Discovery and pharmacological characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation channels

Article abstract of DOI:10.1111/bph.13151

Background and Purpose The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents. Experimental Approach A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by measuring intracellular Ca²⁺ levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated. Key Results SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca²⁺ influx into cells with IC₅₀s of 9.5, 282 and 226 nM, whereas TRPC4 and TRPC5-mediated Ca²⁺ entry was not affected. Patch-clamp experiments confirmed that the compound blocked TRPC6 currents with an IC₅₀ of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive rats (SHR). Conclusions and Implications Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6 channels that opens new opportunities for the investigation of TRPC channel function in vivo.

Full text of DOI:10.1111/bph.13151

DOI:10.1111/bph.13151
www.brjpharmacol.org
British Journal of
BJP Pharmacology
RESEARCH PAPER
Correspondence
Carsten Strübing, Sanofi R&D,
Industriepark Höchst H825,
D-65926 Frankfurt am Main,
Germany. E-mail:
Discovery and
carsten.struebing@sanofi.com
-
---------------------------------------------------------------
pharmacological
*
Present address: Bayer Pharma
AG, Global Drug Discovery.
-
---------------------------------------------------------------
characterization of a novel
potent inhibitor of
diacylglycerol-sensitive TRPC
cation channels
Received
November 2014
Revised
March 2015
Accepted
28 March 2015
7
6
1
1
1
1
1
T Maier , M Follmann *, G Hessler , H-W Kleemann , S Hachtel ,
2
2
1
1
1
1
B Fuchs , N Weissmann , W Linz , T Schmidt , M Löhn , K Schroeter ,
1
1
1
L Wang , H Rütten and C Strübing
1
2
Sanofi R&D, Frankfurt am Main, Germany, and Excellencecluster Cardio-Pulmonary System
ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German
(
Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany
BACKGROUND AND PURPOSE
The cation channel transient receptor potential canonical (TRPC) 6 has been associated with several pathologies including
focal segmental glomerulosclerosis, pulmonary hypertension and ischaemia reperfusion-induced lung oedema. We set out to
discover novel inhibitors of TRPC6 channels and investigate the therapeutic potential of these agents.
EXPERIMENTAL APPROACH
A library of potential TRPC channel inhibitors was designed and synthesized. Activity of the compounds was assessed by
2
+
measuring intracellular Ca levels. The lead compound SAR7334 was further characterized by whole-cell patch-clamp
techniques. The effects of SAR7334 on acute hypoxic pulmonary vasoconstriction (HPV) and systemic BP were investigated.
KEY RESULTS
2
+
SAR7334 inhibited TRPC6, TRPC3 and TRPC7-mediated Ca influx into cells with IC50s of 9.5, 282 and 226 nM, whereas
2
+
TRPC4 and TRPC5-mediated Ca entry was not affected. Patch-clamp experiments confirmed that the compound blocked
TRPC6 currents with an IC50 of 7.9 nM. Furthermore, SAR7334 suppressed TRPC6-dependent acute HPV in isolated perfused
lungs from mice. Pharmacokinetic studies of SAR7334 demonstrated that the compound was suitable for chronic oral
administration. In an initial short-term study, SAR7334 did not change mean arterial pressure in spontaneously hypertensive
rats (SHR).
CONCLUSIONS AND IMPLICATIONS
Our results confirm the role of TRPC6 channels in hypoxic pulmonary vasoregulation and indicate that these channels are
unlikely to play a major role in BP regulation in SHR. SAR7334 is a novel, highly potent and bioavailable inhibitor of TRPC6
channels that opens new opportunities for the investigation of TRPC channel function in vivo.
3
650 British Journal of Pharmacology (2015) 172 3650–3660
© 2015 The British Pharmacological Society

Discovery of the novel TRPC6 inhibitor SAR7334
BJP
Abbreviations
2
+
[
Ca ] , intracellular calcium concentration; FITR, Flp-In T-Rex; FLIPR, fluorometric imaging plate reader; FSGS, focal
i
segmental glomerulosclerosis; HPV, hypoxic pulmonary vasoconstriction; OAG, 1-oleoyl-2-acetyl-sn-glycerol; SHR,
spontaneously hypertensive rats; THF, tetrahydrofuran; TRP, transient receptor potential; TRPC, transient receptor
potential canonical
Tables of Links
TARGETS
LIGANDS
Ion channelsa
TRPC1
TRPC6
TRPC7
Enzymesb
PLC
Econazole
OAG, 1-oleoyl-2-acetyl-sn-glycerol
SKF96365
TRPC3
TRPC4
TRPC5
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://
www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are
a,b
permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 ( Alexander et al., 2013a,b).
tively more active TRPC3 channels that increases basal Ca +
influx into myocytes (Dietrich et al., 2005). Although these
results highlight the interplay and importance of both TRPC3
and TRPC6 channels in the regulation of systemic vascular
tone in mice, it remains unclear to date how TRPC6 channels
modulate BP in a normal genetic context or in disease. In
addition to systemic vasoregulation, experiments in trans-
genic and knock-out mice revealed several other functions of
TRPC6 channels in, for example, cardiac hypertrophic signal-
ling (Wu et al., 2010b; Seo et al., 2014), regulation of lung
endothelial permeability (Weissmann et al., 2012) and plate-
let aggregation (Harper et al., 2013). Moreover, direct evi-
dence for the involvement of TRPC6 channels in human
disease has been found in patients with inherited focal seg-
mental glomerulosclerosis (FSGS). In affected individuals,
activating mutations of TRPC6 channels were identified as
2
Introduction
Since the discovery of transient receptor potential (TRP)
cation channels in the early 1990s, significant efforts have
been directed towards the development of pharmacological
TRP modulators. Such tools are essential for the confirmation
and extension of our current understanding of TRP channel
function that mainly originates from genetic studies (Moran
et al., 2011). Moreover, due to the role of certain TRP chan-
nels in human pathologies (Nilius and Owsianik, 2010; Wu
et al., 2010a), these ion channels represent interesting drug
targets.
A potential candidate for therapeutic intervention is the
TRPC6 channel. This member of the canonical TRP subfamily
is expressed in several tissues, in particular brain, kidney and
vasculature. The TRPC6 channel is activated by the PLC-
generated hydrolysis product diacylglycerol (Hofmann et al.,
2+
the cause of Ca influx into podocytes and subsequent dys-
function of the glomerular filtration barrier (Reiser et al.,
2005; Winn et al., 2005). Therefore, limiting TRPC6 channel
activity may be a novel treatment option for FSGS and other
1
999) and can be modulated by diverse signalling mecha-
nisms including lipid messengers (Fleming et al., 2007; Kwon
et al., 2007) and reactive oxygen species (Ding et al., 2011;
Anderson et al., 2014). Thus, TRPC6 channels serve as signal
integrators, coupling activation of various receptors and
2+
conditions characterized by pathological Ca entry.
It would be highly desirable to validate the therapeutic
value of inhibition of TRPC6 channels using pharmacological
tools. A number of compounds, including SKF96365, econa-
zole, W7 and compound 8009-5364 (Harteneck and Gollasch,
2011; Urban et al., 2012; Bon and Beech, 2013) have been
described as inhibitors of TRPC6 channel activity. However,
the utility of these substances is limited due to their low
potency and poor selectivity. More recently, a series of
anilino-thiazole TRPC3/6 channel blockers have been
described by Washburn et al. (2013). These compounds are
highly potent but have a low oral bioavailability and are not
suitable for chronic pharmacological studies.
2+
intracellular networks to cation – most importantly Ca
–
entry and plasma membrane depolarization. Functional
TRPC channels are most likely tetramers (Mio et al., 2005;
Barrera et al., 2007) and TRPC6 channels have been shown to
form homomers as well as heteromers with other subunits,
especially the closely related TRPC3 and TRPC7 channels
(Hofmann et al., 2002). Such multimeric assembly can create
channel complexes with diverse properties which compli-
cates the elucidation of channel function in native tissues.
Nevertheless, studies in genetically modified animals and
human genetic data have provided important insights into
the (patho)physiological role of TRPC6 channels. TRPC6−
mice were found to have increased vascular contractility
and raised BP. This somewhat unexpected finding can be
explained by the compensatory up-regulation of constitu-
/−
We describe here a novel class of TRPC6 channel inhibi-
tors that combine nanomolar activity with a favourable
pharmacokinetic profile, which allows pharmacological
investigation of channel function in vivo.
British Journal of Pharmacology (2015) 172 3650–3660 3651

T Maier et al.
BJP
Devices, Sunnyvale, CA, USA). Cells grown on black poly-D-
lysine-coated 96-well plates (Greiner, Frickenhausen,
Germany) were washed with standard extracellular solution
Methods
Pharmacophore modelling
(140 mM NaCl, 1 mM MgCl
2
, 5.4 mM KCl, 2 mM CaCl ,
2
The pharmacophore model was built with Catalyst (Accelrys,
San Diego, CA, USA). Up to 250 conformers were generated
for each training molecule. For model generation, the default
parameter settings were used with the following features
allowed for model generation: acceptor, basic, ring aromatic
and hydrophobic features.
10 mM HEPES, 10 mM glucose, pH 7.35) and stained with
dye solution (2 μM Fluo-4 AM, 0.02% pluronic F127, 0.1%
BSA in standard extracellular solution) for 30 min at room
temperature. The cells were rinsed and incubated with stand-
ard extracellular solution supplemented with different con-
centrations of the test compound or vehicle for 10 min.
2+
Ca entry into TRPC3/6/7-expressing cells was elicited
by application of the diacylglycerol, 1-oleoyl-2-acetyl-sn-
glycerol (OAG). For calculation of SAR7334-induced inhibi-
tion, fluorescence values were plotted over time and the AUC
Chemistry
Aminoindanol derivatives with cis or trans geometries were
synthesized (Figure 1B) starting from 2-bromo-1-indanones
by nucleophilic substitution with amines (1.2 eq. amine in
2+
was considered as a measure of Ca influx.
acetone, 1.6 eq. of K
2
CO as base, room temperature, 2 h),
3
subsequent carbonyl reduction (cis: 1.5 eq. L-selectride in
tetrahydrofuran (THF), 0°C – room temperature, 10–48 h;
Electrophysiological techniques
trans: 6.0 eq. NaBH
ture, 3–24 h) and finally O-alkylation/arylation (2.0 eq. alkyl
halide, 5.0 eq. Ag O, toluene, 85°C, 2–5 h / 3.0 eq. aryl fluo-
4
, 1.5 eq. HCl, THF/H
2
O, room tempera-
Whole-cell patch-clamp measurements were performed
essentially as described (Miehe et al., 2012). An intracellular
pipette solution containing 120 mM CsOH, 120 mM glu-
2
ride, 5.0 eq. NaH, DMSO, 150°C, 10–24 h). Trans geometries,
in particular with aryloxy substituents (R5 = aryl), were selec-
tively accessible by epoxide opening of indene oxide with
amines (1.3 eq. amine in MeCN, reflux, 10–24 h) and a Mit-
sunobu reaction with double inversion via an aziridinium
intermediate (Freedman et al., 1991) [1.2 eq R5-OH, 2.5 eq.
conic acid, 2 mM MgCl
10 mM HEPES (pH 7.4 adjusted with gluconic acid) and an
extracellular bath solution containing 140 mM NaCl, 5.4 mM
2
, 3 mM CaCl
2
, 5 mM Cs -BAPTA,
4
KCl, 2 mM CaCl
2
, 1 mM MgCl , 10 mM glucose, 10 mM
2
HEPES (pH 7.4 adjusted with NaOH) were used. Cells grown
on poly-L-lysine-coated coverslips were continuously super-
fused with extracellular solution and compounds were
applied using an ALA BPS-8 perfusion system (ALA Scientific
Instruments, Westbury, NY, USA). Cells were held at −70 mV
and current-voltage relationships were measured every 2 or
3 s by applying voltage ramps (180 ms) from −100 to +80 mV.
All experiments were performed at room temperature.
polymer(polysterene)-bound PPh , 2.0 eq. diisopropyl azodi-
3
carboxylate, THF, room temperature, 10–24 h]. Stereochemi-
cally pure SAR7334 was selectively synthesized starting from
(1R,2S)-indene oxide (Larrow et al., 1999).
Detailed information and an explicit experimental
pathway for the preparation of SAR7334 is provided in Sup-
porting Information Fig. S2.
Ethics statement
Cell culture and cell line generation
All animal care and experimental procedures were performed
according to Sanofi Ethical Committee guidelines and to the
Guide for the Care and Use of Laboratory Animals published
by the National Institutes of Health. Sanofi is an authorized
institution to house and handle laboratory animals according
Cells were grown at 37°C in a humidified atmosphere (5% or
7
% CO ) under standard cell culture conditions. Stable HEK
2
cell lines expressing recombinant hTRPC6 (GenBank acces-
sion number AF080394) or TRPC7 channels (GenBank
accession number AJ272034) under the control of
a
§11 German Animal Welfare Act. Procedures were approved
tetracycline-inducible promoter were generated using the
Flp-In T-Rex (FITR) system (Invitrogen, Karlsruhe, Germany).
TRPC6 and TRPC7 HEK-FITR cells were maintained in DMEM
by the competent authorities (Regierungspräsidium Darm-
stadt or Regierungspräsidium Giessen). All studies involving
animals are reported in accordance with the ARRIVE
guidelines for reporting experiments involving animals
−
1
−1
(
with GlutaMAX I, 4.5 g·L glucose and 110 mg·mL sodium
pyruvate) supplemented with 10% (v/v) FBS (Biochrom,
Berlin, Germany), 1 mM glutamine, 1 mM MEM sodium
(Kilkenny et al., 2010; McGrath et al., 2010). A total of 26
animals were used in the experiments described here.
−1
−1
pyruvate, 40 μg·mL hygromycin and 15 μg·mL blasticidine
HCl. Channel expression was induced by supplementing the
−
1
Measurement of hypoxic pulmonary
growth medium for 18–24 h with 1 μg·mL doxycycline.
hTRPC3 channels (GenBank accession number NM_003305)
were stably expressed in CHO cells using a proprietary high
expression vector (Steinbeis – Transferzentrum für Ange-
wandte Biologische Chemie, Mannheim, Germany) and
maintained in HAM’s F12 supplemented with 10% (v/v)
vasoconstriction (HPV) in isolated perfused
and ventilated lungs
C57/BL6N mice were anaesthetised with xylazine and keta-
mine and anticoagulated with heparin as previously described
(Weissmann et al., 2004; Fuchs et al., 2011). Male 6–8 weeks
old mice were from Charles River Laboratories (Sulzfeld,
Germany). In brief, lungs were explanted during deep anaes-
thesia and artificially ventilated and perfused blood free at
2 mL·min− at 37°C with Krebs-Henseleit buffer containing
−
1
FBS (Biochrom), 1 mM glutamine, 0.6 mg·mL geneticin,
1
−1
1
00 U·mL− penicillin and 100 μg·mL streptomycin.
1
Fluo-4 measurement of intracellular calcium
2
+
concentration ([Ca ]
i
)
120 mM NaCl, 4.3 mM KCl, 1.1 mM KH
1.3 mM MgCl , 13.32 mM glucose, 5% (w/v) hydroxyethyla-
mylopectin and 23.8 mM NaHCO . The left atrial pressure was
2
PO
4
, 2.4 mM CaCl ,
2
2+
Ca measurements were performed at room temperature
using a fluorometric imaging plate reader (FLIPR; Molecular
2
3
3
652 British Journal of Pharmacology (2015) 172 3650–3660

Discovery of the novel TRPC6 inhibitor SAR7334
BJP
A: Pharmacophore model
B: Library design
cis or trans Series
R5
R3
O
O
1. CO reduction
. LG R5
O
N
H
R4
R3
R4
2
R3
R4
R1
Br
R1
N
R1
N
R2
R2
R2
Further derivatization
if R2, R4, R5 = PG
trans Series
R3
R5
R3
R4
O
N
N
H
R4
O
HO R5
R3
R4
R1
R1
R1
OH
N
R2
R2
R2
via
R3
+
R1, R2 = substitued alkyl, aryl, acyl, alkoxy, halogen, etc.
R3, R4 = substituted alkyl, aryl
N
R4
R1
R5 = substituted alkyl, aryl, acyl
R2
LG = leaving group, PG = protecting group
FLIPR screening of > 1200
library compounds
C: Structure of SAR7334
N
SAR7334
Cl
O
N
N
Figure 1
Discovery of SAR7334 using pharmacophore-guided design of focused aminoindanol libraries. Pharmacophoric information derived from a
selection of known analogues of the cation channel inhibitor SKF96365 were translated into library designs and synthesized. Promising
aminoindanol derivatives were further investigated with emphasis on stereochemical positioning of relevant substituents. (A) Mapping of SAR7334
to the TRPC6 pharmacophore model. The red sphere corresponds to a positive ionizable moiety, the light blue sphere indicates lipophilic features,
while brown spheres mark ring aromatic features. The mapping of SAR7334 to the pharmacophore was generated with LigandScout (Inteligand
Software-Entwicklungs und Consulting GmbH, Maria Enzersdorf, Austria). (B) Aminoindanol derivatives with cis or trans geometries were accessed
from 2-bromo-1-indanones by nucleophilic substitution with amines, carbonyl reduction and subsequent O-alkylation/arylation. Trans geometries,
in particular with aryloxy substituents (R5 = aryl) were realized by epoxide opening of indene oxide with amines and a Mitsunobu reaction with
double inversion. (C) Structure of SAR7334.
British Journal of Pharmacology (2015) 172 3650–3660 3653

T Maier et al.
BJP
set at 2.0 mmHg. Positive pressure ventilation (250 μL tidal
of vehicle or SAR7334 (labelled ‘post’). This interval corre-
sponded to the maximal plasma levels of SAR7334 (see
Figure 6). Baseline data (labelled ‘pre’) were sampled over the
same time interval on the day before treatment.
−
1
volume, 90 breath·min
and 2 cm
H
2
O positive end-
expiratory pressure) was performed with a mixture containing
2
1% O
2
, 5.3% CO
2
, balanced with N
2
(normoxia) or 1% O ,
2
5
.3% CO
2
(hypoxia). The pressure in the pulmonary artery and
in the left atrium was measured. Changes in pulmonary artery
pressure directly reflect changes in pulmonary vascular tone as
the lungs are perfused at constant flow. Lungs were ventilated
in an alternating mode with hypoxia (10 min) or normoxia
Data analysis
Averaged data are expressed as means ± SEM. Repeated meas-
ures two-way ANOVA was performed with GraphPad Prism
(GraphPad Software Inc., La Jolla, CA, USA) to analyse treat-
(15 min) to induce acute HPV. In such a sequence of repetitive
ment effects on BP. P-< 0.05 was considered as statistically
significant. IC50 values were calculated with Origin 6.0 soft-
hypoxic ventilation manoeuvres, increasing doses of SAR7334
were applied 5 min prior to the next hypoxic ventilation
manoeuvre. For application the SAR7334 stock solution
ware (Microcal Software Inc., Northampton, MA, USA). Data
2+
from [Ca ]
i
measurements were normalized to the area under
(2 mM in 100% DMSO) was diluted 1:100 in perfusion buffer
the fluorescence curve in the absence of SAR7334 and fitted
using a standard three-parameter logistic model. The relative
current inhibition in patch-clamp experiments was defined as
the decrease of the OAG-induced current after application of
SAR7334 normalized to the maximal OAG-induced current
before drug application. Dose response curves were fitted to
and appropriate amounts were cumulatively added to the
recirculating perfusate (15 mL). The first application was per-
formed after the second hypoxic ventilation period. The
strength of acute HPV is given as the maximum increase of
pulmonary arterial pressure in each hypoxic ventilation
period, referenced to the strength of the second hypoxic
manoeuvre (set at 100%).
n
the equation: f = 100 − (100/(1 + (C/IC50) )); where f is the
relative inhibition, C is the applied drug concentration and n
is the Hill coefficient. For isolated perfused lung experiments,
statistical analysis was performed by Student’s t-test.
In vivo determination of
SAR7334 pharmacokinetics
Plasma concentrations of SAR7334 were determined in a serial
sampling study after single oral administration of the com-
pound (250 g) in 30% glycopherol/cremophor (75/25) 70%
glucose (5%) solution to male Sprague Dawley rats (Harlan
Winkelmann, Borchen, Germany). From each animal, eight
plasma samples (approximately 200 µL blood were taken by tail
tip sampling) were collected over 24 h and stored below −15°C
until analysis. After addition of the precipitant solution (ace-
tonitrile) containing an analogous internal standard, the test
item SAR7334 was detected by LC-MS/MS, using an Agilent LC
Materials
DMEM, HAM’s F12, glutamine, MEM sodium pyruvate, hygro-
mycin B, blasticidin HCl, geneticin, Fluo-4 AM, Pluronic F127
and Cs
4
-BAPTA were from Invitrogen; MgCl
2
and MgSO were
4
from Merck (Darmstadt, Germany); doxycycline was from BD
Biosciences (Heidelberg, Germany), and OAG was from Avanti
Polar Lipids Inc. (Alabaster, AL, USA). All other chemicals were
from Sigma-Aldrich (Munich, Germany).
(
Series 1200; Agilent Technologies, Santa Clara, CA, USA) with
Results
CTC HTC PAL auto sampler (CTC Analytics AG, Zwingen,
Switzerland) and a Sciex API4000 (AB Sciex, Toronto, Canada)
mass spectrometer in the positive ion mode. Using a sample
volume of 50 μL, the lower limit of quantitation was 2.0 ng·mL
and the linear range was between 2.0 and 2000 ng·mL .
We identified several close analogues of the cation channel
inhibitor SKF96365 by similarity and substructure searches as
potential inhibitors of TRPC6 channels and investigated the
-1
−1
2
+
ability of these compounds to block TRPC6-mediated Ca
entry in TRPC6-HEK-FITR cells. For the substructure search,
the relative position and type of aromatic rings were system-
atically varied. Examples of identified molecules with activi-
ties in the μM range are given in Supporting Information
Fig. S1. Validated actives were subsequently used to derive a
pharmacophore model with Catalyst (Kurogi and Guner,
2001; Guner et al., 2004). Next, several scaffolds and geom-
etries were evaluated for their potential to inhibit TRPC6
activity using this pharmacophore. One of the best matching
structures was the aminoindanol scaffold as illustrated with
the derivative SAR7334 (Figure 1A).
Capitalizing on the variable chemistry of this structural
class, we designed a library of more than 1200 diversely
substituted aminoindanol derivatives with a special emphasis
on the stereochemical positioning of substituents (Figure 1B).
While a cis relationship at the indane was realized starting
from the bromoketone, the eventually more interesting trans
configuration was quantitatively accessed using indene oxide
as starting material. Potential asymmetric centres in the sub-
stituents (R1-5) were introduced with the corresponding
Telemetric assessment of BP
Adult male (6 months old) spontaneously hypertensive rats
(SHR; Harlan Winkelmann) were treated on two consecutive
-1
days. On day one, the animals received 1 mL·kg vehicle by
oral gavage. After 24 h, rats received either vehicle alone or
-1
were treated with 10 mg·kg SAR7334. Telemetric measure-
ment of BP was performed as described (Lohn et al., 2009). In
brief, a telemetric device (TL11M2-C50-PXT PMP, Data Sci-
ences International, St. Paul, MN, USA) was placed between
the aorta and the vena cava and the catheter tip of the
transmitter was inserted into the aorta. Systolic BP, diastolic
BP and heart rate were acquired continuously at a sampling
rate of 500 Hz and data were stored as 5 min averages. Mean
arterial pressure was calculated from systolic and diastolic
pressure and low-pass filtered using the fast Fourier transform
function of the vendor software (Dataquest A.R.T. V4.0, Data
Sciences International) for better visualization of time-
dependent BP variations. For statistical analysis, raw data
were averaged over a 6 h period starting 2 h after application
3
654 British Journal of Pharmacology (2015) 172 3650–3660

Discovery of the novel TRPC6 inhibitor SAR7334
BJP
starting materials. FLIPR screening of this library identified
several compounds with TRPC6 channel blocking activity.
Among the most potent inhibitors was SAR7334 (Figure 1C)
featuring a trans relationship of the substituents at the indane
five-membered ring. Stereochemically defined access to
SAR7334 is possible in four steps starting from indene (Sup-
porting Information Fig. S2).
SAR7334 reduced TRPC6-mediated Ca influx with an
IC50 of 9.5 nM. TRPC3 and TRPC7 channels, the two closest
TRPC6 channel homologues, were also inhibited although at
significantly higher concentrations (IC50 = 282 nM and 226
nM respectively) (Figure 2).
By contrast, the compound did not have appreciable
effects on TRPC4 or TRPC5 channels (IC50 > 10 μM, see Sup-
porting Information Fig. S3). To verify the channel-inhibiting
activity of SAR7334, we performed patch-clamp experiments
on TRPC6-HEK-FITR cells. Application of 50 μM OAG stimu-
lated typical non-selective cation currents through TRPC6
channels in these cells. SAR7334 dose-dependently reduced
TRPC6 currents with an IC50 of 7.9 nM (Figure 3).
confer sensitivity to SAR7334. In addition to compound
selectivity, we were interested in the pharmacokinetic prop-
erties of SAR7334. Measuring plasma concentrations in rats
revealed that pharmacologically effective concentrations of
the substance were reached and maintained for several hours
after oral administration (Figure 6).
Taken together, the high potency, good selectivity profile
and appropriate pharmacokinetic properties suggest that
SAR7334 is a suitable molecule for the investigation of
TRPC6-mediated processes in vivo. Therefore, we decided to
use the compound to study the effect of inhibiting TRPC6
channels on BP in SHR. For this purpose, BP was monitored
telemetrically in conscious animals. Our results illustrated in
Figure 7 did not show an effect of SAR7334 on arterial pres-
sure, questioning the role of TRPC6 channels in regulation of
systemic vascular tone in rodents.
2
+
Discussion and conclusions
Thus, SAR7334 blocked TRPC6-mediated ion currents and
Ca + influx with similar potency. Likewise, recordings from
TRPC3- and TRPC5-expressing cells (Supporting Information
Fig. S4) confirmed the substantially lower potency of the
compound towards these ion channels, as seen in the FLIPR
experiments.
In this work, we used a rational drug design approach to
synthesize libraries of novel small-molecule inhibitors of
TRPC6 channels. The aminoindanol derivative SAR7334 was
among the most potent TRPC6 channel blockers identified by
FLIPR screening. Patch-clamp experiments confirmed that
SAR7334 suppressed TRPC6 currents with an IC50 < 10 nM.
Thus, the potency of the compound is comparable with that
of the recently identified anilino-thiazole series of TRPC6/3
channel inhibitors (Washburn et al., 2013).
2
As physiological activation of TRPC6 channels is triggered
by PLC-coupled receptors, we further investigated how
receptor-stimulated TRPC6 responses were affected by
SAR7334. For this purpose, we challenged TRPC6-HEK-FITR
cells with trypsin which had been shown to activate PLC-
linked proteinase-activated receptors 2 and to robustly induce
Previous studies in TRPC6− animals, as well as pharma-
cological studies, have demonstrated that acute HPV in mice
is mediated specifically by TRPC6 channels (Weissmann et al.,
2006; Urban et al., 2012). The results of our experiments in
the isolated perfused lung model were consistent with these
data. Importantly, these results also demonstrated that
SAR7334 was able to suppress native TRPC6 channel activity.
It is not yet clear why inhibition of HPV required significantly
higher concentrations of the compound than blockade of
recombinant TRPC6 channel responses. An explanation for
the shift in dose dependence often seen in intact lung experi-
ments compared with cellular assays might be that com-
pound access to the target arterial muscle cells is limited by
the endothelial barrier. However, it is equally possible that
native TRPC6 channel complexes incorporate other TRPC
subunits or auxiliary proteins such as ORAI1 and STIM1 that
modify channel pharmacology. The TRPC1 channel is
co-expressed with TRPC6 channels in precapillary pulmonary
artery smooth muscle cells, whereas all other TRPC channel
isoforms including TRPC3 and TRPC7 are only present at very
low levels (Weissmann et al., 2006). Although the biochemi-
cal evidence for the formation of TRPC1/TRPC6 channel
complexes is contradictory (Xu et al., 1997; Hofmann et al.,
2002; Strubing et al., 2003), functional TRPC1/TRPC6 heter-
omers have been described in heterologous systems (Storch
et al., 2012). Therefore, such channels could potentially exist
in pulmonary arteries and account for the observed attenu-
ated sensitivity to SAR7334. In any case, it will be interesting
to examine the effect of SAR7334 in models that co-express
TRPC6 channels together with putative binding partners in
order to compare the pharmacology of such heteromeric
assemblies with that of native TRPC6 channel complexes.
/−
2+
intracellular Ca release in HEK cells (Kawabata et al., 1999).
In most TRPC6-HEK-FITR cells, trypsin evoked large, rapidly
declining TRPC6 currents that reached an almost steady-state
level within 30–60 s. This current was substantially reduced
2
+
by 100 nM SAR7334 (Figure 4), whereas Ca release and,
hence, the underlying PLC activity was not attenuated by up
to 10 μM of the inhibitor (Supporting Information Fig. S5).
The reduction of trypsin-induced TRPC6 currents by
1
00 nM SAR7334 amounted to 88 ± 3% (n = 5) which was not
different from the corresponding effect on OAG-induced
current responses (see Figure 3C).
Having observed inhibition of both OAG- and receptor-
induced TRPC6 currents by SAR7334, we next wanted to test
the activity of the compound in a relevant physiological
model. Acute HPV has been shown to exclusively depend on
the activity of TRPC6 channels (Weissmann et al., 2006). We,
therefore, investigated the effect of SAR7334 on hypoxic vaso-
constriction in perfused isolated lungs from mice and found
that this compound abolished hypoxia-induced increases in
pulmonary arterial pressure. Half maximal inhibition was
achieved at about 100 nM indicating that SAR7334 is able to
efficiently block native TRPC6 channels in situ (Figure 5).
Selectivity testing showed that the compound had little
effect on other ion channels or receptors (Supporting Infor-
2
+
mation Fig. S3). Notably, store-operated Ca entry driven by
ORAI1/STIM1 was also largely resistant to SAR7334 (Support-
ing Information Fig. S6). Thus, despite the reported interac-
tion of ORAI1 and STIM1 with TRPC6 channels (Liao et al.,
2
007; Jardin et al., 2009), it seems unlikely that they directly
British Journal of Pharmacology (2015) 172 3650–3660 3655

T Maier et al.
BJP
A
B 1.0
TRPC6
TRPC3
TRPC7
3
3
2
2
2
1
1
60
+
OAG
20
80
40
00
60
20
0
.8
0.6
7
.5 nM
0
0
.4
.2
15 nM
IC = 9.5 nM
5
0
8
0
0
0
3
0 nM
4
0.0
75 nM
0
.001
0.01
0.1
con
–40
[SAR7334] (µM)
1
min
C
D 1.0
+
OAG
2
2
2
1
1
1
70
40
10
80
50
20
75 nM
0
.8
.6
150 nM
0
300 nM
0.4
IC = 282 nM
5
0
90
60
30
0
0
.2
.0
750 nM
0
con
0.01
0.1
1
–
30
[
SAR7334] (µM)
1
min
E
F 1.0
3
3
2
2
1
1
50
00
50
00
50
00
+ OAG
0
.8
.6
75 nM
0
150 nM
3
00 nM
0.4
IC = 226 nM
5
0
0
.2
.0
5
0
750 nM
con
0
0
–
50
0.01
0.1
1
–
100
[
SAR7334] (µM)
1
min
Figure 2
Inhibition of Ca2 influx via diacylglycerol-sensitive TRPC channels by SAR7334. Time-dependent changes of intracellular Ca2+ concentration in
+
Fluo-4-loaded induced TRPC6- HEK-FITR cells (A), TRPC3-CHO cells (C) and TRPC7 HEK-FITR cells (E) were measured using FLIPR. TRPC-mediated
2+
Ca influx following application of 30 μM OAG (+OAG) was dose-dependently reduced in cells pre-incubated with the indicated concentrations
of SAR7334. Control traces (con) were recorded during application of extracellular solution. Representative traces are shown. The relative
inhibition of TRPC6 (B), TRPC3 (D) and TRPC7 channels (F) was calculated from the AUC for each concentration SAR7334 and IC50s were derived
from the best fit of the data (solid lines) with a logistic model. Data are given as means ± SEM (n = 5–10).
3
656 British Journal of Pharmacology (2015) 172 3650–3660

Discovery of the novel TRPC6 inhibitor SAR7334
BJP
I (nA)
1
00 nM SAR7334
1
0
0
.2
.8
.4
A
A
Trypsin
1
0
3
4
–0.2
2
–
0.4
con
5
0 µM OAG
–0.6
5
0 µM OAG + 1 µM SAR7334
–
–
0.8
1.0
–100 –80 –60 –40 –20
20
40
60
80
V (mV)
m
–
0.4
0.8
–
–
–
–
1.8
2.0
2.2
2.4
–
1
µM
–2.6
1
00 nM
10 nM
0
30
60
90
120 150 180 210 240 270 300 330 360
B
0.2
Time (s)
1
nM
0
.0
I (pA)
9
6
3
00
00
00
2
4
B
–
–
–
–
–
0.2
0.4
0.6
0.8
1.0
3
1
–
100 –80 –60 –40 –20
–300
20
40
60
80
V (mV)
m
0
30
60
90
120 150 180 210 240 270 300
–
600
Time (s)
C 1
Figure 4
00
Inhibition of receptor-stimulated TRPC6 currents by of SAR7334.
Whole-cell currents were measured in TRPC6-HEK-FITR cells. (A) The
time course of TRPC6 currents at −70 mV during application of
8
6
4
2
0
0
0
0
0
2
00 nM trypsin and 100 nM SAR7334 is depicted. Trypsin and
SAR7334 were added to the extracellular solution during the time
indicated by the bars. (B) Current responses measured during
voltage ramps at the time points indicated in A are shown.
IC = 7.9 nM
50
The expression of TRPC6 channels in vascular myocytes
and its demonstrated role in receptor and pressure-induced
2+
Ca influx support the idea that the channel may be involved
1
10
100
1000
in BP regulation. Consistent with this proposal, up-regulation
of TRPC6 channels has been detected in several hypertensive
animal models (Bae et al., 2007; Mita et al., 2010; Zulian et al.,
[
SAR7334] (nM)
Figure 3
2010). However, the study of vascular effects of these chan-
nels in TRPC6−
/−
mice is obscured by compensatory
SAR7334 potently inhibits TRPC6 whole-cell currents. TRPC6 currents
were elicited in TRPC6-HEK-FITR cells by application of 50 μM OAG.
up-regulation of TRPC3 channels in some, but not all, vascu-
(A) Current traces measured during voltage ramps before and after
lar beds (Dietrich et al., 2005; Weissmann et al., 2006). The
application of the TRPC6 channel activator OAG and after consecutive
additions of SAR7334. (B) Time course and dose dependence of
SAR7334-induced TRPC6 inhibition. Whole-cell currents were meas-
ured at −70 mV during voltage ramps and plotted versus time. For
better comparison of responses, current amplitudes were normalized
to the maximal current after addition of OAG and maximal currents
were superimposed. The arrow indicates application of SAR7334. (C)
TRPC6 currents were measured as in (B) and inhibition was deter-
mined in steady state after application of the respective concentration
of SAR7334. The dose–response relationship was fitted to a logistic
function. Data are given as means ± SEM (n = 3–12).
−/−
hypertensive phenotype of TRPC6 animals, caused by exag-
2+
gerated TRPC3-mediated Ca entry into smooth muscle cells,
does not allow us to draw conclusions about the vascular
function of TRPC6 channels in wild-type animals. Therefore,
we set out to clarify the effect of acute inhibition of TRPC6
channels in SHR, an established rodent hypertension model.
Telemetric assessment of BP did not show any effect of
SAR7334 application despite the fact that circulating com-
pound levels were sufficient to achieve complete inhibition of
TRPC6 channels. This result indicates that TRPC6 channels
British Journal of Pharmacology (2015) 172 3650–3660 3657

T Maier et al.
BJP
1
A
1
1
20
00
Hypoxia control (n=5)
8
6
4
2
0
0
0
0
0
0.1
Hypoxia + SAR7334 (n=5)
*
**
***
0
200
400
600
800
1000
0.01
Concentration (nM)
0
4
8
12
16
20
24
Time (h)
B
Hypoxia control
Hypoxia + SAR7334
Figure 6
Hypoxia
Hypoxia
Pharmacokinetic profile of SAR7334. Plasma concentrations of
SAR7334 were determined after single oral administration of
1
2
1
2
SAR7334
−1
11
11
10
9
10 mg·kg of the compound to male Sprague Dawley rats. Data are
control
given as means ± SEM (n = 3).
1
0
9
0
0
Acknowledgements
0
5
10 15 20 25
0
5
10 15 20 25
Time (min)
Time (min)
We would like to thank Andrea Leonhardt, Stefan Müller, Tim
Koschella and Karin Quanz for expert technical assistance as
well as Markus Rupp for performing isolated lung experi-
ments. We also thank Dr. Werngard Czechtizky and Dr. Klaus
Steinmeyer for critical reading of the manuscript.
Figure 5
Effect of SAR7334 on hypoxia-induced vasoconstriction in isolated
perfused and ventilated lungs. (A) Acute hypoxic pulmonary vaso-
constriction (HPV) was assessed as the increase in pulmonary artery
pressure (ΔPAP) upon hypoxic ventilation in isolated perfused mouse
lungs. Data were normalized to the reference pressure response in
the absence of SAR7334 for each experiment. SAR7334 dose-
dependently reduced the strength of HPV. SAR7334 had no signifi-
cant effect on normoxic vascular tone (not shown). Data shown are
means ± SEM (n = 5). ***P < 0.001, significantly different from
control response. (B) Original recordings of PAP in the absence (left)
or presence (right) of 1 μM SAR7334. Hypoxic challenge is indicated
by the bars.
Author contributions
T. M., M. F., G. H., H.-W. K., S. H., B. F., N. W., W. L., T. S.,
M. L., K. S., L. W. and C. S. performed the research and
analysed the data. T. M., M. F., G. H., H.-W. K., S. H., N. W.,
W. L., T. S., M. L., K. S., L. W., H. R. and C. S. designed the
research. T. M. and C. S. wrote the manuscript.
do not play a major role in systemic BP regulation in SHR.
Nevertheless, it remains to be seen whether the same holds
true for other hypertensive models, for example those with
increased levels of vascular TRPC6 channels, and whether
local haemodynamics are modulated by TRPC6 channel
inhibition.
SAR7334 combines high potency with a good oral phar-
macokinetic profile and reasonable selectivity versus TRPC3
and TRPC7 channels which make this compound a valuable
tool for further evaluation of TRPC channel pharmacology in
vivo. Clearly, more work is needed to fully explore the poten-
tial therapeutic utility of SAR7334 and other TRPC channel
blockers. Given the compelling data associating TRPC6
channels with diseases such as FSGS or lung ischaemia
reperfusion-induced oedema (Reiser et al., 2005; Winn et al.,
Conflicts of interests
T Maier, M Follmann, G Hessler, H-W Kleemann, S Hachtel,
W Linz, T Schmidt, M Löhn, K Schroeter, L Wang and C
Strübing were employees of Sanofi-Aventis Deutschland
GmbH at the time this study was conducted. T Maier, M
Follmann, G Hessler, H-W Kleemann, S Hachtel, L Wang and
C
Strübing are inventors on
a
patent application
(WO2011107474 A1) related to this work. T Schmidt is a
contributor to WO2011107474 A1 according to applicable
German law. N Weissmann was funded by a grant from
Sanofi-Aventis Deutschland GmbH.
2
005; Weissmann et al., 2012), we believe that such efforts are
worthwhile pursuing.
3
658 British Journal of Pharmacology (2015) 172 3650–3660

Discovery of the novel TRPC6 inhibitor SAR7334
BJP
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70
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(
average MAP in vehicle-treated (n = 8) and SAR7334-treated (n = 5)
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