Discovery of a Highly Selective and Potent TRPC3 Inhibitor with High Metabolic Stability and Low Toxicity

Article abstract of DOI:10.1021/acsmedchemlett.0c00571

The overactivation of transient receptor potential canonical 3 (TRPC3) is associated with neurodegenerative diseases and hypertension. Pyrazole 3 (Pyr3) is reported as the most selective TRPC3 inhibitor, but it has two inherent structural limitations: (1) the labile ester moiety leads to its rapid hydrolysis to the inactive Pyr8 in vivo, and (2) the alkylating trichloroacrylic amide moiety is known to be toxic. To circumvent these limitations, we designed a series of conformationally restricted Pyr3 analogues and reported that compound 20 maintains high potency and selectivity for human TRPC3 over its closely related TRP channels. It has significantly improved metabolic stability compared with Pyr3 and has a good safety profile. Preliminary evaluation of 20 demonstrated its ability to rescue Aβ-induced neuron damage with similar potency to that of Pyr3 in vitro. Collectively, these results suggest that 20 represents a promising scaffold to potentially ameliorate the symptoms associated with TRPC3-mediated neurological and cardiovascular disorders.

Full text of DOI:10.1021/acsmedchemlett.0c00571

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Letter
Discovery of a Highly Selective and Potent TRPC3 Inhibitor with
High Metabolic Stability and Low Toxicity
Sicheng Zhang,∇ Luis O. Romero,∇ Shanshan Deng, Jiaxing Wang, Yong Li, Lei Yang, David J. Hamilton,
Duane D. Miller, Francesca-Fang Liao, Julio F. Cordero-Morales,* Zhongzhi Wu,* and Wei Li*
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ABSTRACT: The overactivation of transient receptor potential canonical 3 (TRPC3) is associated with neurodegenerative diseases
and hypertension. Pyrazole 3 (Pyr3) is reported as the most selective TRPC3 inhibitor, but it has two inherent structural limitations:
(1) the labile ester moiety leads to its rapid hydrolysis to the inactive Pyr8 in vivo, and (2) the alkylating trichloroacrylic amide
moiety is known to be toxic. To circumvent these limitations, we designed a series of conformationally restricted Pyr3 analogues and
reported that compound 20 maintains high potency and selectivity for human TRPC3 over its closely related TRP channels. It has
significantly improved metabolic stability compared with Pyr3 and has a good safety profile. Preliminary evaluation of 20
demonstrated its ability to rescue Aβ-induced neuron damage with similar potency to that of Pyr3 in vitro. Collectively, these results
suggest that 20 represents a promising scaffold to potentially ameliorate the symptoms associated with TRPC3-mediated
neurological and cardiovascular disorders.
KEYWORDS: transient receptor potential canonical 3, pyrazole 3, selective inhibitor, high metabolic stability, low toxicity
postulated as a critical contributor to Alzheimer’s disease.11
ransient receptor potential canonical (TRPC) channels
1
Tare ubiquitously expressed in vertebrate cells.
TRPC3 is also expressed in cardiomyocytes, and its over-
expression has been found to be involved in adverse stress
responses, hypertrophy, and heart failure.12 In the immune
system, it has been suggested to contribute to the restoration
of Ca2+ influx and activation of T-cells, facilitating the response
to antigen stimulation.13,14 Additionally, TRPC3 is involved in
the proliferation and migration of a variety of tumor cells
including melanoma, lung, and breast cancers.15−17
The
TRPC channel subfamily consists of seven members
designated as TRPC1−7. Except for pseudogene TRPC2, the
other channels are further divided into two groups, TRPC1/4/
5 and TRPC3/6/7, based on amino acid sequence homology
and functional similarities.2 As nonselective cationic channels,
they control the influx of Ca2+ and other cations like Na+ in
response to activation of phospholipase C-coupled plasma
membrane receptors and thus play critical roles in the
regulation of intracellular Ca2+ concentration by hormones
and growth factors.3,4 Among the TRPC family, TRPC3 plays
a prominent functional role in basic cellular responses
including proliferation, differentiation, and death in response
to various environmental stimuli,5−7 implying a variety of
diverse biological functions.3 TRPC3 is the most abundant
TRPC channel in the brain.8 Recent studies have suggested
that TRPC3 channels are critical for the signaling cascade of
brain-derived neurotrophic factors,9,10 which has been
Current understanding of the role of TRPC3 in pathological
studies suggests that selective TRPC3 channel inhibitors may
be suitable for the treatment of diseases.15,18 Over the past
Received: November 2, 2020
Accepted: February 26, 2021
Published: March 5, 2021
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© 2021 American Chemical Society
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decade, several TRPC3 inhibitors have been reported and are
summarized in Figure 1. Among these inhibitors, Pyr3 is the
most selective for TRPC3 inhibition, with a reported IC50
value of 0.7 μM, since most of the other inhibitors possess
comparable or higher potency against both TRPC3 and
TRPC6.19 Pyr3 has been reported in several pharmacological
studies for the treatment of TRPC3-related diseases such as
cardiac hypertrophy19 and smooth muscle proliferation.18
However, Pyr3 has two structural liabilities. First, while
structure−activity relationship (SAR) studies showed that the
trichloroacrylic amide group in Pyr3 (tail moiety in Figure 2) is
critical for its TRPC3 inhibition specificity,20 it is a reactive
group with high toxic liabilities. Second, the ester head moiety
in Pyr3 can be quickly hydrolyzed in vivo, leading to the
inactive acid derivative Pyr8.21 To circumvent these two
structural limitations, we hypothesized that incorporating the
tail group in a conformationally restricted ring will significantly
reduce the alkylating toxicity, and isosterically replacing the
ester with a stable linker will improve the metabolic stability.
Toward this direction, we designed and synthesized a series of
modified pyrazoles as illustrated in Figure 2. Among these
molecules, compound 20 shows a significantly improved
metabolic stability and safety profile, while maintaining high
potency and selectivity for TRPC3 inhibition similar to that of
Pyr3. Preliminary evaluation of compound 20 demonstrated its
ability to rescue Aβ-induced neuron damages with similar
potency to Pyr3.
As the first step of modification, we only replaced the tail
moiety with a 6-membered pyridone ring while keeping the
rest of the Pyr3 structure intact, to determine whether
restricting the conformation in the tail region still maintains
TRPC3 inhibition activity at all. To synthesize compounds 2−
8, we started from the condensation of ethyl-2-(ethoxy-
methylene)-4,4,4-trifluoro-3-oxobutanoate and 4-bromophe-
nylhydrazine hydrochloride to produce the pyrazole inter-
mediate 1. Coupling of 1 with 4-chloropyridone or pyridone
afforded 2 or 3, respectively (Scheme 1a). Treatment of 2 with
N-chlorosuccinimide (NCS) gave the multichlorinated com-
Figure 2. Design of metabolically stable and low toxic selective
TRPC3 inhibitors by addressing structural liabilities in Pyr3.
Scheme 1. Synthesis of Ester Compounds 2−8 with
a
Different Chlorine Substitution Patterns
a
Reagents and conditions: (i) EtOH, 60 °C, 6 h; (ii) CuI, DMCDA,
K2CO3, toluene, reflux, 12 h; (iii) NCS, DMF, 100 °C, 8 h.
a
Scheme 2. Synthesis of Amide Compounds 17−22
Figure 1. Chemical structures of reported TRPC3 inhibitors.
a
Reagents and conditions: (i) selectfluor, MeCN, 50 °C, 3 h; (ii)
NBS, DMF, r.t., 8 h. (iii) 1 M KOH (aq.), EtOH, H2O, rt, 8 h; (iv)
SOCl2, CH2Cl2, reflux for 4 h; (v) ethylamine, TEA, r.t., 12 h.
pounds 4−6 (Scheme 1b). Finally, 3,5-dichlorinated 7 and 5-
chlorinated 8 were obtained using copper catalyst mediated
coupling reactions as shown in Scheme 1c.
To make compounds 9 and 10, fluorine or dibromine was
incorporated to compound 2 by the treatment with selectfluor
or N-bromosuccinimide, respectively (Scheme 2a and b). The
corresponding carboxylic acids 11−16 were obtained through
hydrolysis of the parent ester compounds before they were
converted to their corresponding amides 17−22 (Scheme 2c).
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Figure 3. Inhibition of TRPC3-mediated currents by synthesized compounds. (a) Cartoon depicting the experimental conditions used to test the
effect of the synthesized compounds on HEK293 cells overexpressing hTRPC3. TRPC3 currents were measured in the whole-cell configuration of
the patch-clamp technique, and cells were challenged with agonists/antagonists through perfusion. (b) Percentage of hTRPC3 currents elicited by
GSK170 and inhibited by the synthesized compounds in transfected HEK293 cells. Bars represent mean SD; n is denoted above the x-axis. (c)
Representative traces of hTRPC3 currents activated by 1 μM GSK170 and inhibited at different concentrations of compound 20 (blue traces). Each
concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (d) Dose−
response profile of hTRPC3 currents elicited by 1 μM GSK170 and inhibited at different concentrations of compound 20. Circles represent mean
SD. (e) Representative traces of hTRPC3 currents activated by 1 μM GSK170 and inhibited at different concentrations of Pyr3 (gray traces). Each
concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (f) Dose−
response profile of hTRPC3 currents elicited by 1 μM GSK170 and inhibited at different concentrations of Pyr3. Circles represent mean SD. (g)
Representative traces of hTRPC3 currents activated by 1 μM GSK170 and inhibited at different concentrations of compound 20 (blue traces) in the
presence of extracellular Ca2+ (2 mM). Each concentration was tested on independent cells and normalized against its internal control (maximum
amplitude) to avoid tachyphylaxis. (h) Dose−response profile of hTRPC3 currents elicited by 1 μM GSK170 and inhibited at different
concentrations of compound 20 in the presence of extracellular Ca2+ (2 mM). Circles represent mean SD. (i) Representative traces of hTRPC3
currents activated by 1 μM GSK170 and inhibited at different concentrations of Pyr3 (gray traces) in the presence of extracellular Ca2+ (2 mM).
Each concentration was tested on independent cells and normalized against its internal control (maximum amplitude) to avoid tachyphylaxis. (j)
Dose−response profile of hTRPC3 currents elicited by 1 μM GSK170 and inhibited at different concentrations of Pyr3 in the presence of
extracellular Ca2+ (2 mM). Circles represent mean SD. GSK170: GSK1702934A.
Other compounds 27, 32, 37, and 38 were synthesized by
following the procedures shown in Supporting Information
(Schemes S1−S3). All compounds have purities ≥ 95% based
on analytical chemistry characterization (Supporting Informa-
tion).
We first evaluated the ability of the Pyr3 analogues to inhibit
TRPC3 using the patch-clamp technique in the whole-cell
configuration (Figure 3a). We activated human TRPC3
(hTRPC3) with a specific agonist, GSK1702934A (GSK170),
in HEK293 cells and determined the inhibitory effect of each
compound at 10 μM concentration in the presence of the
agonist (Figures 3b and S1a). We recorded TRPC3 currents in
the absence of Ca2+ to evaluate compound inhibition with a
minimal contribution of Ca2+-desensitized channels. As
expected, Pyr3 inhibited 91 4% (mean SD) of hTRPC3
currents. Notably, compound 20 was as efficient as Pyr3
inhibiting hTRPC3 (89
(84 2%) and 2 (71
3%), followed by compounds 17
5%). Further modification of
compound 20 by replacing 3-chlorine with 3-fluorine to
produce 21 or replacing the amide by a sulfonamide moiety to
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Figure 4. Compound 20 is a selective inhibitor of the TRPC subfamily. (a) Representative whole-cell recordings of HEK293 cells overexpressing
hTRPA1, rTRPV1, rTRPV4, hTRPC3, mmTRPC6, hTRPC7, and hTRPM8. Currents were evoked with high concentrations of the agonist (red)
and challenged with 20 (10 μM) in the presence of the respective agonist (blue). (b) Percentage of peak current blocked by 20 (10 μM) in the
presence of each agonist. AITC, Allyl isothiocyanate; Cap, capsaicin; GSK101, GSK1016790A. Bars are mean SD. n is denoted above the x-axis.
chlorines at C4 in Pyr3), the overall SAR for this new scaffold
is largely consistent with that of the Pyr3 scaffold.20 It is also
interesting to note that the chlorine at C4 in the compound 20
scaffold plays a more important role than the chlorine at C3 or
C5, since analogues lacking this C4 chlorine (e.g., 3, 7, and 8)
are significantly less potent. As an added benefit, the amide
analogues which are predicted to be more metabolically stable
also showed higher potency than the corresponding ester
Figure 5. LC/MS analyses of the purified 20−TRPC3 complexes
(right) together with the prepared control (left) support the direct
binding of 20 to TRPC3 proteins. Spectra of MS data collected from
2.5 min to avoid buffer salts to MS.
analogues (Figure 3b).
Since compound 17 had a slower time course of inhibition
than those of Pyr3 and compound 20 (Figure S1b and c), we
focused our subsequent studies using compound 20. We
determined the IC50 of compound 20 for hTRPC3 inhibition
in the absence of extracellular Ca2+ as 0.37 0.03 μM (mean
SD), which is slightly lower than that of Pyr3 (0.53 0.05
μM, Figure 3c−f). We also compared their inhibition potency
in the presence of extracellular Ca2+ (Figures 3g−j and S2e,f).
As expected, 20 showed a comparable potency with Pyr3 at a 2
mM Ca2+ concentration. To further confirm the inhibitory
activity of compound 20, we performed a separate experiment
with a commonly used TRPC3 agonist, 1-oleoyl-2-acetyl-sn-
glycerol (OAG, a membrane-permeable analogue of the
endogenous agonist diacylglycerol). Compound 20 and Pyr3
showed similar inhibitory effects and a similar trend in the
dose−response profile compared to that of GSK170 (Figures
S1d−f and S2a−d). We next determined the ability of
compound 20 to inhibit members of the TRP channel
superfamily hTRPA1, rat TRPV1 (rTRPV1), rTRPV4,
hTRPC3, mouse TRPC6 (mm TRPC6), hTRPC7, or
hTRPM8 overexpressed in HEK293 cells (Figure 4a). The
experiments were performed using solutions without Ca2+ to
avoid TRP channel desensitization.22 After fully activating
these channels with their respective agonists, 10 μM of
Figure 6. Compound 20 shows a good safety profile in mice. (a)
Mouse survival curves. (b) Mean percent change in mouse body
weight SD relative to body weight at the time of initiating drug
treatment. All mice received a daily dose at 200 mg/kg body weight of
compounds 20, 27, or Pyr3 (n = 5) for 5 consecutive days. Dashed
lines indicate mouse weight relative to its respective baseline weight at
day 0 and 15% weight loss.
provide 32 failed to further improve the inhibitory potency.
Since compound 20 best mimics the structure of Pyr3 (the C5
carbon in compound 20 occupies the position for one of the
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Figure 7. Effects on neuron dendritic morphology. (a) Compound 20 and Pyr3 protect against Aβ-induced damage to dendrites. Mature
hippocampal neurons (DIV = 14) were treated with CHO control medium, or naturally secreted Aβ-containing conditioned medium (7PA2)25 or
in combination with compound 20 or Pyr3 at different concentrations, for 16 h, followed by immunocytochemistry using an antibody against
MAP2 (green) and DAPI (blue). Scale bar: 100 μm. (b) Lack of toxicity of the inhibitor compounds in neuronal culture. Hippocampal neurons
(DIV = 14) were treated with compound 20 or Pyr3 alone at different concentrations, followed by immunocytochemistry staining with MAP2
(green) and DAPI (blue). Scale bar: 100 μm. (c) Quantification of dendritic length. Bars are mean SEM. **p < 0.01 (one-way ANOVA). ****p
< 0.0001 (one-way ANOVA). n = 50 dendrites.
compound 20 was perfused together with the agonists. At this
saturating concentration (10 μM), compound 20 failed to
inhibit hTRPA1, rTRPV1, rTRPV4, and hTRPM8 currents
(Figure 4a and 4b). On the other hand, compound 20
inhibited approximately 39 5% of hTRPC6 and 26 1% of
hTRPC7 (Figure 4a and 4b), which are closely related to
TRPC3.
stability and safety profile of compound 20. As expected, the
half-life was extended substantially from less than 15 min for
Pyr3 to more than 4 h for compound 20 in mouse, rat, and
human liver microsomes, consistent with our design to replace
the metabolically labile ester headgroup in Pyr3. A preliminary
in vivo toxicity study was subsequently performed in mice.
Because Pyr3 undergoes fast hydrolysis to its inactive
metabolite Pyr8 and both showed rapid blood clearance in
mice,21 compound 27, bearing the same trichloroacrylic amide
group as Pyr3 but with a stable amide bond, was used together
with Pyr3 as the comparison treatments for the purpose of this
toxicity study. All mice (n = 5) were treated daily with
compounds 20, 27, or Pyr3 at 200 mg/kg by intraperitoneal
injection for 5 consecutive days, and their survival was
monitored. Compound 27 resulted in 100% mortality within
48 h in treated mice. The mice in the Pyr3-treated group with
80% survival dropped by roughly an average of 15% body
weight compared to their weights at the start of the
experiment, indicating the potential toxicity of Pyr3. In
contrast, compound 20 displayed neither mortality nor
significant weight loss (Figure 6). Since the only difference
between compounds 20 and 27 in structure is their tail moiety,
this study provided preliminary evidence that our rational
structural modification can significantly decrease the safety
liability associated with the trichloroacrylic amide group in
Pyr3. It should be noted that since Pyr3 rapidly hydrolyzed in
vivo to its much polar acid derivative Pyr8 which may have a
much faster in vivo clearance, the appearance of a lower
mortality in Pyr3 treatment group than its metabolically stable
analogue 27 is likely due to the fast metabolism and
elimination of Pyr3.
Several studies found that Pyr3 inhibits Ca2+ release-
activated Ca2+ channel 1 (Orai1) with comparable blocking
potency.23 Similarly, we found that compound 20 inhibits
STIM/Orai mediated Ca2+ influx as Pyr3 when activated with
thapsigargin (Figure S3). Future modifications could be
introduced to compound 20 to discriminate between TRPC3
and STIM/Orai channels. Despite the side effect of pyrazoles
at STIM/Orai channels, our results demonstrate that
compound 20 is a potent and selective inhibitor of hTRPC3
against other members of the TRP channel superfamily.
To preliminarily confirm the direct on-target engagement of
compound 20 with hTRPC3, we performed LC-MS analyses.
After incubation of compound 20 with the hTRPC3 protein2
for 1 h, we performed repeated size-exclusion chromatography
to completely remove free compound 20 from the protein-
bound fraction. Methanol was then added to this complex to
denature the protein and extract the bound compound 20. As
shown in Figure 5, LC-MS detected the presence of compound
20 in the extract solution while no peaks were detected in the
control. Since unbound compound 20 was washed out during
the complex preparation, this result strongly suggests the direct
binding of compound 20 to hTRPC3. Future structural and
functional studies with Cryo-EM will reveal the molecular
interactions of the complex.
Having confirmed the selective hTRPC3 inhibition and
direct molecular interactions, we next evaluated the metabolic
Finally, to explore the potential biological effects of 20 in a
TRPC3 related disease model, we evaluated its neuroprotective
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ability in primary cultured neurons (14 DIV prepared from
E17 rats as described24). Compound 20 displayed similar
protective effects as Pyr3 in preventing soluble oligomeric Aβ-
induced synaptic toxicity on dendritic spine morphology as
evidenced by MAP-2 staining (Figure 7).
In summary, we describe here the rational design and
preliminary characterization of a new metabolically stable, low-
toxic, and selective hTRPC3 inhibitor compound 20. We also
demonstrated the direct binding of compound 20 to hTRPC3
protein and its potential neuroprotective effects. These results
indicate that further characterization and optimization of
compound 20 may lead to a selective TRPC3 inhibitor as a
potentially viable agent for diseases where overexpression of
TRPC3 is involved.
Tennessee Health Science Center, Memphis, Tennessee 38163,
United States
Duane D. Miller − Department of Pharmaceutical Sciences,
College of Pharmacy, the University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States;
orcid.org/0000-0002-6093-0985
Francesca-Fang Liao − Department of Pharmacology,
Addiction Science, and Toxicology, the University of
Tennessee Health Science Center, Memphis, Tennessee 38163,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00571
Author Contributions
∇S.Z. and L.O.R. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00571.
Notes
The authors declare the following competing financial
interest(s): W.L., Z.W., and F.L. are listed as inventors for a
filed provisional patent application covering these compounds.
Whole-cell recording, experimental procedures and
1
methods, characterization data, and H and 13C NMR
spectra (PDF)
ACKNOWLEDGMENTS
■
AUTHOR INFORMATION
Corresponding Authors
■
We thank Dr. Valeria Vásquez and Dr. Lubin Lan (retired) for
technical support. We thank Dr. Amanda Clarke at UTHSC
for editorial assistance. We thank Dr. Salvatore Mancarella for
experimental advice. This work was partially supported by NIH
Grants R01GM125629 (to J.F.C.-M.) and R01AG049772 (to
F.-F.L.) and funds from a UTHSC CORNET award and the
UTCoP Drug Discovery Center (to W.L.). The contents of the
article are solely the responsibility of the authors and do not
necessarily represent the official views of the NIH.
Wei Li − Department of Pharmaceutical Sciences, College of
Pharmacy, the University of Tennessee Health Science Center,
Memphis, Tennessee 38163, United States; orcid.org/
0000-0002-9522-4474; Email: wli@uthsc.edu
Zhongzhi Wu − Department of Pharmaceutical Sciences,
College of Pharmacy, the University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States;
Email: jimwu@uthsc.edu
Julio F. Cordero-Morales − Department of Physiology, the
University of Tennessee Health Science Center, Memphis,
Tennessee 38163, United States; Email: jcordero@
uthsc.edu
ABBREVIATIONS
■
AITC, allyl isothiocyanate; Cap, capsaicin; GSK101
,
GSK1016790A; GSK170, GSK1702934A; NCS, N-chlorosucci-
nimide; OAG, 1-oleoyl-2-acetyl-sn-glycerol; Pyr3, pyrazole 3;
SAR, structure−activity relationship; TRPC, transient receptor
potential canonical.
Authors
Sicheng Zhang − Department of Pharmaceutical Sciences,
College of Pharmacy, the University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States;
orcid.org/0000-0003-3798-7550
Luis O. Romero − Department of Physiology, the University of
Tennessee Health Science Center, Memphis, Tennessee 38163,
United States; Integrated Biomedical Sciences Graduate
Program, College of Graduate Health Sciences, Memphis,
Tennessee 38163, United States
Shanshan Deng − Department of Pharmaceutical Sciences,
College of Pharmacy, the University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States
Jiaxing Wang − Department of Pharmaceutical Sciences,
College of Pharmacy, the University of Tennessee Health
Science Center, Memphis, Tennessee 38163, United States
Yong Li − Department of Chemical Biology and Therapeutics,
St. Jude Children’s Research Hospital, Memphis, Tennessee
38105, United States
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