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.
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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.
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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
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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
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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