Identification of potent and selective small molecule inhibitors of the cation channel TRPM4

Article abstract of DOI:10.1111/bph.14220

Background and Purpose: TRPM4 is a calcium-activated non-selective cation channel expressed in many tissues and implicated in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and selective inhibitors. We sought to discover a novel series of small-molecule inhibitors by combining in silico methods and cell-based screening assay, with sub-micromolar potency and improved selectivity from previously reported TRPM4 inhibitors. Experimental Approach: Here, we developed a high throughput screening compatible assay to record TRPM4-mediated Na⁺ influx in cells using a Na⁺-sensitive dye and used this assay to screen a small set of compounds selected by ligand-based virtual screening using previously known weakly active and non-selective TRPM4 inhibitors as seed molecules. Conventional electrophysiological methods were used to validate the potency and selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied using Western blots and electrophysiology experiments. Key Results: A series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub-micromolar potency and adequate selectivity. We also showed for the first time that a naturally occurring variant of TRPM4, which displays loss-of-expression and function, is rescued by the most promising compound 5 identified in this study. Conclusions and Implications: The discovery of compound 5, a potent and selective inhibitor of TRPM4 with an additional chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates.

Full text of DOI:10.1111/bph.14220

Identification of potent and selective small molecule inhibitors of the cation
channel TRPM4
Lijo Cherian Ozhathil*1, Clémence Delalande*2, Beatrice Bianchi1, Gabor Nemeth2, Sven
Kappel1, Urs Thomet1, Daniela Ross-Kaschitza1, Céline Simonin2, Matthias Rubin1, Jürg
Gertsch1, Martin Lochner1,2, Christine Peinelt1, Jean-Louis Reymond2, Hugues Abriel1
Running Title: Potent TRPM4 inhibitors
Affiliations:
1: Institute of Biochemistry and Molecular Medicine, National Center of Competence in Research
NCCR TransCure, University of Bern, Bühlstrasse 28, 3012 Bern (Switzerland)
2: Department of Chemistry and Biochemistry, National Center of Competence in Research NCCR
TransCure, University of Bern, Freiestrasse 3, 3012 Bern (Switzerland)
* Co-first authors.
Corresponding authors:
Hugues Abriel
Jean-Louis Reymond
Institute of Biochemistry
and Molecular Medicine
Department of Chemistry and Biochemistry
National Center of Competence in Research
National Center of Competence in Research NCCR TransCure
NCCR TransCure
University of Bern, Freiestrasse 3, 3012 Bern
University of Bern, Bühlstrasse 28, 3012 Bern (Switzerland)
(Switzerland)
E-mail: jean-louis.reymond@dcb.unibe.ch
E-mail: hugues.abriel@ibmm.unibe.ch
Acknowledgements:
We thank Jean-Sebastien Rougier, PhD and Sarah Vermij for helpful discussions and critical proof
reading. This work was supported by NCCR Transcure 51NF40-160620 and a grant by the Swiss
Heart Foundation to Hugues Abriel.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.14220
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BACKGROUND AND PURPOSE
TRPM4 is a calcium-activated nonselective cation channel expressed in many tissues and implicated
in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and
selective inhibitors. We sought to discover a novel series of small-molecule inhibitor by combining in
silico methods and cell based screening assay, with sub-micromolar potency and improved selectivity
from previously reported TRPM4 inhibitors.
EXPERIMENTAL APPROACH
Here, we developed a HTS compatible assay to record TRPM4-mediated Na+ influx in cells using a
Na+-sensitive dye and used this assay to screen a small set of compounds selected by ligand-based
virtual screening using previously known weakly active and non-selective TRPM4 inhibitors as seed
molecules. Conventional electrophysiological methods were used to validate the potency and
selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously
expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied
using western blots and electrophysiology experiments.
KEY RESULTS
A series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with
sub-micromolar potency and adequate selectivity. We also show for the first time that a naturally
occurring variant of TRPM4, which display loss-of-expression and function, is rescued by the most
promising compound 5 identified in this study.
CONCLUSIONS AND IMPLICATIONS
The discovery of compound 5, the most potent and selective inhibitor of TRPM4 with an additional
chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human
diseases and developing clinical drug candidates.
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KEYWORDS:
TRP channels, TRPM4, drug discovery, ligand based virtual screening, 9-phenanthrol, chemical
chaperone, prostate cancer.
ABBREVIATIONS:
TRPM4
Transient receptor potential cation channel subfamily M member 4
HTS
High throughput screening
HEK 293
Human embryonic kidney cells 293
LNCaP
Lymph node carcinoma of the prostate
INTRODUCTION
The ion channel TRPM4 belongs to the 28-member TRP (transient receptor potential) channel family.
It is activated upon increase of intracellular Ca2+ and conducts monovalent cations such as Na+, K+,
and Cs+, hence modulating the transmembrane electrical potential. TRPM4 is expressed in many cell
types and tissues, but its role in physiology is still poorly understood (Launay et al., 2002; Nilius et
al., 2003; Fonfria et al., 2006). With regard to its role in human diseases, more than 20 TRPM4
genetic variants have been described in families with cardiac conduction alterations such as
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progressive conduction block and congenital atrioventricular block (AVB) (Kruse and Pongs, 2014;
Syam et al., 2016). A significant number of these pathogenic TRPM4 variants leads to gain-of-
expression and function, while others to loss-of-expression and function (Kruse et al., 2009; Liu et al.,
2010, 2013; Syam et al., 2016). Based on studies with experimental autoimmune encephalitis (EAE)
mice model, TRPM4 has been suggested to be a key player in the neurodegenerative process of
multiple sclerosis (Schattling et al., 2012; Malhotra et al., 2013; Makar et al., 2015). Interestingly
TRPM4-deficient mice with EAE displayed much milder clinical manifestations than wild-type (WT)
EAE mice (Schattling et al., 2012). Recently, TRPM4 has also been shown to be involved in
migration and proliferation of human prostate cancer cells (Ashida et al., 2004; Singh et al., 2006;
Suguro et al., 2006; Prevarskaya et al., 2007; Schinke et al., 2014; Holzmann et al., 2015; Berg et al.,
2016; Sagredo et al., 2017). Taken together, these recent findings strongly suggest that TRPM4 is a
potential pharmacological target for treating neurological and cardiovascular disorders, as well as
certain types of cancer.
Most of the characterizations of TRPM4 in different tissues have heavily relied on patch clamp
technique with pharmacological inhibitors that have several issues. Until now, 9-phenanthrol has been
the most commonly used TRPM4 inhibitor to dissect its roles in physiology and diseases (Figure 1)
(Grand et al., 2008; Guinamard et al., 2014). However, the low potency and lack of selectivity of 9-
phenanthrol on TRPM4 limit its usefulness in studies using animal models or primary cell lines
(Burris et al., 2015; Garland et al., 2015). Other reported TRPM4 inhibitors include flufenamic acid
(FFA), (Figure 1) acting similarly weakly on TRPM4 but with a longer list of documented off-target
effects that are related to its primary role as a non-steroidal anti-inflammatory drug (Gardam et al.,
2008). While glibenclamide, an even weaker TRPM4 inhibitor also inhibit ATP-dependent K+
channel, which are its primary targets in treatment of type II diabetes (Demion et al., 2007; Alexander
et al., 2013).
Here, we developed a fluorescence cell-based screening assay monitoring TRPM4-induced Na+ influx
using an intracellular Na+ specific dye (Asante Natrium Green-II, ANG-II). This assay allowed
reliable assessment of TRPM4 activity modulation by small molecules. In the absence of structural
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information on inhibitor binding sites on TRPM4, we used a ligand-based virtual screening (LBVS)
approach (Scior et al., 2012) to search commercially available compounds for analogs of the above
mentioned weak and non-selective TRPM4 inhibitors (Sterling and Irwin, 2015). We performed
LBVS with the 3D-shape and pharmacophore similarity algorithm xLOS (atom category eXtended
Ligand Overlap Score) as similarity measure. xLOS is a scaffold-hopping algorithm (Schneider et al.,
1999) that recently proved its validity in related searches for selective Ca2+ channel (Simonin et al.,
2015) and kinase inhibitors (Kilchmann et al., 2016). Activity screening of a small series of
compounds selected by LBVS procedure allowed us to rapidly identify anthranilic amides 4 and 5,
two analogs of FFA with a 10-fold stronger and more selective inhibition of TRPM4 compared to 9-
phenanthrol (Figure 1). Structure-activity relationship (SAR) study further uncovered 6 as the first
selective sub-micromolar TRPM4 inhibitor. These TRPM4 blockers reversibly inhibit the
overexpressed TRPM4 channels in HEK293 cells and block TRPM4-mediated currents in human
prostate cancer cells. Their direct and specific interaction with TRPM4 is further exemplified by their
activity as chemical chaperone to rescue the expression and activity of a loss-of-function TRPM4
variant. These results prepare the way for more potent and specific TRPM4 inhibitors.
RESULTS
Development and validation of a TRPM4 dependent Na+ influx-screening assay
First, we developed a Na+ influx assay to screen large libraries of chemically diverse small molecules.
We used a cell line stably expressing TRPM4 under a tetracycline-inducible promoter (Amarouch et
al., 2013). Functional membrane expression of TRPM4 in the tetracycline-induced cells was
compared with the non-induced cells and non-transfected HEK293 cells. Furthermore, endogenous
expression of TRPM4 in HEK293 cells as reported in our previous studies (Amarouch et al., 2013)
was knocked down by CRISPR/Cas9 technology (KD) (Figure 2A and Supporting information
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Figure S1). In order to perform FLIPR (Fluorescent Imaging Plate Reader) based screening of
compounds, the cells were pre-incubated with Na+-free assay buffer to deplete intracellular Na+,
followed by loading the cells with a Na+-sensitive ANG-II dye. Reintroducing Na+ along with
ionomycin in the stimulus buffer for Ca2+-dependent activation of TRPM4 resulted in an increase of
fluorescence signal over the baseline (Figure 2B). We used the area under the curve (AUC) (marked
area, Figure 2B) to quantify the signal intensity with or without ionomycin. Upon TRPM4
overexpression, the fluorescence signal increased three fold compared with non-transfected HEK293
or KD cells (Figure 2B). The increase in fluorescence in non-transfected HEK293 cells and non-
induced TRPM4 cells could be due to Na+ influx via various endogenous Na+ transporters (Figure
2B); however, the effect is significantly smaller than in TRPM4-overexpressing cells (Figure 2B).
Following the demonstration that the ANG-II dye could detect Na+ influx, which was largely
mediated through TRPM4 channel activation, we further assessed the dependence of the fluorescence
signal on the extracellular Na+ and Ca2+ concentration in the stimulus buffer. We determined Na+
dependence with stimulus buffer containing different concentrations of Na+ by replacing the ion with
equimolar N-methyl-d-glucamine (NMDG). ANG-II dye presented an affinity for Na+ ions with a
Michaelis-Menten constant Km of 76.6 ± 6.3 mM (Figure 2C). Next, we determined Ca2+ dependence
using stimulus buffer containing 140 mM Na+ and varying concentrations of extracellular Ca2+
without any chelator. The increase in fluorescence signal showed a dependence on Ca2+ concentration
in the stimulus buffer with an EC50 of 0.2 ± 0.1 mM (Figure 2D). We finally assessed the robustness
of the assay by monitoring the statistical Z` factor, which always scored 0.5 or more in all our
experiments, while the ratio of AUC in presence and absence of ionomycin remained above 2
(Supporting information Figure S2, A and B). Finally, the assay developed here tolerated DMSO up
to 1%, the highest concentration used to dissolve the test compounds (Supporting information Figure
S2, C and D).
Discovery of small molecule TRPM4 inhibitors
To identify potent TRPM4 inhibitors, we performed a focused screening campaign using LBVS with
xLOS (Simonin et al., 2015) to search a catalog of 900,000 commercially available drug-like small
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molecules for analogs of the known but relatively weak TRPM4 inhibitors 9-phenanthrol, FFA and
glibenclamide (Figure 1). We selected 214 compounds among the top scoring virtual hits, purchased
1 mg solid samples which we conditioned as 10 mM stock solution in DMSO, and screened these
compounds for inhibition of TRPM4 at 10 µM using 9-phenanthrol (25 µM) as positive control
(Figure 3Ai). Initial screen revealed three hits for which we purchased and tested an additional 247
analogs selected by LBVS with xLOS as well as other fingerprint similarity search methods (Awale
and Reymond, 2014). As a result of these screening efforts, we identified four hits, compounds 4, 5, 8
and 9, repurchased 25 mg solid samples, and confirmed their inhibitory activity at 5 µM concentration
(Figure 1 and Figure 3 Aii). These compounds inhibited the initial rate of TRPM4-mediated Na+
influx in a concentration-dependent manner. The best TRPM4 inhibitors were the aryloxyacyl-
anthranilic acids 4 (IC50 = 1.6 ± 0.3 µM) and 5 (IC50 = 1.5 ± 0.1 µM, ligand efficiency (LE) = 0.371),
showing a 20 times stronger inhibition than 9-phenanthrol (IC50 = 29.1 ± 5.8 µM) (Figure 3B - D).
Structure-activity relationship (SAR) study of TRPM4 inhibitors
For SAR studies, we initially focused on anthranilic inhibitor 5 and devised a synthetic route for
varying substituents on both aromatic rings and within the acetyl linker and prepared 49 analogs.
(Figure 4A, Table 1, and Supporting information Table S1-S3). Changing the substituents of the
aromatic ring often abolished the activity of the compound. However, 13 anthranilic acid derivatives
showed TRPM4 inhibition with IC50 values in the range 1-10 µM, confirming the robustness of the
identified scaffold (Table 1, Figure 1). Overall the results obtained with the different analogs
indicated that TRPM4 inhibition required the presence of the anthranilic acid moiety with a halogen
substituent at position 4 and a defined substitution pattern on the aryloxy group (Figure 4B). The
SAR profile uncovered the 1-naphthyloxy analogs 6 as the first submicromolar inhibitor in the series
with an IC50 = 0.4 ± 0.3 µM (LE = 0.345) (Table 1). However, when considering its LE (Hopkins et
al., 2014), compound 6 does not bring additional improvement over compound 5. On the other hand,
pure enantiomers (>99% ee) of 4 (52 and 53) were obtained using preparative chiral-phase HPLC and
analyzed for purity using the same method. Both enantiomers were similarly active and chirality of
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the linker did not significantly influence activity,(Supporting information Table S3), while other
modifications of the linker were detrimental to activity (54 to 58, Supporting information Table S3).
Compound 5 selectively inhibits TRPM4 overexpressed in HEK293 cells
To gain further insight into the three most potent inhibitors found here 4, 5 and 6 , we analyzed their
predicted off-target effects using the polypharmacology browser (Awale and Reymond, 2017), an
online tool that predicts possible biological activity by multi-fingerprint comparisons with bioactive
compounds from the ChEMBL database (Gaulton et al., 2012). While 4 and 5 were essentially free of
off-target predictions, we found that 6 had a reported activity at 10 M on GLPR1 (glucagon-like
peptide receptor 1) (https://pubchem.ncbi.nlm.nih.gov/bioassay/624417 (accessed July 6, 2017)).
We further confirmed the inhibitory activity of compounds 4 and 5 using classical patch-clamp
electrophysiology recordings on TRPM4 overexpressing cell line. In excised membrane patches, we
quantified TRPM4 inhibition using the generic TRPM4 inhibitors 9-phenanthrol (Figure 5A) and
FFA (Figure 5B), and compared it with our newly identified hit compounds 4 (Figure 5C) and 5
(Figure 5D). Maximal current at +100 mV was used to construct concentration-response curves
(Average current traces: Supporting information Figure S3). Interestingly, both compounds
inhibited TRPM4 with an IC50 of 1.0 ± 0.2 and 1.8 ± 0.1 µM for 4 and 5 respectively, which are
several times more potent than IC50 values of 17.0 ± 2.8 and 9.2 ± 1.2 µM for 9-phenanthrol and FFA,
respectively. In addition, we also observed reversible inhibition by 4 and 5 when applied to the
cytosolic side of the ruptured membrane patches. (Supporting information Figure S4).
Further, to evaluate the selectivity of the newly identified hits against TRPM5, the closest homolog of
TRPM4, we performed excised membrane voltage clamp recordings on HEK293 cells expressing
TRPM5. As a positive control, we used triphenylphosphine oxide (TPPO), a known TRPM5 inhibitor
at 20 µM. (Palmer et al., 2010). TPPO inhibited TRPM5 current but did not affect TRPM4 current
(Figure 5Ei and Eii). However, compound 4 had a variable potentiating effect on TRPM5, while
compound 5 had no significant effect on the TRPM5 current (Figure 5Ei). In addition, we also tested
the selectivity of compound 5 against other TRP family members including TRPM7, TRPM8, TRPV1
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and TRPV6 using whole-cell patch clamp recordings. At 10 or 100 M, compound 5 did not show
any inhibition of currents from these targets (Supporting information Figure S5).
Next we investigated if compound 5 had any off target effects on 17 different ion channel proteins and
membrane receptors, including Gamma-aminobutyric acid receptor subunit alpha-1(GABA(A1)), N-
methyl-D-aspartate (NMDA) receptor, Ca2+ channel and voltage gated K+ channels, which are targets
of the parent drug FFA. At 10 M compound 5 did not show any significant effect on these targets,
and showed only < 5% inhibition of dofetilide binding to the cardiac hERG channel. (Supporting
information Table S4).
Compound 5 inhibits endogenous TRPM4 currents in LNCaP prostate cancer cells
We next assessed if compound 5 also inhibits endogenous TRPM4 currents. We recorded TRPM4
whole- cell currents with 10 µM Ca2+ in the patch pipette in the prostate cancer cell line LNCaP as
described previously (Holzmann et al., 2015; Kilch et al., 2016). The currents activated slowly
(Figure 6A) with the typical TRPM4 current-voltage relationship (I-V) shown in Figure 6B. After
TRPM4 currents were activated, we applied compound 5 at different concentrations. Compound 5
inhibited TRPM4 currents reversibly in a concentration dependent manner with an IC50 of 1.1 ± 0.3
µM (Figure 6C). This is consistent with our findings in a TRPM4 overexpression system (Figure 3
and 5), where compound 5 also inhibits TRPM4 currents with an IC50 of ̴ 1 µM.
Compound 5 rescues functional expression of A432T, a loss-of-expression TRPM4 variant
Based on the TRPM4 selectivity of compound 5, we investigated whether compound 5 can rescue
membrane expression of TRPM4 A432T, a loss-of-expression variant found in cardiac AVB patients
(Syam et al., 2016). It is reported that decreased expression and altered trafficking of ion channel
disease causing variants, such as in CFTR and hERG, can be corrected either by incubation at a lower
temperature or by using selective chemical compounds (Denning et al., 1992; Zhou et al., 1999). We
followed a similar strategy and observed that the expression of A432T was increased after a 24-hour
long incubation of the cells at 28 °C (Supporting information Figure S6 A).
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Next we pre-incubated cells expressing A432T with 50 µM compound 5 overnight and evaluated any
change in A432T total and surface expression using western blot (Figure 7Ai). Compound 5 showed
only ̴ 30% cytotoxicity at 100 µM, making it feasible for longer incubation with cells (Supporting
information Figure S7). Densitometric quantification of the upper fully glycosylated (FG) and the
lower core glycosylated (CG) bands showed partial rescue of total and surface expression(biotinylated
fraction) of A432T by compound 5 (Figure 7Aii) . We further evaluated if the rescued expression of
A432T with pre-incubation of compound 5 also led to increase in current density in excised
membrane patches. As illustrated in Figure 7 B and C, A432T showed a significant decrease in the
Ca2+ sensitive outward current in comparison to WT. Pre-incubation with compound 5 at 50 µM
overnight and followed by a washout before current recordings, partially rescued the functional
expression and the Ca2+ sensitive outward current of A432T. Conversely, an inactive congener of
compound 5, i.e. compound 51, did not rescue the expression of A432T at 50 µM (Figure 7D).
Selectivity of compound 5 for rescuing A432T was further evaluated by incubating the loss-of-
expression hERG variant named Dupl hERG, which was found in a congenital long-QT syndrome
°
patient (Grilo et al., 2010), both with 50 μM compound 5 and with lower-temperature (28 C)
incubation. As presented in (Supporting information Figure S6 B), Western blot experiments
showed that a reduction of incubation temperature rescued the trafficking defect of Dupl hERG,
whereas no effect was observed after incubation with compound 5. However, the rescuing property of
compound 5 was not limited to A432T but also increased WT TRPM4 expression similar to
incubation with lower temperature (Supporting information Figure S6 C and D) .
DISCUSSION AND CONCLUSION
Human mutations in the TRPM4 gene have been implicated in cardiac conduction disorders (Kruse et
al., 2009; Liu et al., 2010, 2013; Stallmeyer et al., 2012; Syam et al., 2016). So exploring the role of
TRPM4 in cardiac physiology and pathophysiology upholds great clinical relevance. However, most
of these physiological characterizations relied upon patch clamp studies with TRPM4 KO mice
models combined with pharmacological inhibitors. Notably, the most reported TRPM4 inhibitor, 9-
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phenanthrol, did show some selectivity for TRPM4 compared to other TRP channels. However, it is
not sufficiently potent, and high concentrations reversibly inhibit delayed outward rectifying K+ and
voltage-gated Ca2+ currents in mouse ventricular myocytes (Simard et al., 2012). A recent study
showed that 9-phenanthrol inhibits TMEM16A, a Ca2+-activated Cl- channel in arterial smooth
myocytes (Burris et al., 2015). Furthermore, 9-phenanthrol belongs to the class of polycyclic aromatic
hydrocarbons, which contribute to its cellular toxicity (Feng et al., 2012). The non-steroidal anti-
inflammatory drug FFA and glibenclamide are also used as TRPM4 inhibitors (Ullrich et al., 2005;
Demion et al., 2007). Although these compounds inhibit TRPM4 current reversibly, they also have
many off target effects on other ion channels (Gardam et al., 2008; Alexander et al., 2013). These
findings support the need for more potent and selective TRPM4 inhibitors. In this study, we
developed a novel Na+-sensitive screening assay and performed a focused screening campaign using
LBVS with xLOS, which lead to the identification of compounds 4, 5 and 6, as potent TRPM4
inhibitors. In addition, we found that compound 5 possessed a dual function as a TRPM4 inhibitor and
also as a chemical chaperon rescuing the membrane expression of a loss-of-expression TRPM4
variant.
Compound 5 as a potential inhibitor of TRPM4
Recently, virtual screening methods have emerged as an efficient approach for identifying potent and
specific inhibitors for different ion channels (Langer and Krovat, 2003; Fernández-Ballester et al.,
2011 Simonin et al., 2015). In this study we carried out an initial virtual screening using an in-house
developed pharmacophore similarity search algorithm: xLOS, which compared the spatial overlap
between a catalog of commercially available drug-like small molecules to the three reference TRPM4
inhibitors, 9-phenanthrol, glibenclamide and FFA. The purchased compounds selected from the top
scoring list were further validated using a new fluorescence based Na+ sensitive screening assay,
which measured the activity of TRPM4 in a stably expressing cell line. This screening identified two
aryloxyacyl-anthranilic acid compounds 4 and 5 as the most potent compounds with IC50 in ~1 µM
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range, which is several fold lower compared to all the reference inhibitors (Abriel et al., 2012). It is
noteworthy that compound 5 inhibited TRPM4 current in both overexpressed cell line and
endogenously expressed in prostate cancer cell line with similar potency and presented selectivity
over other TRP family members including TRPM5, TRPM7, TRPM8, TRPV1, TRPV3, TRPV6 .
Analysis of our in silico and cell based results highlighted compound 5 as having a good balance of
potency and selectivity which encouraged us for further optimization of this compound through
chemical modifications. Although, altering substituents on aromatic rings and within the acetyl linker
often abolished the inhibition activity, we did identified compound 6 with increased potency of < 1
µM IC50 inhibition (Table 1, and Supporting information Table S1-S3). However, due to off target
effect of compound 6 on GLPR1 receptor as predicted by polypharmacology browser, we did not
perform any further experiments with this compound. Future optimization studies on compound 6
could provide a promising tool compound for TRPM4 pharmacology.
Success in finding these inhibitors demonstrates the capability of combining in silico and Na+
sensitive screening assay to explore focused chemical landscapes to identify potent and specific
inhibitors of TRPM4. The chemical diversity surveyed here in the virtual screening and subsequent
SAR study is summarized in Figure 8 with a color-coded map of the chemical space covered by the
compounds tested, plotted with our recently reported web-based tool WebMolCS (Awale et al., 2017)
using the principle of similarity mapping (Awale and Reymond, 2015). This 3D-map positions
molecules as individual spheres in a space where distances represent the relative similarities of
molecules to one another, as measured by the Tanimoto coefficient of a 1024-bit binary Daylight type
substructure fingerprint, which perceives detailed features of the molecules (Hagadone, 1992). Each
sphere is color-coded according to the observed TRPM4 activities (inactive = blue, most active = red).
This representation illustrates that the anthranilic acid TRPM4 inhibitors occupy a chemical space
significantly shifted from the reference inhibitors 9-phenanthrol, FFA and glibenclamide used for
LBVS. Furthermore, the map shows that only very few analogs of 9-phenanthrol were actually tested
due to limited commercial availability.
Compound 5 as a potential chemical chaperone for TRPM4 loss-of-expression variant
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A number of mutations in the human TRPM4 gene linked with various cardiac pathologies have led to
reduced membrane expression and function either due to misfolding or improper assembly of the
channel protein (Stallmeyer et al., 2012; Syam et al., 2016). In past decade, several studies used in
silico and in vitro screening to identify small molecules that can partially rescue the trafficking defect
of many proteins (Naik et al., 2012) including ion channels such as CFTR (Van Goor et al., 2006;
Rowe and Verkman, 2013). Most of these chemical chaperones had shown certain selectivity toward
the target either by direct interactions or via other accessory proteins. We reasoned that, since
compound 5 outlays better potency, selectivity and lower cytotoxicity, it could be a potential
candidate to be used as chemical chaperone for A432T-TRPM4 mutant channel. Indeed compound 5
rescued the total and surface expression of both the core and fully glycosylated forms of the A432T
variant channel. This chemical chaperoning effect may be related to a compound-dependent increase
in protein stability. Moreover, the observed increase in A432T-TRPM4-mediated current upon
compound 5 pre-incubation, followed by a washout protocol, is consistent with an increased number
of channels at the cell surface. The partial functional rescuing may be explained by either mutation-
dependent altered gating or only partial washout of the compound 5. However, it is notable that this
rescuing effect was specific to the structure of compound 5, since an inactive congener of 5 with no
inhibitory activity, i.e., 51, did not show any rescuing effect. It was also channel specific since 5 did
not rescue a loss-of-expression hERG channel variant. These results suggest that this positive effect
could be from a direct and selective interaction of compound 5 with the TRPM4 protein and hence
reducing its degradation by the endoplasmic reticulum-associated degradation process. Note that at
this stage, there is no evidence that the protein binding of 5 leading to TRPM4 inhibition is identical
to the one resulting in its rescuing. Nevertheless, to the best of our knowledge, this is the first report
of a small molecule compound functionally rescuing the membrane expression of a TRP channel loss-
of-expression variant.
In conclusion, this study demonstrates that using the newly developed xLOS method linked to a Na+-
sensitive screening assay, a series of halogenated anthranilic amide compounds with potent and
selective TRPM4 inhibition properties was discovered. The highly potent compounds 4, 5 and 6 that
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we identified in this study will open new ways in TRPM4 pharmacology. In the context of recent
structural data of TRPM4 (Guo et al., 2017; Winkler et al., 2017; Autzen et al., 2018), future
structure-function relationship studies coupled with our selective and potent inhibitor compound 5
will benefit for better understanding of channel function. Moreover, compound 5 demonstrated not
only selective inhibition but also chemical chaperone property, which provide a good starting point
for developing clinical drug candidates for rescuing loss-of-expression variants.
MATERIALS AND METHODS
Ligand-based virtual screening
First round of virtual screening (VS) was performed in the Princeton database, which contains
900,000 purchasable compounds, using three different reference compounds, namely 9-phenanthrol,
glibenclamide and flufenamic acid. The VS was carried out with an in-house developed extended
Ligand Overlap Score (xLOS) method, which computes the 3D-shape and –pharmacophore similarity
between any two compounds (typically reference and database compounds)(Simonin et al., 2015).
The single lowest energy 3D-models of reference and database molecules were generated using
CORINA program available from Molecular Networks Pvt. Ltd. XLOS used these 3D-models to
calculate a 3D-similarity score between the reference compound and the database molecules.
Afterward, for each of the three reference molecules, 235 molecules were visually selected from the
top 1000 xLOS scoring compounds, 214 of those were purchased from Princeton Biomolecular
Research. These compounds were dissolved in DMSO solution and tested on TRPM4 activity. Next,
three hits from this first round of VS were used for a second round of VS in the Princeton database
using xLOS. From this second round, 247 compounds were selected and purchased for biological
testing.
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Visualization of screening library in 3D
The visualization of screening library was facilitated by an in-house developed webMolCS web server
(www.gdb.unibe.ch). WebMolCS takes a user-defined list of molecules (in SMILES format) as input
and produces interactive color-coded 3D-maps using either principal component analysis or similarity
mapping of any of the six different molecular fingerprints used for molecular representation. In the
present study, similarity mapping (Sim map) and substructures fingerprint (sFP) were used to generate
the 3D-maps. The colors of the 3D-maps shown in Figure 1 represent the similarity (calculated using
sFP fingerprint) of the screened compounds to the best compound 6 from synthetic optimization; from
low to high similarity: blue-cyan-green-yellow (Awale and Reymond, 2017).
Chemical synthesis of compounds
The synthesis and characterization of all compounds are described in the Supplementary methods.
Cell culture
HEK293 and tetracycline-inducible HEK293 Flag-TRPM4-expressing cells were used. The HEK293
cells were a gift from Dr. R.S. Kass, Columbia University, New York, USA. These cells were given
to the group of Dr. R.S. Kass by Dr. B. Stillman, Cold Spring Harbor Laboratories, Cold Spring,
USA. Tetracycline-inducible HEK293 Flag-TRPM4 cells were a gift from Dr. P. Bouvagnet
(University of Lyon, France). These cells were cultured at 37 °C in DMEM supplemented with 10%
fetal bovine serum (FBS, Invitrogen, CA, USA), 4 mM glutamine (Sigma), 5 μg/mL S-blasticidin
(Invitrogen) and 0.4 mg/mL Zeocin (Invitrogen). TRPM4 expression was induced by adding 1 µg/mL
of tetracycline to induction medium (DMEM without phenol red (Gibco 31053, Paisley, UK)
supplemented with 10% FBS (Invitrogen) and 2% L-Glutamin (Sigma)) 15-20 hours before the
experiment. For TRPM4 variant studies, HEK293 cells cultured at 37 °C in DMEM supplemented
with 10% FBS and 4 mM glutamine were transiently transfected with 250 ng of HA-tagged TRPM4
WT or HA-tagged TRPM4 p.A432T in a 100 mm dish, mixed with 4 µL of JetPEI (Polyplus
transfection, Illkirch, France) and 46 μL of 150 mM NaCl. The cells were incubated for 24 h at 37 °C
This article is protected by copyright. All rights reserved.

with 5% CO2. All transfections included 100 ng of eGFP as a reporter gene. Cells were used 24 hours
after transfection. Lymph node carcinoma of the prostate (LNCaP) cells was purchased from the
American Type Cell Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI Medium
1640 (Gibco) supplemented with 10% FCS and 1% penicillin/streptomycin (Invitrogen).
TRPM4 KD cell line has been provided by Transposagen Biopharmaceuticals (Lexington, KY, USA)
and generated using CRISPR/Cas9 technique. The cells had been transfected with both the Cas9
vector and the corresponding U6 expressed sgRNA vector in a 1:1 molar ratio. Transfection was done
with Lipofectamine 2000 (ThermoFischer Scientific, MA, USA) following the manufacturer’s
protocols. Loss of TRPM4 gene function and protein was then checked in single clone`s amplified
using qPCR and western blot. The selected clone were cultured and expanded in condition similar for
HEK293 as mentioned above.
sgRNA targets:
TRPM4 TS1
TRPM4 TS2
GCCACCTCGCCGCTCTCGC
GCTCCAGGGGGCCTGCCCG
Compound cytotoxicity assay
Cytotoxicity of compounds 4, 5 and 6 were studied in RAW 264.7 and HeLa cells for anti-
proliferative effects. The cells were seeded at 2’000 cells per well in 100 µL culture media in a 96-
well plate. Following overnight incubation different concentrations of the compounds were added and
incubated together with the solvent control for 72 hour at 37 °C. After that, the cells were incubated
with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, final concentration 0.5
mg/mL) for 4 hour at 37 °C and solubilized with DMSO. The plates were measured at 550 nm
(absorbance) and the IC50 value related to cell survival/proliferation was calculated and plotted.
Paclitaxel was used as positive control. Experiments were performed in at least three independent
experiments, each in triplicate.
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Western blot
To detect TRPM4 protein expression, whole-cell lysates were prepared by lysing cells in lysis buffer
(50 mM HEPES pH 7.4; 150 mM NaCl; 1.5 mM MgCl2: 1 mM EGTA pH 8.0; 10 % glycerol; 1%
Triton X-100; 1X Complete protease inhibitor cocktail (Roche, Mannheim, Germany)) for 1 hour at 4
°C. Cell lysates were centrifuged for 15 minutes at 16,000 g at 4 °C and protein concentration was
evaluated using Bradford assay. 60 µg of each protein sample was run on 9 % polyacrylamide gels,
later transferred with the TurboBlot dry blot system (Biorad, Hercules, CA, USA). After transfer
membrane were blocked with 0.1% BSA in PBS and incubated with Primary antibodies anti-TRPM4
(generated by Pineda, Berlin, Germany) and anti-α-actin A2066 (Sigma-Aldrich, St. Louis, Missouri,
USA) using SNAP i.d. system (Millipore, Billerica, MA, USA), followed by an incubation with
secondary antibody, IRDye 800CW, (LI-COR Biosciences, Lincoln, NE, USA). Membrane were
scanned using LiCor Odyssey Infrared imaging system ( LiCor Biosciences, Lincoln, NE, USA) and
protein band intensity was quantified using Image Studio Lite software from LiCor Biosciences.
For detecting TRPM4 WT and A432T variant whole-cell (total) and surface expression after
incubation with compound 5, the cells were washed twice with cold 1X PBS and treated with
EZlinkTM Sulfo-NHS-SS-Biotin (Thermo Scientific, Waltham, MA, USA) 0.5 mg/mL in cold 1X
PBS for 15 minutes at 4°C. Subsequently, the cells were washed twice with 200 mM Glycine in cold
1X PBS and twice with cold 1X PBS to inactivate and to remove the excess biotin, respectively. The
cells were then lysed with 1X lysis buffer for 1 hour at 4°C. Cell lysates were centrifuged at 16,000 g
at 4°C for 15 minutes. Two milligrams of the supernatant were incubated with 50 µL Streptavidin
Sepharose High Performance beads (GE Healthcare, Uppsala, Sweden) for 2 hours at 4°C, and the
remaining supernatant was kept as the input. The beads were subsequently washed five times with 1X
lysis buffer before elution with 50 µL of 2X NuPAGE sample buffer (Invitrogen, Carlsbad, CA, USA)
plus 100 mM DTT at 37°C for 30 minutes. These biotinylated fractions were analyzed as TRPM4
expressed at the cell surface. The input fractions, analyzed as total expression of TRPM4, were
resuspended with 4X NuPAGE Sample Buffer plus 100 mM DTT to give a concentration of 1 mg/mL
and incubated at 37 °C for 30 minutes.
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Na+-influx screening assay
TRPM4-expressing cells were plated in 96-well black-walled clear bottomed poly-D-lysine coated
plates (Corning, NY, USA) at a density of 30,000 cells / well in 100 µL of induction medium
(described above). After plating, cells were incubated for 48 hours at 37 °C in a 5% CO2 incubator.
On the day of assay, the induction medium was replaced with 100 µL/well of the assay buffer (in
mM: 140 NMDG-Cl, 10 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES (pH 7.2) with NMDG-OH and 290 to 300
mOsM) and incubated for 45 minutes at room temperature (RT). Later, the assay buffer was replaced
with 95 µL/well of dye loading solution prepared in assay buffer supplemented with 0.1% pluronic
acid F-127 (Teflabs, TX, USA) and 5 µM of Na+ sensitive dye, Asante Natrium Green – II (ANGII)
(Teflabs). The cells were incubated in the dark at RT for 45 minutes. After incubation, the dye loading
solution was completely drained and replaced with 72 µL assay buffer and incubated in the dark for
20 minutes, followed by fluorescence readout on FLIPR TETRA® (Molecular devices, CA, USA).
For evaluation of the effects of TRPM4 inhibitors using the Na+ influx assay, a 10X compound plate
were prepared in assay buffer in 96-well polystyrene plates (Corning, NY, USA). All stocks were
dissolved in DMSO. To initiate Na+ influx through activation of TRPM4, a 5X stimulus buffer plate
containing (in mM) 700 NaCl, 4 CaCl2, 4 MgCl2, 40 HEPES, (pH 7.2 with NaOH) along with 50 µM
of ionomycin prepared in a 96-well polystyrene plate (Corning, NY, USA). The background control
wells contained stimulus buffer without ionomycin (W/O).
Fluorophores were excited by the 488 nm line of an argon laser. Emission was filtered with a 540 ±
30 nm bandpass filter. Initial baseline fluorescence was measured at 1 Hz from the plate loaded with
ANGII dye for 1 minute. Followed by addition of 8 µL solution from 10X compound plate,
fluorescence was monitored at an interval of 1 Hz for 5 minutes. Finally, to activate TRPM4, 20 µL of
solution from the stimulus buffer plate was added, and fluorescence was acquired at 0.5 Hz intervals
during and after addition for 5 minutes. Data from individual assay wells were normalized to initial
baseline fluorescence. The initial rise in fluorescence was measured as area under the curve, and the
counts were normalized using the formula {[(Ionomycin - Compound)/ (Ionomycin – W/O
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Ionomycin)] * 100} to plot concentration-response curves for inhibitor hits. The experimenter for the
screening experiments (L.C.O) was blinded and was not aware of any chemical properties of the
compounds until the best hits were validated. The steps are summarized as workflow mentioned in
table 2.
Electrophysiology
Cells were plated in 35 mm poly-D-lysine coated dishes (Corning, NY, USA) in induction medium
(phenol red free DMEM medium (GIBCO-31053)) supplemented with 10 % FBS and 1 µg/mL
tetracycline (Invitrogen, USA). After plating, cells were incubated for 48 hours at 37 °C in a 5% CO2
incubator. Electrophysiological recordings were performed in the inside-out patch clamp
configuration with patch pipettes (1-2 µm tip opening) pulled from 1.5 mm borosilicate glass
capillaries (World Precision Instruments, Inc. Fl, USA) using DMZ Universal puller (Zeitz-
Instruments, GmbH, München, Germany). Pipette tips were polished to have a pipette resistance of 2–
4 M Ω in the bath solution. The pipette solution contained (in mM) 150 NaCl, 10 HEPES, 2 CaCl2
(pH 7.4 with NaOH). The bath solution contained (in mM) 150 NaCl 10 HEPES, 2 HEDTA (pH 7.4
with NaOH) as Ca2+-free solutions. Solutions containing 0.1 to 2 mM Ca2+ were prepared by adding
the appropriate concentration of CaCl2 without a Ca2+ chelator to a solution containing (in mM) 150
NaCl, 10 HEPES (pH 7.4 with NaOH) as reported previously (Zhang et al., 2005). Bath solutions with
different Ca2+ concentrations were applied to cells by a modified rapid solution exchanger (Perfusion
Fast-Step SF-77B; Warner Instruments Corp. CT, USA). Inhibitors in DMSO stock were diluted to
appropriate concentrations in the pipette solution and applied on extracellular side of the cells.
Membrane currents were recorded with a Multiclamp 700B amplifier (Molecular devices, CA, USA)
controlled by Clampex 10 via a Digidata 1332A (Molecular devices, CA, USA). Data were low-pass
filtered at 5 kHz and sampled at 10 kHz. Experiments were performed at RT (20–25 °C). For I-V
relations, currents were recorded using a stimulation protocol consisting of voltage steps of 200 ms
from a holding potential of 0 mV ranging from -80 mV to +100 mV, followed by a tail voltage at -100
mV. For constructing concentration-response curves, steady-state current at +100 mV recorded in 300
µM Ca2+ were normalized to current in absence of any inhibitor. For rescue experiments with
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compound 5, before performing patch clamp recordings the culture medium was replaced with bath
solution without any added inhibitor.
For whole-cell patch clamp on LNCaP cells, 50 ms voltage ramps spanning -100 to +100 mV were
delivered every 2 s from a holding potential of 0 mV. Whole-cell currents were recorded with a
HEKA patch clamp system (EPC-10, HEKA Elektronik Dr. Schutze GmbH, Rhein, Germany). Series
resistance (Rs) was monitored in response to a 5 mV voltage step and compensated online up to 80%
if needed. Bath solution contained (in mM): 140 NaCl, 0.5 CaCl2, 3 MgCl2, 10 HEPES. Glucose was
added to adjust osmolarity to 330 mOsmol/L and pH was adjusted to 7.2 with NaOH. Pipette solution
contained (in mM): 140 Cs-glutamate, 10 HEDTA, 10 HEPES, 8 NaCl, 12.24 MgCl2, and 0.00049
CaCl2. Free Mg2+ of
3
mM and 10 µM Ca2+ was calculated according to
http://web.stanford.edu/~cpatton/webmaxcS.htm. Whole-cell currents at -80 and +80 mV were
extracted, normalized to the cell capacity, and plotted versus time.
Data analysis
To quantify the Na+ influx after addition of stimulus buffer, the area under the curve (AUC) was
calculated using the ScreenWorksTM software (Molecular devices, CA, USA). Data points acquired
from the Na+ influx assay from FLIPR or electrophysiology data were exported and analyzed using
IGOR PRO 6 (Wavematrix, OR, USA). Z` factor was calculated as described earlier (Zhang et al.,
1999), using the following equation: Z` = 1 – [ (3SDp + 3SDn) / (3Meanp – 3Meann) ] where SD is
standard deviation, and p and n are positive and negative control, respectively. Michaelis-Menten
constant for Na+ dependence of the signal was deduced using the following equation: V = [Vmax [S] /
(Km + [S])], where Vmax is maximal fluorescence signal, S is Na+ concentration and Km is Michaelis-
Menten constant. Concentration-response curves were fitted using the Hill equation fit parameter of
IGOR (NC = NCmax [Cmpd]nH / {[Cmpd]nH + EC50nH}), where NC is normalized current or counts,
[Cmpd] is compound concentration and nH is the Hill coefficient. Data are presented as mean ± SEM
except for Figure 2Ai and Aii, where each data point n = 2 and data are presented as mean ± SD.
Statistically significant difference between means were determined using students`s t test for
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comparison between two means with n ≥ 5. P ≤ 0.05 were accepted as significant and represented as *
in respective figure panels. The data and statistical analyses comply with the recommendations on
experimental design and analysis in pharmacology (Curtis et al., 2015).
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in
http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to
PHARMACOLOGY(Harding et al., 2018), and are permanently archived in the Concise Guide to
PHARMACOLOGY 2017/18 (Alexander Stephen PH et al., 2017).
Author Contributions: L.C.O, C.D, B.B, G.N, S.K, M.R, J.G, M.L, C.P, J.L.R, H.A designed the
experiments. L.C.O developed and validated screening assay. L.C.O and M.R performed compound
screening. L.C.O and S.K performed and analyzed patch clamp experiments. B.B performed western
blots for chemical chaperone experiments. U.T performed additional compound selectivity profiling
and D.R-K performed biotinylation experiments. C.D, G.N, C.S performed virtual screening,
purchased compounds. C.D, G.N performed chemical synthesis for SAR studies. L.C.O, J.L.R, H.A
wrote the manuscript with extensive comments from C.D, B.B, G.N, M.R, J.G, S.K, M.L, C.P, J.L.R,
H.A.
Conflict of interest: The authors declare no conflicts of interest.
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TABLE:
TRPM4 inhibitors
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ID
Na+ influx Electrophysiology R1 R2
IC50 (µM) IC50 (µM)
R3
1.6 ± 0.3
1.5 ± 0.1
1.0 ± 0.2
1.8 ± 0.2
1.8 ± 0.1
3.4 ± 1.1
2.6 ± 0.3
2.6 ± 0.4
0.4 ± 0.3
2.2 ± 0.4
8.3 ± 0.7
5.9 ± 0.5
1.2 ± 0.5
1.0 ± 0.2
1.8 ± 0.1
1.6 ± 0.5
Cl CH3 2-CH3-4-Cl
4
5
Cl
Br
H
H
2-Cl
2-Cl
11
12
13
14
15
16
6
7
8
9
10
9P
Cl CH3 2,4-diCl
Cl
Cl
Cl
Cl
H
H
H
H
2,4-diBr
2,4-diF
2-Cl-5-nitro
3-phenyl
0.2 ± 0.07
29.1 ± 5.8 17.0 ± 2.8
Table 1. IC50 values of TRPM4 inhibitors tested using the Na+ influx screening assay and
electrophysiology recordings.(9P- 9 Phenanthrol)
ASSAY WORKFLOW
No.
1
Step
Value
Detail
30,000 cells/well
Incubation for 48 hours
Cell plating and induction
100 µL
Drain the content
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Assay buffer
Incubation for 45 mins
Na+ depletion
Dye loading
100 µL
2
3
Drain the content
ANGII dye prepared in assay buffer
Incubation in dark for 45 mins at RT
95 µL
Drain the content
Assay buffer
72 µL
Store plate in dark at RT
4
5
Baseline readout (FLIPR)
540 nm
8 µL
Fluorescence readout 1 Hz for 1 min
10X compounds in assay buffer plate
Incubation for 5 mins
Test compound
Stimulus
6
7
FLIPR readout 1 Hz
5X stimulus buffer plate
Incubation for 5 mins.
FLIPR readout at 0.5 Hz.
20 µL
Table 2. Steps for Na+ influx screening assay campaign.
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Figure 1. Chemical structures of TRPM4 inhibitors used in this study.
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Figure 2. Development and validation of a Na+ influx screening assay: A) Western blot analysis
showing TRPM4 expression as detected by anti-TRPM4 antibody from whole-cell extracts prepared
from HEK293 cells, TRPM4-expressing cells either induced with tetracycline or non-induced and
TRPM4 CRISPR-Cas9 knocked-down cell line (KD). Alpha-actin served as a loading control. B)
Averaged traces obtained on the FLIPR with Na+-sensitive dye ANG-II in different cellular conditions
as mentioned (panel A). The initial rate of fluorescence increase in either absence (black) or presence
(red) of 10 µM ionomycin was deduced by measuring the area under the curve (AUC) from the
shaded portion for different cellular conditions (n ≥ 5). Ci) Averaged traces obtained on the FLIPR
from TRPM4-expressing cells, with varying concentration of extracellular Na+. Cii) The initial rate of
fluorescence increase measured as AUC in different Na+ concentrations were fitted using the
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