 and 15 in the Ca2+ flux assay, with IC50 values in the ≤ 10 nM range in human TRPM8-
2
+
transfected cells, exceeds or is comparable to the Ca flux activity of reported TRPM8 antagonists
9
with in vivo activity, including PF-05105679 (Pfizer, Phase 1, hTRPM8 IC50 = 181 nM), AMG
3
33 (Amgen, Phase 1, hTRPM8 IC50 = 13 ± 4 nM),13 AMG2850 (Amgen),10-11 RQ-00203078
4
3
12
(
RaQualia Pharma), AMTB (Bayer), arylglycine derivative 12 and vinylcycloalkyl-substituted
benzimidazole 25 (Janssen),52-53 DFL23448 and DFL23693,14 M8-B,15 M8-An,54 tryptophan
derivative 1438 and others. None of the analogs with hTRPM8 IC50 values ≤ 100 nM in the Ca2+
flux assay (5, 7, 10-15), were able to stimulate the channel alone at a single high concentration of
10 µM (Figure S7), in agreement with results obtained in our antagonist assays.
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