Synthesis and characterization of store-operated calcium entry inhibitors active in the submicromolar range

Article abstract of DOI:10.3390/ijms21249777

The store-operated calcium entry, better known as SOCE, forms the main Ca²⁺ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki ≈ 75 nM. Furthermore, below 1 μM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.

Full text of DOI:10.3390/ijms21249777

International Journal of
Molecular Sciences
Article
Synthesis and Characterization of Store-Operated
Calcium Entry Inhibitors Active in the
Submicromolar Range
Camille Le Guilcher 1,2, Tomas Luyten 3 , Jan B. Parys 3 , Mathieu Pucheault 4 and
Olivier Dellis 1,2,
*
1
Physiopathogénèse et Traitements des Maladies du Foie, Université Paris-Saclay, Rue des Adeles,
91405 Orsay, France; camille.le-guilcher@inserm.fr
INSERM U1193, Rue des Adeles, 91405 Orsay, France
Laboratory for Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine & Leuven
Kanker Instituut, B-3000 Leuven, Belgium; tomas.luyten@kuleuven.be (T.L.); jan.parys@kuleuven.be (J.B.P.)
Institute of Molecular Science, CNRS, Université de Bordeaux, 33400 Talence, France;
mathieu.pucheault@u-bordeaux.fr
2
3
4
*
Correspondence: olivier.dellis@u-psud.fr; Tel.: +33-169-154-959
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 17 November 2020; Accepted: 18 December 2020; Published: 21 December 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The store-operated calcium entry, better known as SOCE, forms the main Ca2+ influx
pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation
and the immune response. During the past decades, several inhibitors were developed, but they
lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate
(2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them,
our compound P11 showed the best inhibitory capacity with a Ki
≈
75 nM. Furthermore, below 1 µM,
P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB,
the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under
phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in
the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new
and potent family of immunosuppressors.
Keywords: store-operated calcium entry; inhibitor; synthesis; apoptosis
1. Introduction
The control of Ca2+ homeostasis is a challenging process for the cell. Indeed, many cellular
functions, including contraction, differentiation, and proliferation, are regulated by an increase of the
intracellular Ca2+ ion concentration [ ]. These increases are commonly due to the release of Ca2+ ions
1
from internal stores (mainly the endoplasmic reticulum (ER)) and to an influx of Ca2+ ions from the
extracellular space. The diversity of the Ca2+-release and Ca2+-influx mechanisms in various cell types
is responsible for the variety of Ca2+ responses occurring after cell stimulation.
In leukocytes in general, and in lymphocytes in particular, cell stimulation induces the synthesis of
inositol 1,4,5-trisphosphate (IP3) allowing the release of Ca2+ ions out of the ER through the IP3 receptor
(IP3R). This depletion activates a Ca2+ influx known as store-operated calcium entry (SOCE) [
2].
During the last 15 years, the two main proteins responsible for SOCE have been unveiled: STIM1, the
Ca2+-sensor protein localized in the ER membrane, and Orai1, the pore-forming protein localized in
the plasma membrane. The importance of Orai1 for lymphocyte function appears from the fact that
the protein was identified after the discovery of a point mutation creating a non-functional channel,
Int. J. Mol. Sci. 2020, 21, 9777; doi:10.3390/ijms21249777
www.mdpi.com/journal/ijms

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inducing a severe combined immunodeficiency [3]. Thus, even if Orai1 channels are present and the
leukocyte repertoire unchanged, the immune system remains deficient.
For more than 30 years, several compounds able to block SOCE have been characterized and
developed [4–6]. Indeed, a SOCE inhibitor could represent a new way to impair the activation
of immune cells and could be of interest in the treatment of chronic inflammatory diseases and
leukemias/lymphomas as well as to impair graft rejection or failure.
Among all these molecules, 2-aminoethoxy diphenylborinate (2-APB) has emerged as an
interesting compound due to its special properties and the ease to modify its basal structure.
Furthermore, 2-APB has intriguing properties: at a concentration between 1 and 5
the leukocyte SOCE but is a full blocker at concentrations above 20-30 M [
characterized several 2-APB analogs having either this bimodal function or displaying only inhibiting
or only potentiating effects [ ]. The group of Mikoshiba on the other hand has developed tandem
2-APB molecules with very potent SOCE inhibition capacity, called DPB162-AE and DPB163-AE [ ],
] while DPB162-AE also affects ER
µM, 2-APB potentiates
µ
7,8]. We previously
7,9
6
though DPB163-AE at low concentrations activates SOCE [
6
Ca2+-store content in various cell types [10]. Very recently, asymmetrical 2-APB analogues obtained
after the exchange of one of the two phenyl groups, or the replacement of the ethanolamine, were tested
on MDA-MB-231 cells, which exert an Orai1-dependent SOCE [11]. However, none of these molecules
were able to totally block the MDA-MB-231 cells SOCE at 10
from internal stores, demonstrating a lack of specificity [11].
µ
M, and some could induce Ca2+ release
Importantly, 2-APB also has a similar biphasic effect on the IP3R [12
Ca2+ pumps (SERCA [14
15]) and can directly open Orai3 channels, allowing an Orai3-dependent
SOCE [16 18]. These data all underscore that one limit to the use of 2-APB and its analogs is their lack
,13] but also inhibits the ER
,
–
of specificity, implying that any new interesting molecules derived from them must be tested for its
effects on other potential targets like IP3R and SERCA.
In this new study, we designed and synthesized 2-APB together with 16 new, non-commercially
available 2-APB analogs. In an attempt to get more potent SOCE inhibitors, only the phenyl rings
were slightly modified or replaced (Figure 1). Indeed, we previously showed that the presence of
the ethanolamine group is associated with the inhibitory capacity of the molecule and that groups
larger than phenyl rings impair the potentiation ability [7]. Among these molecules, our interest was
particularly drawn to the molecule we called P11 as it is more specific for the SOCE over IP3R and
SERCA, and inhibits the SOCE with a Ki << 1 µM. This most potent SOCE inhibitor induces apoptosis
of phytohemagglutinin (PHA)-stimulated Jurkat cells and impairs the synthesis of interleukin-2,
clearly blocking T cell activation. This 2-APB analog P11 could, therefore, represent a new important
step in the creation of more potent and specific SOCE inhibitors.

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Figure 1. Synthesis of aminoethyl diphenylborinate (APB) analogs using amine borane complex,
isolated yields are given after recrystallization.
2. Results
In our previous work, we used commercially available 2-APB analogs containing various types
of modifications of the phenyl groups of the molecule. Replacement of the two phenyl groups by
larger groups or the impairment of the free rotation of the phenyl groups by the creation of a carbon
bridge between them totally impaired the potentiation ability and gave rise to several interesting,
inhibitory molecules [7]. On the contrary, decreasing the size of the two aryl groups only shifted the
potentiation/inhibition capacity to higher concentrations [7].
In an attempt to identify and characterize new, stronger SOCE inhibitors, we decided to perform
our synthesis of 2-APB analogs by increasing the size of the two aryl groups by adding small groups
like methyl or methoxyl groups, or larger ones like other aromatic rings (molecules are depicted
in Figure 1).
Using a previously described methodology, these compounds were prepared in a one-pot
procedure using air-stable amine borane complexes and Grignard reagents formed under Barbier
conditions. The amine borane complexes have been shown to provide easy access to borinic acids and
their derivatives. Indeed, by direct addition on the diisopropylamine borane, the diarylaminoborane
can rapidly be obtained without undergoing a third addition leading to the triarylborane. In all
cases, the diarylaminoborane was readily transformed into the corresponding 2-aminoethylborinate,
via simple methanolysis followed by transesterification using 2-aminoethanol.
This approach allowed us to introduce alkyl chain in para (P3, 90%; P8, 85%; P9, 70%) and/or
meta position (P5, 89%; P7, 79%, P10, 85%). Other functional groups were perfectly tolerated such as
methoxy (P2, 81%; P4, 79%) halides (P13, 80%; P14, 80%, P15, 79%) but also larger (hetero)aromatics
(P6, 88%; P11, 79%, P12, 68%, P17, 79%).

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To visualize SOCE, we performed a classical protocol: Jurkat cells in Ca2+-free HBS medium are
stimulated by 1 µ
M thapsigargin (TG, black arrow, Figure 2A) during 600 s to allow the release of Ca2+
ions by the ER, and the subsequent opening of the store-operated Ca2+ channels (SOCC). Addition of
1 mM CaCl2 allowed for a massive entry of Ca2+ ions through SOCC leading thus to an increase of the
cytosolic calcium concentration ([Ca2+
]cyt). To test 2-APB and its analogues, different concentrations of
compounds were added 30 s prior to Ca2+ addition.
Figure 2. 2-APB has a dual effect on Jurkat T cell store-operated calcium entry (SOCE), while the other
2-APB analogs have only an inhibiting effect. (
measurement in Jurkat cells using Indo-1 fluorescence. Cells were treated for 10 min with thapsigargin
(1
M, black arrow) to allow Ca2+ release from endoplasmic reticulum (ER) and opening of the SOCE
channels. After 10 min, 1 mM CaCl2 was added, allowing Ca2+ entry through the SOCE channels.
2-APB ( ) or P11 ( ) at the indicated concentrations was applied 30 s prior to CaCl2 (red arrow). Results
are representative for five experiments. ( ) Dose-response curves of compounds P1 (= 2-APB), P3, P7,
P9, and P11 on Jurkat cell SOCE. Experiments were performed as in Figure 2A, and the peak [Ca2+
A
) and (
B
) cytosolic Ca2+ concentration ([Ca2+
]
cyt
)
µ
A
B
C
]
cyt
was expressed as % of the peak [Ca2+
]cyt recorded in the absence of any compounds. Compounds were
added 30 s prior to CaCl2. Results are expressed as mean
molecule).
±
SEM (n = 3 to 5 according to the tested

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As shown in Figure 2A, our 2-APB had the typical dual effect on SOCE amplitude: a large
potentiation at 5 increase) and an almost total inhibition at 50 M.
M (> 200% of control [Ca2+
No significant differences were found between commercial and our 2-APB (not shown).
µ
]
cyt
µ
Figure 2B depicted the effect of increasing compound P11 concentration: noteworthy the SOCE
was totally blocked for concentrations > 100 nM.
We realized the same kind of experiments with all our 2-APB analogues (only four molecules were
shown on Figure 2C) and all the molecules have lost the possibility to increase the [Ca2+
]
cyt, confirming
that enlarging the two phenyl groups impairs the potentiation process, resulting in inhibitors with a Ki
ranging 1000-fold (between 75 nM and 75 M, Table 1). Noteworthy, we also tested all these molecules
on a lymphoblast cell lines devoid of any Orai1 expression due to a double mutations [19] and none
was able to induce a [Ca2+
increase due to the activation of Orai3 channels as 2-APB can do.
µ
]
cyt
Table 1. Inhibition constants of the various 2-APB analogs used in this study (left, compounds with a
nanomolar Ki, right, compounds with micromolar Ki).
Compound
Ki (nM)
Compound
Ki (µM)
P11
P6
P15
P8
75 ± 21
275 ± 17
294 ± 74
350 ± 40
374 ± 96
405 ± 23
484 ± 116
641 ± 103
751 ± 97
P13
P5
P16
P3
P2
P14
P4
1.8 ± 0.1
2.1 ± 0.6
3.1 ± 0.6
3.5 ± 0.6
3.5 ± 0.65
5.4 ± 0.3
75 ± 21
P12
Dibenzothienyl-APB [7]
P10
P9
P7
P17
>> 100
2.1. Addition of Methyl and/or Methoxyl Groups
The addition of one methyl on both phenyl rings in para and meta positions respectively for
compound P3 (and P5) allowed the creation of inhibitors with an inhibition constant of 2–3 M (Table 1,
µ
Figure 2B). Interestingly, the addition of a second methyl to both phenyl rings significantly increased the
inhibition efficacy of the molecule (p > 0.05, n = 3, compounds P7 (and P10), Figure 2B). Among these
two molecules, the compound P10 was the most active with a Ki
inhibition was reached at 3 µM.
≈
0.5 µM (Table 1), and complete
We also created 2-APB analogs with one methoxy group on the two aromatic rings (compounds
P2 and P4). Even if the presence of a methoxy group on the aromatic rings in para position had the
same effect as a methyl group (compound P3 ≈ P2), the replacement in meta drastically impaired the
inhibitory capacity of the compound P4 (Ki = 75
(not shown).
±
21
µM, Table 1) with a maximal inhibition of
≈
80%
Compound P16 represents a chimera between P3 and P2 (Figure 1), bearing one methyl and one
methoxy group on either aromatic ring. As compounds P3 and P2 had similar properties, unsurprisingly
P16 had a similar Ki (3.1 ± 0.6 µM, Table 1).
From these molecules, it seems that the more the two rings are ramified, the more they inhibit the
Jurkat cell SOCE.
2.2. Fluorine- and Chlorine-Containing Analogs
Compounds P13 and P14 are, respectively, analogs of compound P3 where the methyl groups
were replaced by chlorine or fluorine. The presence of a halide only weakly modified the Ki
and 5 M respectively for compound P13 (Cl) and compound P14 (F) vs. 3.5 M for compound P3
(Table 1).
≈
2 µM
µ
µ

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Interestingly, the compound P15 which has one methyl + one chlorine on both phenyl rings had a
large increase in its inhibitory capacity (Ki ≈ 0.3 µM, Table 1).
These results confirm that the more ramified is the phenyl group, the more efficient the molecule
inhibits the SOCE. However, the use of chlorine or fluorine did not hugely improve the inhibition
capacity of the molecules.
2.3. Compounds P8 and P9
These two compounds are analogs of P3 with a bulky alkyl residue (tBu, P8) or a linear alkyl
chain (nBu, P9). P8 and P9 are better inhibitors with Ki = 350
±
40 nM and 641
±
103 nM, respectively
(n = 3 for both, Table 1 and Figure 2B). Thus, the addition of larger groups increases the efficacy of
the molecules. Noteworthy, compound P7, bearing a 3,4-dimethyl substituent, displayed the same
behavior as P9 (Figure 2B).
2.4. Fused Ring Analogs
The best inhibitor we previously described was a compound in which the two phenyl groups
were replaced by 2 benzothienyl groups attached to the central boron atom by the thienyl parts
(
Ki ≈ 0.4 µM, full inhibition at concentration > 3
µM [7]). Our compound P12 is very similar, except that
the benzothienyl groups are attached to the boron atom by the benzyl parts. The reversal of this group
had no real effect as the Ki of compound P12 was very similar at 374 ± 96 nM (n = 3, Table 1).
The naphtyl analog P6 had a slightly better Ki, 275
±
17 nM (n = 3, Table 1). However, the best
inhibitor was obtained when the group was formed by two phenyls bound by a single C-C linker.
This compound P11 had a Ki < 100 nM (75 21 nM, n = 3, Table 1) and fully inhibited the SOCE at
concentrations below 1 M (Figure 2B). Interestingly, when we added a gem-dimethyl between the
±
µ
two phenyl groups (compound P17), no effect on the SOCE amplitude was seen for concentrations
>3 µM, and higher concentrations disrupted the fluorescence and impaired the measurement.
Thus, the free rotation of the two phenyl groups on the same side seems important for the high
efficacy of compound P11.
2.5. P11 Specifically Inhibits Store-Operated Calcium Entry (SOCE) but Does Not Affect the
Ca2+-Extrusion Mechanisms
As shown in Figure 2A, after TG treatment and addition of extracellular Ca2+, the [Ca2+
]
cyt
rapidly increased and reached in control conditions a peak of about 1
µM followed by a decay to
a new equilibrium around 500 nM. As [Ca2+ results from a balance between Ca2+ influx and
]
cyt
efflux, this decay was due to the activity of the plasma membrane Ca2+ ATPases (“PMCA”) and the
Na+/Ca2+ exchanger (“NCX”) counteracting the SOCE (TG has blocked the SERCA activity, impairing
Ca2+ uptake in the ER). To ensure that compound P11 acted on the SOCE and not on the Ca2+-efflux
mechanisms, we next performed a Mn2+-quenching experiment with Indo-1. Mn2+ ions enter the
cells through SOCC but cannot be pumped outside the cells by PMCA and NCX; once in the cells,
Mn2+ bound to Indo-1 and quenched its fluorescence measured at 430 nm. Thus, an increase of the Mn2+
influx amplitude was associated with an increase of the Indo-1 quenching speed, and reversely [7,9].
After a 10 min TG treatment to open the SOCC, 100 µ
M MnCl2 was added. Mn2+ induced a
quenching rate of
−
0.89
±
0.05% Fo/s (n = 3, Figure 3A). In presence of increasing P11 concentrations
added 30 s prior to the Mn2+ ions, the Indo-1 quenching rate decreased to reach a
≈
90% blockade at
1 µM (-0.09 ± 0.01, n = 3, Figure 3A).

Int. J. Mol. Sci. 2020, 21, 9777
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Figure 3. Compound P11 inhibits Mn2+ influx in Jurkat cells as well as SOCE in Jurkat cells and other
cell types. (
) Mn2+ quenching of Indo-1 in Jurkat cells is inhibited by increasing P11 concentration.
Cells were pre-treated for 10 min with thapsigargin (TG, 1
M) to allow Ca2+ release from the ER and
opening of the SOCE channels. Thirty seconds before addition of MnCl2, the different [P11] (were added
to the bath medium. At t = 0 s, 100 M MnCl2 was added. Fluorescence of Indo-1 was recorded at 430 nm
after excitation at 360 nm. Results are representative for three experiments and express the fluorescence
relative to that prior to MnCl2 addition. ( ) Dose-response curves for compound P11 on SOCE
A
µ
µ
B
measured in Jurkat, DG75 (human Burkitt lymphoma cell line), U937 (human monocytic lymphoma
cell line), and MDA-MB231 (breast cancer) cells. Experiments were performed for each cell line as in
Figure 2A, and the peak [Ca2+
]
cyt
was expressed as % of the peak [Ca2+
]
cyt
recorded in the absence
of any compounds. P11 was added 30 s prior to 1 mM CaCl2. Results are expressed as mean ± SEM
(n = 3 except n = 5 for the Jurkat cells) ( cyt) measurement
) Cytosolic Ca2+ concentration ([Ca2+
in Orai1-expressing lymphoblastoid cell line (“LCL Orai1+”) and Orai1-deficient lymphoblastoid
cell line (“LCL Orai1 “) using Indo-1 fluorescence. Cells were treated for 10 min with thapsigargin
(1
C
]
−
µ
M, black arrow) to allow Ca2+ release from ER and opening of the SOCE channels. After 10 min,
1 mM CaCl2 was added, allowing Ca2+ entry through the SOCE channels. 1
µM P11 was added at
t = 800s. Results are representative for five experiments.

Int. J. Mol. Sci. 2020, 21, 9777
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From this result, it is clear that the compound P11 targeted the SOCE.
2.6. P11 Is Also Able to Block SOCE in Other Cell Types
We also tested P11 on the DG75 (Burkitt B lymphoma), U937 (monocytic lymphoma),
and MDA-MB231 (breast carcinoma) cell lines. With the SOCE measured in the same conditions,
compound P11 was significantly more efficient on these three cell lines than on Jurkat cells with
Ki = 32 ± 2 nM, 40
±
5 nM, and 50
±
5 nM, respectively, for DG75, U937, and MDA-MB231 cells vs.
75 ± 21 nM for Jurkat cells (Figure 3B). Thus, in several cell lines, full inhibition of the SOCE was
already obtained at a concentration of 100 nM (Figure 3B).
We next tested P11 efficacy on two lymphoblastoid cell lines (“LCL”) established from
an Orai1-deficient with a severe combined immunodeficiency (“LCL Orai1 ”) and a healthy
−
(“LCL Orai1+”) patients (Figure 3C [19]). As shown in Figure 3C, in absence of extracellular
CaCl2, TG induced the Ca2+ release from the ER in the two types of cells. However, CaCl2 addition
after 10 min of TG treatment was only able to induce a massive [Ca2+
]
rise in LCL expressing Orai1
cyt
proteins, confirming the SOCE default of the SCID patient. Noteworthy, 1
µM P11 only induced an
immediate decrease of [Ca2+
]cyt in Orai1-expressing LCL and was inefficient in Orai1-deficient LCL.
These results confirm that Orai1 is the target of P11 in these cells.
2.7. Selectivity of Compounds P11 and P9
Despite interesting properties, 2-APB is a complex molecule that lacks specificity. Thus, 2-APB was
firstly described as an IP3R inhibitor [20] but it blocks also SERCA [14] and some TRP channels (TRPC3
for example [21]). Thus, on Jurkat cells, 2-APB has three main targets that play an important role in
Ca2+ homeostasis. As our molecules are analogs of 2-APB, it was obvious to check whether they also
affected SERCA and IP3R activity.
Among our various molecules, we selected to perform experiments on P11 and two structurally
close compounds, P8 (Ki ≈ 300 nM) and P9 (Ki ≈ 600 nM).
Measurements of SERCA-mediated 45Ca2+ store loading and IP3-dependent release from the
ER Ca2+ store were performed on permeabilized cells in unidirectional conditions. As this approach
required the use of cells forming strongly adherent monolayers, we used L15 fibroblasts stably
overexpressing IP3R1, similarly to our previous work [9]. Unfortunately, P8 induced already a strong
Ca2+ leak from the ER by an IP3-independent pathway, impairing the experiment.
As shown in Figure 4, both P11 and P9 displayed some inhibitory effects on the activity of SERCA
and/or IP3R.
Compound P9 did not significantly inhibit IP3R activity but was only a partial inhibitor of the
SERCA activity:
−
52
±
3% (n = 4) at 100
µ
M with a Ki value of 6 0.3 µM (n = 4). Interestingly, below 3 µM,
±
compound P9 thus only inhibited SOCE and quickly lost its efficacy at decreasing concentrations
(Figure 4).
In contrast, compound P11 was a weak inhibitor of the IP3R (
calculation of the Ki was however impossible because too high P11 concentrations would be needed to
determine this value with sufficient precision. This molecule was also a more complete inhibitor of the
−
29
±
9% at 100 µM, n = 3);
SERCA activity, with an almost total inhibition at concentrations > 30
compound P11 was therefore still a hundred times less efficient for inhibiting SERCA activity than for
inhibiting SOCE (Ki = 7.4 M (n = 3) vs. 75 21 nM (n = 3). Thus, at concentrations < 1 M, P11 is
a specific SOCE inhibitor without any effect on SERCA or IP3R in Jurkat cells.
µM (−
89
±
1%, n = 3). However,
±
2
µ
±
µ

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Figure 4. Dose-response curves for compounds P9 (A) and P11 (B) indicating a preferential inhibition of
SOCE over the loading of the ER Ca2+ stores by SERCA pumps (“Loading”) and the Ca2+ release from
the ER by IP3Rs (“IICR”). The effects of P9 and P11 on the IP3–induced Ca2+ release (“IICR”) and the
SERCA-mediated Ca2+ loading (“loading”) were investigated in permeabilized L15 fibroblasts. The ER
Ca2+ stores of saponin-permeabilized L15 fibroblasts were loaded until steady-state with 45Ca2+ and
Ca2+ release was induced by incubation in a Ca2+-free efflux medium in the presence of TG (10
The indicated concentration of P9/P11 were added either during the loading phase for assessing SERCA
activity or during the efflux phase before addition of IP3 (0.7 M) to assess IP3R activity. Activity was
µM).
µ
expressed as % of the activity measured in the absence of any compounds. Results are expressed as
mean ± SEM (n = 3 for “loading, n = 5 for “SOCE” and “IICR”).
All our subsequent experiments were therefore performed only with compound P11 as it is a
specific inhibitor of SOCE that can be used at concentrations <1 µM.
2.8. Compound P11 Impairs the Activation of Jurkat Cells
Activation of T cells needs a [Ca2+
]
increase for several minutes to allow the synthesis of
cyt
interleukin-2 (“IL-2”) and the further activation of the immune functions. Therefore, inhibition of the
[Ca2+
cyt rise due to SOCE impairs T cell activation and proliferation [7,22].
]
Having shown that compound P11 was able to block the Jurkat cell SOCE, we next performed
experiments using phytohemagglutinin (“PHA”). PHA is commonly used to cross-link the T cell
receptor allowing the synthesis of IL-2 and thereafter the T cell activation [22]. As shown in Figure 5A,
in presence of 1 mM extracellular CaCl2, PHA induced a Ca2+ mobilization which was largely inhibited
by increasing [P11]. Noteworthy, PHA was able to induce a weak Ca2+ release from internal stores
(orange curve). One micromolar of P11 did not affect this Ca2+ release as the PHA-induced Ca2+ rise
was very similar to the one recorded in absence of extracellular Ca2+ ions, showing that P11 only

Int. J. Mol. Sci. 2020, 21, 9777
10 of 23
acted on the SOCE and not the ER Ca2+ release. However, after reaching a peak at 180 s, the [Ca2+
]
cyt
started to decay and decreased to a value beneath the [Ca2+
]cyt before cell stimulation. Thus, high [P11]
concentrations not only blocked the SOCE of PHA-stimulated cells, but also emptied the cytosolic
Ca2+ content.
Figure 5. Compound P11 decreases phytohemagglutinin (PHA)-induced Ca2+ mobilization and the
synthesis of interleukin-2 (IL-2) and increases cell death and decreases by PHA-stimulated Jurkat cells.
(
A
) Cytosolic Ca2+ concentration ([Ca2+
]
cyt) measurement in Jurkat cells using Indo-1 fluorescence.
g/mL PHA plus increasing [P11] concentrations in presence of 1 mM
extracellular CaCl2, except “PHA 0Ca” which was obtained by stimulating cells only with PHA in
absence of extracellular CaCl2. These traces are representative of three repeats. ( ) Jurkat cells were
stimulated by 10 g/mL PHA in the presence of P11 at the indicated concentrations, UT = untreated
cells. IL-2 concentration was measured by ELISA in the cell supernatants. ( ) Dead cells were estimated
after 24 h using trypan blue staining. ( ) The values of produced IL-2 were corrected for the number of
Cells were treated with 10
µ
B
µ
C
D
living cells, showing that addition of increasing P11 concentrations at the time of PHA stimulation,
progressively decreased the IL-2 synthesis by living cells. Statistically significant differences are
indicated by: * p < 0.05, ** p < 0.01. n = 6.
We next studied the effect of increasing [P11] on the neo-synthesized IL-2 concentration in the
medium of PHA -stimulated Jurkat cells after 24 h.
In the absence of PHA stimulation, Jurkat cells did not produce any IL-2 (“UT”, Figure 5B).
On the contrary, under PHA stimulation, approximately 100 pg/mL IL-2 was detected in the medium
(
103 ± 4 pg/mL, n = 3, Figure 5B) after 24 h. Addition of increasing P11 concentrations at the time of
PHA stimulation progressively decreased the IL-2 synthesis to 26 6 pg/mL at 1 M P11 ( 74%, n = 3,
Figure 5A). By itself, 1 µM P11 was not able to induce IL-2 synthesis (not shown).
±
µ
−

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As this diminution in IL-2 synthesis can both be explained by the decrease of its synthesis by
all cells, or by a decrease in the number of cells that can synthesize IL-2, we next evaluated cell
death by trypan blue staining in the presence of PHA stimulation with and without the addition of
compound P11.
Under resting culture conditions, the percentage of dead cells measured was lower than 5%
(
3.4 ± 0.4%, n = 5, Figure 5C). After 24h PHA stimulation, the number of dead cells did not significantly
increase (4.4 1.0%, n = 4). Furthermore, compound P11 by itself was not toxic as no significant
increase in dead cells was noticed at a maximal concentration of the compound (at 1
n = 5).
±
µM: 4.0
±
0.4%,
Addition of increasing concentrations of compound P11 simultaneously with PHA, however
strongly increased the proportion of dead cells up to 33.2 1.3% at 1 M (n = 4, p < 0.05, Figure 5C).
By calculating the amount of IL-2 synthetized by the remaining living cells, it was clear that increasing
concentrations of compound P11 decreased this synthesis capacity by 63% (39.2 8.6 pg/mL vs.
±
µ
≈
±
107.6 ± 4.1 pg/mL for PHA-stimulated cells in the absence of P11, Figure 5D).
These results clearly show that compound P11 has a dual effect on activated Jurkat cell, on the
one hand reduction of IL-2 synthesis and on the other an induction of their death.
2.9. Compound P11 Induces Apoptosis of PHA-Stimulated Jurkat Cells
To verify the mechanism of cell death induced by compound P11, we next performed caspase-3
activity assays using the fluorogenic Ac-DEVD-AFC substrate as well as a TUNEL assay.
As shown in Figure 6, compound P11 alone, even at a concentration of 1
increase in capase-3 activity. However, caspase-3 activity was increased by
PHA stimulation during 24 h (1836 132 AU vs. 1037 100 AU, n = 5). Interestingly, the additional
µM, did not induce any
≈
twofold after 10 g/mL
µ
±
±
presence of 10 nM compound P11 already induced a maximal activating effect on caspase-3 activity
with a ≈ threefold increase (3332 ± 113 AU at 1 µM, n = 5).
Figure 6. Compound P11 increases the caspase-3 activity specifically in PHA-stimulated Jurkat cells.
Caspase-3 activity assays were performed using fluorogenic Ac-DEVD-AFC. Cells were then incubated
for 24 h with 0, 1, or 10
µg/mL PHA and 0 to 1
µM P11 in complete culture medium. Caspase-3 activity
was about twofold increased at 10
µg/mL PHA stimulation for 24 h. Moreover, the addition of 10 nM
compound P11 to PHA-stimulated Jurkat cells already produced a maximal activation of caspase-3.
Statistically significant differences are indicated by: ns = non-significant, * p < 0.05, ** p< 0.01, n = 6.
Moreover, similar results were obtained when the percentage of dead cells was measured with
the TUNEL assay (Figure 7). In resting conditions, less than 5% of the cells had fragmented DNA
(
4.2 ± 0.3, n = 3). PHA alone induced a threefold increase in the number of cells with fragmented
DNA (13.3 1.6%, n = 3). At concentrations > 10 nM, compound P11 induced a 4.5-fold increase
(17.9 ± 1.2%, n = 3 at 1 µM).
±
≈

Int. J. Mol. Sci. 2020, 21, 9777
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Figure 7. Compound P11 increases DNA fragmentation in PHA-stimulated Jurkat cells. (A) Cells were
stained with DAPI (blue, top) to visualize the nuclei and with the TUNEL method (green, bottom)
to visualize apoptotic cells. Each staining was merged with the phase contrast image to visualize
whole cells. In control conditions or after stimulation by 1
cells were detectable (~5%). PHA (10 g/mL)-stimulated cells demonstrated a significant increase in
apoptotic cells. Costimulation with concentrations > 10 nM of P11 compound induced a 1.5-fold
increase. Representative pictures of cells after 24 h of treatment with 10 g/mL PHA alone or with
1–1000 nM P11, or with 1 M P11 alone. Scale bar = 50 m. ( ) The number of apoptotic cells (ratio of
µM P11 for 24 h, only few apoptotic
µ
≈
µ
µ
µ
B
TUNEL-positive cells/DAPI-positive cells, expressed in percent) was counted after 24 h of treatment.
Statistically significant differences are indicated by: ns = non-significant, ** p < 0.01, n = 6.
Taken together, these results demonstrate that compound P11 is not toxic for the cells when added
alone but could profoundly impair Jurkat cell activation by inducing their apoptosis.
3. Discussion
Since the discovery of the molecular partners implied in SOCE in the middle of the 2000s,
the characterization of their modulators has emerged as attracting enough for pharmaceutical companies
like GSK and Hoffmann-La Roche or newcomers like Calcimedica. Indeed, it appeared that aberrant
SOCE could be associated with autoimmune disorders, inflammation, or cancers [23]. Inhibiting SOCE
appears as a new and promising way to treat this kind of disease or to ease it.
SOCE was functionally characterized during the 1980s (formerly named capacitative calcium
entry by Jim Putney [24]), but its molecular mechanism was only unveiled in the last decade [
3,25].
After store depletion, the ER proteins STIM1 interact with and activate the plasma membrane Ca2+

Int. J. Mol. Sci. 2020, 21, 9777
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channels mainly consisting of Orai1 proteins [
3
]. Noteworthy, loss-of-function mutations in the Orai1
gene, rendering the Orai1 protein unfunctional or absent in the plasma membrane, via SOCE inhibition,
mainly impair the immune system [26]. On the opposite, some gain-of-function mutations in Orai1
or STIM1, allowing a constitutive SOCE or an increased SOCE amplitude are also associated with
diseases like the York platelet syndrome or the Stormorken syndrome [27]. From this fact, the control
of Orai1 channels (directly or indirectly via STIM1) and the SOCE emerged as a new way to control the
immune system, but also the proliferation of cancerous cells or other diseases. Noteworthy, two Orai1
paralogues exist, Orai2 and Orai3 with “less global” roles: thus, Orai2 regulates the SOCE amplitude by
forming heteromeric channels with Orai1 with reduced conductance in some T cells [28]. About Orai3;
there exists a dichotomy between SOCE observed in breast cancer cell lines and in cells from patient
samples. In estrogen-receptor expressing cells, Orai3 channels are responsible for the SOCE, while Orai1
are the ones responsible in estrogen-receptor-negative cells [29]. Thus Orai3 channels could also
represent a target of interest for the treatment of the estrogen-receptor-positive breast cancer cells
which represent 80% of breast cancers [29].
However, in the vast majority of the cell types, the spatial emplacement of the three Orai proteins
in the cells seem to be different: Orai1 mainly is located in the plasma membrane [
3
,30], Orai2 in the
internal compartments, and Orai3 in the ER membrane [31
participates in SOCE [3,9,19].
–33]. Thus, in T and B cells only Orai1
Despite huge therapeutic potential, the characterization of new SOCE modulators is impaired
by the limited knowledge of the Orai1 structure. Indeed, only the crystal structure of the Drosophila
melanogaster dOrai, which shares 73% sequence homology with hOrai1, has been published at a 3.35 Å
resolution [34]. Furthermore, no hOrai1 structure has yet been established when bound to STIM1 in its
active conformation. A controversy also still exists on the number of Orai proteins needed to form a
functional channel, tetramer [35] or hexamer [36,37], although the latter seems to be the most accepted
currently. Therefore, the development of new SOCE modulators is still not possible by docking work
but powered by the characterization of analogs of existing modulators.
Among SOCE modulators, we focused our work on 2-APB, based on its following three properties:
(i) 2-APB is a small molecule; (ii) 2-APB potentiates SOCE amplitude at low concentrations (
and inhibits SOCE at higher concentrations (> 30 M); (iii) the relative ease to synthesize analogs.
In three previous studies, we already showed that the central boron-oxygen-carbon plays a role in
the potentiation capacity and the ethanolamine and the two phenyl groups in the inhibition [ 38].
≈
5 µM)
µ
7,9,
Thus, by modifying one or the other part of the 2-APB molecule, it is possible to create analogs with
better potentiation or inhibition ability. Furthermore, our 2-APB analogs request Orai1 expression to
act on the T cell SOCE [9].
In the present work, we only modified the two phenyl rings by adding simple radicals like
methyl or by replacing them with more complex cycles to develop new SOCE inhibitors (Figure 1).
Interestingly, all our molecules modified on the two phenyl groups lost their potentiation capacity.
In Djillani et al. 2014 [7], we showed that two analogs where one (nicknamed “methoxy-APB”) or two
methoxyl (“dimethoxy-APB”) groups were added on the same phenyl, keeping the second one intact,
still had a potentiation capacity (even if it was decreased). On the opposite, our new compounds
with added methoxyl groups on the phenyl groups have only inhibiting properties, confirming that
increasing the size of both phenyl groups allows the creation of new SOCE inhibitors.
Among our new molecules, compound P11 was the most potent SOCE inhibitor. With an inhibitory
constant between 32 and 75 nM depending on the cell type used and a complete SOCE inhibition at
a concentration well below 1 µM, P11 is one of the best SOCE inhibitors described in the literature.
The twofold difference in Ki between cell types could reflect a difference in the Store-Operated Calcium
Channels (SOCC) composition. Indeed, it has been shown that the expression ratio of Orai1/STIM1
modulates the sensitivity of the SOCE to 2-APB [39]. Furthermore, Orai1 can be associated with
some TRP channel subunits like TRPC1, TRPC3, and TRPC6 [40] which can also interact with 2-APB,
and thus potentially with its analogs.

Int. J. Mol. Sci. 2020, 21, 9777
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The free rotation between the two phenyl groups on the same side seems to play an important
role in the inhibition capacity of compound P11. When this rotation is impaired by a second carbon
“bridge,” like in compound P24, the molecule was not able to inhibit, even partially, the SOCE at
concentrations below 1 µM. Thus, we assume that when 2-APB or its analogs enter the SOCC pore,
the two “wings” (phenyl or larger/longer aryls) need to tether and reach the hypothetic inhibitory
binding site(s).
However, the great advantage of compound P11 is that it avoids the major side effects of 2-APB,
as it did neither inhibit the IP3R nor the SERCA pumps of the ER. Furthermore, it did not induce a Ca2+
leak from the ER by an unknown pathway, for example, as our compound P8 or as DPB162-AE [10
]
did. This absence of side effects could be related to the apparent non-toxicity of the molecule on resting
cells: compound P11 by itself at 1 µM did neither increase DNA fragmentation nor cell death.
Jim Putney showed more than 30 years ago that the Ca2+ influx was activated by the ER depletion,
and that this Ca2+ entry he called “capacitative calcium entry,” eventually named store-operated
Ca2+ entry (SOCE), allowed the refilling of the ER [24]. More recently, Zheng et al. [41] showed
that the refilling of ER Ca2+ is fully impaired in cells that do not express all three Orai isoforms.
However, after 24 h in culture, Ca2+ stores became eventually refilled, meaning that SOCE is critical
for the immediate refilling of the ER Ca2+ stores. It is known that ER Ca2+ store depletion leads to ER
stress induction that, if not counteracted by the unfolded protein response, will provoke apoptosis.
In our experiments, the cells were stimulated with PHA and different [P11] for 24 h to study the
apoptosis induction and to measure IL-2 synthesis and caspase-3 activity. Remarkably, P11 was not by
itself cytotoxic, and it exerted a cytotoxic effect only when the cells were stimulated and the [Ca2+
]
cyt
rise impaired (Figure 5A). Surprisingly P11 induced a decrease of [Ca2+
]cyt beneath pre-stimulation
values. As the cells were still under PHA stimulation, we assume that there was a constant ER Ca2+
release and that the SOCE blockade impaired the refilling of the ER Ca2+ content and could induce an
ER stress leading to the apoptosis.
Enlarging the 2-APB molecule on the two phenyl groups allows the development of increasingly
efficient inhibitors. We anticipate that our compound P11 could also be further modified and are
confident that molecules with an inhibition capacity in the low nanomolar range could be obtained.
Thus, from our studies and those of Mikoshiba’s group, it appears possible to create 2-APB analogs
with higher potency and selectivity. Due to its dual effect on SOCE, the advantage of 2-APB over other
SOCE modulators is that, depending on the substitution performed, it is possible to create inhibiting or
potentiating agents. Our compound P11 could, therefore, represent a new promising branch of the
2-APB family that could help to cure human disorders in which Ca2+ homeostasis is modified.
4. Materials and Methods
4.1. Cell Lines
Jurkat (acute T cell leukemia from ATCC), DG75 (Burkitt B lymphoma, kind gift of Martin
Rowe, University of Birmingham, UK), and U937 (monocytic cell line from ATCC) cell lines were
basically maintained in RPMI-1640 medium (Lonza, Levallois-Perret, France) supplemented with 10%
heat-inactivated fetal calf serum and 2 mM L-glutamine, at 37 ◦C in a 5% CO2 humidified atmosphere.
The MDA-MB231 breast cancer cell line (form ATCC) was maintained in same conditions as the
leukocyte cell lines except that DMEM (Lonza, Levallois-Perret, France) medium was used instead of
RPMI1640. Epstein-Barr virus immortalized B cells from Orai1-deficient and healthy patients were a
kind gift of Capucine Picard and Alain Fischer (study center of primary immunodeficiencies, AP-HP,
Hopital Necker, Paris France [19]) and maintained as the leukocytes cell lines. The L15 fibroblast cell
line stably overexpressing IP3R1 [42] was a generous gift of Katsuhiko Mikoshiba (now at Shanghai Tech
Univ., China). The cells were maintained in DMEM medium (Gibco®, Life Technologies, Gent, Belgium)
supplemented with 10% heat-inactivated fetal calf serum, 3.8 mM L-glutamine, 1% non-essential amino

Int. J. Mol. Sci. 2020, 21, 9777
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acids, 85 IU mL−1 penicillin, 85
(pH 7.4) at 37 ◦C and 10% CO2.
µg/mL streptomycin, 400
µg/mL geneticin, and 25 mM Na-Hepes
4.2. Cytosolic Ca2+ Concentration and SOCE Measurement
[Ca2+
was recorded by a fluorimetric ratio technique as previously described [7,43,44].
]
cyt
Leukocytes were spun and resuspended at a density of 106 cells/mL in Hepes buffered saline
(HBS; 135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 11.6 mM Hepes, 11.5 mM glucose adjusted
to pH 7.3 with NaOH). Due to the presence of 0.002% Ca2+ in the NaCl, this HBS medium contained
0.1 mM of Ca2+. Then, cells were incubated in the dark with 4
µ
M Indo-1-AM for one hour at room
temperature under slow agitation. Cells were then centrifuged and resuspended in HBS medium
before measurement. To perform the experiments, 0.5 to 1
106 cells were suspended in 2 mL HBS in
×
a quartz cuvette and inserted into a spectrofluorophotometer (RF-1501 Shimadzu Corporation, Kyoto,
Japan) connected to a PC computer (Dell Computer Corp., Montpellier, France). A temperature of 37◦C
was maintained by circulating water from a bath. Ultraviolet light of 360 nm was used for excitation
of Indo-1, and emissions at 405 and 480 nm were recorded. Background and autofluorescence of the
cell suspension were subtracted from the recordings. The maximum Indo-1 fluorescence (Rmax) was
obtained by adding 1
Minimum fluorescence was determined following the depletion of external Ca2+ with 5 mM EGTA.
[Ca2+ cyt was calculated according to the equation [Ca2+
cyt = Kd (R-Rmin)/(Rmax-R), where Kd is the
µM ionomycin to the cell suspension in the presence of 10 mM extracellular CaCl2.
]
]
apparent dissociation constant of Indo-1-calcium complex (230 nM), and R is the ratio of fluorescence
values recorded at 380 nm in absence and presence of 10 mM CaCl2 [43].
To visualize the SOCE, cells were placed into the quartz cuvette and 0.5 EGTA was added
just before the beginning of the recordings. To induce the SOCE, the cells were treated with 1
µM
thapsigargin (TG) during 10 min in Ca2+-free HBS to induce Ca2+ release from the ER and the opening
of SOCE channels [7, ]cyt subsequently
45]. Then, 1 mM CaCl2 was added to measure the change in [Ca2+
to Ca2+ influx [45]. In the dose-response experiments, different concentrations of 2-APB and its analogs
were added 30 s prior to Ca2+ readdition [7,9].
In experiments with PHA, cells were placed in the cuvette, and 0.9 mM CaCl2 was added to
reach a 1 mM extracellular concentration. Free Ca2+ concentrations were calculated using the Bad4
software [46]
4.3. Measurement of Mn2+ Influx by Indo-1 Quenching
To study directly the divalent ion influx through the SOCC with selected 2-APB analogs,
we performed Mn2+ quenching experiments. Mn2+ ions bind to Indo-1 and quench the 430 nm
emission wavelength fluorescence after excitation at 360 nm. The decrease of fluorescence is directly
dependent on Mn2+ entry through SOCC. For measurements, cells were treated for 10 min with 1
µM
TG to open SOCC, whereupon 100 µM MnCl2 was added instead of CaCl2. After 60 s a 2-APB analog
was added as indicated and the change in the curve slope was measured.
4.4. Unidirectional 45Ca2+ Flux Experiments
The L15 fibroblasts stably overexpressing IP3R1 were seeded in gelatin-coated 12-well plates
(Greiner, Frickenhausen, Germany) at a density of 60,000 cells per well. Unidirectional 45Ca2+ flux
experiments on confluent cell monolayers were performed at 30 ◦C after membrane permeabilization
with saponin (40
the non-mitochondrial Ca2+ stores were loaded for 45 min in 120 mM KCl, 30 mM imidazole–HCl
(pH 6.8), 5 mM MgCl2, 5 mM ATP, 0.44 mM EGTA, 10 mM NaN3, and 150 nM free 45Ca2+ 28 µCi mL−1).
Subsequently, efflux was initiated by incubation in efflux medium (120 mM KCl, 1 mM EGTA, 10 M TG
µ
g/mL), exactly as previously described [
9,
47].
After permeabilization,
(
µ
and 30 mM imidazole–HCl pH 6.8). The latter medium was replaced every two minutes for 18 minutes.
The exact amount of Ca2+ at each time point is calculated by summing in retrograde order the amount
of radioactivity remaining in the cells at the end of the efflux and the radioactivity collected during

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the successive time intervals. This allows to calculate the steady-state content of the ER Ca2+ stores
(indicative for the SERCA activity, after subtraction of the loading achieved in the presence of 2
TG) the basal leak rate of Ca2+ out of the stores and the IP3-induced Ca2+ release after addition of IP3
(0.7 M) [14]. These various parameters were measured both in control conditions or in the presence of
µM
µ
various 2-APB analogs as indicated. To assess the effect of the 2-APB analogs on the SERCA-mediated
loading of the ER Ca2+ stores, the analogs were added during the 45Ca2+-loading phase as described
previously [9
]. Obviously, compounds that strongly stimulated the basal Ca2+ leak out of the stores
(as P8) could not be investigated for their effect on SERCA activity (as Ca2+ loading was already
compromised by the increased leak) or on IP3-induced Ca2+ release (as the decreased luminal Ca2+
level impacted both the driving force for Ca2+ release and the sensitivity of the IP3R) [48].
4.5. Interleukin-2 (IL-2) Assay
Forty-eight-well plates were seeded with 106 Jurkat T cells per well. Cells were then incubated
for 24 h with or without 10 µg/mL PHA and 0 to 1 µM P11 in complete culture medium. After 24 h,
supernatants were collected, and IL-2 amounts were quantified using a Quantikine Human IL-2
Immunoassay (R&D Systems Europe, Lille, France). Optical density (OD) was measured at 450 nm
using a Victor3 plate reader (Perkin Elmer, Courtaboeuf, France). The concentration of supernatant
IL-2 was determined from the OD curve obtained with the standard. Results were the mean of four
repeats. In parallel plates, cell viability was accessed after treatment with trypan blue and counting
with a Malassez hematimeter (Sigma-Aldrich, Saint Quentin Fallavier, France).
4.6. Fluorometric Assay for Caspase-3 Activity
Caspase-3 activity was measured using the fluorogenic substrate Ac-DEVD-AFC (Tebu-bio,
Le Perray-en-Yvelines, France) according to the manufacturer’s instructions. Briefly, 48-well plates
were seeded with 106 Jurkat T cells per well. Cells were then incubated for 24 h with 0, 1, or 10
µg/mL
PHA and 0 to 1
µM P11 in complete culture medium. After 24 h, cells were collected and lysed in RIPA
lysis buffer supplemented with protease inhibitor cocktails (Life Science Biorad, Marnes la Coquette,
France). The protein samples were quantified using the DC protein assay reagent (Biorad, Les Ulis,
France). Cell lysates (50
µg) were diluted with reaction buffer (200 mM Hepes pH 7.4, 1 mM EDTA,
20% sucrose and 20 mM dithiothreitol) and incubated with the fluorogenic substrate (20
µ
M final
concentration) for 45 min up to 7 h at 37 ◦C. The release of 7-amino-4-trifluoromethylcoumarin (AFC)
was measured by fluorescence (excitation at 405 nm, emission at 505 nm) using a Victor3 plate reader
(Perkin Elmer, Courtaboeuf, France).
4.7. Apoptosis Detection by the Terminal Transferase dUTP Nick End Labelling (TUNEL) Method
We measured the apoptosis levels in Jurkat cells by the “in situ cell death detection” kit (Roche
Applied Science, Meylan, France). Briefly, 50,000 cells/well were seeded in a 96-well plate and
treated with or without 10 µg/mL PHA and 0 to 1 µM P11 for 6, 12, or 24 h. Cells were spun, fixed,
and permeabilized according to the manufacturer’s instructions and the terminal transferase dUTP
Nick end labelling reaction was performed in the dark during 60 min at 37 ◦C. For visualization of
the results, an epifluorescence microscope (Axioskop, Karl Zeiss, Le Pecq, France) was used at an
excitation wavelength of 488 nm, and an emission wavelength of 545 nm. Cells were also loaded
with 4’,6’ –diamidino-2-phenylindole (DAPI) to visualize the nuclei. Total cell number (>200) and
TUNEL-positive cells were counted in several fields (>5) and the ratio of apoptotic cells was calculated.
This experiment was repeated three times.
4.8. Chemicals
TG and ionomycin were purchased from Calbiochem-Merck (Nottingham, UK).
Synthesis of 2-APB and its analogs were done using previously described methodologies [49].
General procedure for APB analog synthesis: To a solution of arylbromide (3 eq), magnesium (3.5 eq),

Int. J. Mol. Sci. 2020, 21, 9777
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and diisopropylamine borane complex (DIPAB, 1 eq) in dry THF (1 mL/mmol) was added at room
temperature to a solution of PhMgBr (2 M in THF, 0.05 eq). After 10 min, (or the end of gas evolution
on large scale), the reaction mixture was heated to 70◦C until no arylbromide remained in the reaction
mixture (8 to 16 h) as witnessed using TLC monitoring. At 0 ◦C, 1 mL/mmol of dry MeOH was added
(caution, exothermic reaction). After 1 h, the reaction mixture was concentrated under reduced pressure,
and diluted in a mixture of 1N HCl/MeOH (7:3, 10 mL/mmol), after 1 h, the product was extracted with
Et2O (3
brine (3
×
10 mL/mmol) and the combined organic phases washed with 1N HCl (10 mL/mmol) and
10 mL/mmol). Organic phases were then dried over anhydrous Na2SO4 and concentrated
×
under reduced pressure. The residue was dissolved in Et2O (4 mL/mmol) and 1.2 eq of ethanolamine
was added. Depending on substituents, precipitation occurs spontaneously or requires addition of
pentane. Crystals were filtered, washed with pentane, and dried under vacuum to yield the pure
borinic acid 2-aminoethyl ester.
DIPAB Diisopropylamine-borane complex (55124-35-1)
A 3000 mL three-necked round-bottomed flask equipped with a mechanical stirrer, a thermometer,
and a dropping funnel was charged with THF (1500 mL) and NaBH4 (56.75 g, 1.5 mol) The heterogeneous
mixture was vigorously agitated using a mechanical stirrer and cooled with an ice/salt bath below 0 ◦C.
The dropping funnel was charged with 37.8 mL of H2SO4 (0.71 mol) The H2SO4 was added dropwise
maintaining the internal temperature below
5 ◦C over 3 h. An iPr2NH (140 mL, 1 mol) solution in
−
THF (100 mL) was added dropwise maintaining the temperature below 0 ◦C over 1 h 30 m. The mixture
was vigorously agitated during 20 h at room temperature. The mixture was filtrated over a N◦3 fritted
funnel and the resulting solid was triturated with THF (3
under reduced pressure the residue was taken with CH2Cl2, and then filtrated to eliminate all solid
residues. The filtrate was washed with water (4 200 mL). The organic phase was dried over Na2SO4
×
400 mL). THF filtrate was concentrated
×
and concentrated under reduced pressure to give DIPAB as colorless oil which solidified upon cooling
1
(102 g, 90%). H NMR (400 MHz, CDCl3):
δ (ppm) 2.96 (s, 1H), 2.89–2.75 (m, 2H), 1.95–1.48 (m, 13H),
1.3–1.01 (m, 7H). 11B NMR (128 MHz, CDCl3): δ (ppm)−20.32 (q).
P1, 2-APB, ((diphenylboryl)oxy)ethanamine (15614-89-8) [50]
1.02 g of ((diphenylboryl)oxy)ethanamine were obtained following the general procedure using
1.73 g of bromobenzene as a white solid (92% yield). 1H NMR (300 MHz, DMSO-d6):
δ
(ppm)
7.40 (d, J = 7.2 Hz, 4H), 7.13 (t, J = 7.2 Hz, 4H), 7.03 (t, J = 7.2 Hz, 2H), 6.07 (bt, 2H), 3.77 (t, J = 6.35 Hz,
2H), 2.90 – 2.75 (p, J = 6.35 Hz, 2H). 11B NMR (96 MHz, DMSO-d6):
δ
(ppm) 4.1 (s). 13C NMR (75 MHz,
DMSO-d6): δ (ppm) 131.5, 126.6, 124.9, 62.4, 41.3. Melting point: 192.5 ◦C.
P2, 2-((bis(4-methoxyphenyl)boryl)oxy)ethanamine (37763-62-5) [50]
1.21 g of 2-((bis(4-methoxyphenyl)boryl)oxy)ethanamine were obtained following the general
1
procedure H using 2.06 g of 3-bromoanisole as a white solid (81% yield). H NMR (300 MHz, CDCl3):
δ (ppm) 7.26 (d, J = 8.5 Hz, 4H), 6.72 (d, J = 8.5 Hz, 4H), 5.88 (bt, 2H), 3.74 (t, J = 6.35 Hz, 2H),
3.67 (s, 6H), 2.80 (t, J = 6.35 Hz, 2H). 11B NMR (96 MHz, DMSO-d6):
δ
(ppm) 10.3 (s). 13C NMR
(101 MHz, DMSO-d6):
δ
(ppm) 148.2, 133.3, 131.6, 127.3, 62.4, 41.3, 20.9. Melting point: 181.7 ◦C. LC-MS
(ESI): C16H20BNO3 570.70 [2M+H]+.
P3, 2-((di-p-tolylboryl)oxy)ethanamine (19565-45-8) [51]
1.04 g of 2-((di-p-tolylboryl)oxy)ethanamine were obtained following the general procedure using
1
1.88 g of 4-bromotoluene as a white solid (90% yield). H NMR (300 MHz, DMSO-d6):
δ (ppm) 7.25
(
d, J = 7.7 Hz, 4H), 6.94 (d, J = 7.7 Hz, 4H), 5.92 (bt, 2H), 3.73 (t, J = 6.4 Hz, 2H), 2.80 (t, J = 6.4 Hz, 1H),
2.20 (s, 6H). 11B NMR (96 MHz, DMSO-d6):
δ
(ppm) 5.7 (s) 13C NMR (75 MHz, DMSO-d6): δ (ppm) 148.2,
133.3, 131.6, 127.3, 62.4, 41.3, 20.9. Melting point: 185.3 ◦C. LC-MS (ESI): C16H20BNO [2M+H]+: 506.80.
P4, 2-((bis(3-methoxyphenyl)boryl)oxy)ethanamine
1.12 g of 2-((bis(3-methoxyphenyl)boryl)oxy)ethanamine were obtained following the general
1
procedure using 2.06 g of 3-bromoanisole as a white solid (83% yield). H NMR (300 MHz, DMSO-d6):
δ
(ppm) 7.06 (t, J = 7.8 Hz, 2H), 6.96 (m, 4H), 6.60 (dd, J = 7.8, 1.7 Hz, 2H), 6.05 (bt, 2H), 3.76 (t, J = 6.4 Hz,
2H), 3.68 (s, 6H), 2.81 (p, J = 6.4 Hz, 2H). 11B NMR (96 MHz, DMSO-d6): (ppm) 5.3 (s). 13C NMR
δ

Int. J. Mol. Sci. 2020, 21, 9777
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(75 MHz, DMSO-d6):
δ
(ppm) 158.2, 127.6, 123.9, 116.9, 110.1, 62.4, 54.5, 41.3. Melting point: 137.7 ◦C.
LC-MS (ESI): C16H20BNO3 570.70 [2M+H]+.
P5, 2-((di-m-tolylboryl)oxy)ethanamine (97979-11-8) [52]
1.05 g of 2-((di-m-tolylboryl)oxy)ethanamine were obtained following the general procedure
1
using 1.88 g of 3-bromotoluene as a white solid (82% yield). H NMR (300 MHz, DMSO-d6): δ (ppm)
7.26–7.10 (m, 4H), 7.01 (t, J = 7.4 Hz, 2H), 6.84 (d, J = 7.4 Hz, 2H), 6.00 (bt, 2H), 3.74 (t, J = 6.4 Hz, 2H),
2.80 (p, J = 6.4 Hz, 2H), 2.22 (s, 6H) 11B NMR (96 MHz, DMSO-d6):
DMSO-d6):
(ESI): C16H20BNO [2M+H]+: 506.8.
δ
(ppm) 4.8 (s). 13C NMR (75 MHz,
δ
(ppm) 135.2, 132.7, 129.1, 126.9, 125.9, 62.80, 41.8, 21.9. Melting point: 193.4 ◦C LC-MS
P6, 2-((di(naphthalen-2-yl)boryl)oxy)ethanamine (515157-62-7) [53]
1.50 g of 2-((di(naphthalen-2-yl)boryl)oxy)ethanamine were obtained following the general
procedure using 2.28 g of 2-bromonaphtalene as a white solid (92% yield). 1H NMR (300 MHz,
DMSO-d6):
δ (ppm) 7.95 (s, 2H), 7.76 (d, J = 7.7 Hz, 4H), 7.73–7.61 (m, 4H), 7.44–7.30 (m, 4H),
6.34 (bt, 2H), 3.88 (t, J = 6.3 Hz, 2H), 3.02–2.88 (p, J = 6.3 Hz, 2H). 11B NMR (96 MHz, DMSO-d6):
δ (ppm) 6.0 (s). 13C NMR (75 MHz, DMSO-d6):
δ (ppm) 132.9, 131.9, 130.8, 129.7, 127.4, 127.2, 125.4,
124.9, 124.3, 62.6, 41.6. Melting point: 206.2 ◦C. LC-MS (ESI): C22H20BNO 650.80 [2M+H].
P7, 2-((bis(3,4-dimethylphenyl)boryl)oxy)ethanamine (99269-70-2)
1.11 g of 2-((bis(3,4-dimethylphenyl)boryl)oxy)ethanamine were obtained following the general
1
procedure using 2.04 g of 4-bromo-o-xylene as a white solid (79% yield). H NMR (300 MHz, DMSO-d6):
δ
(ppm) 7.12 (s, 2H), 7.06 (d, J = 7.4 Hz, 2H), 6.87 (d, J = 7.4 Hz, 2H), 5.87 (bt, 2H), 3.71 (t, J = 6.3 Hz, 2H),
2.78 (p, J = 6.3 Hz, 2H), 2.13 (s, 6H), 2.12 (s, 6H). 11B NMR (96 MHz, DMSO-d6): (ppm) 5.9. 13C NMR
δ
(100 MHz, DMSO-d6):
δ (ppm) 133.4, 133.1, 131.9, 129.2, 127.8, 62.30, 41.3, 19.7, 19.2. Melting point:
209.2 ◦C. LC-MS (ESI): C18H24BNO 562.75 [2M+H]+.
P8, 2-((bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine (1379680-33-7)
1.24 g of 2-((bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine were obtained following the general
1
procedure using 2.34 g of 4-terbutyl-bromobenzene as a white solid (85% yield). H NMR (300 MHz,
DMSO-d6):
δ
(ppm) 7.32 (d, J = 8.2 Hz, 4H), 7.15 (d, J = 8.2 Hz, 4H), 5.98 (bt, 2H), 3.75 (t, J = 6.25 Hz,
(ppm) 6.1 (s). 13C NMR
(ppm) 146.7, 131.2, 123.3, 62.4, 41.4, 33.9, 31.4. Melting point: 255.5 ◦C. LC-MS
2H), 2.80 (p, J = 6.25 Hz, 2H), 1.23 (s, 18H). 11B NMR (96 MHz, DMSO-d6):
δ
(100 MHz, DMSO-d6):
δ
(ESI): C22H32BNO 674.90 [2M+H]+.
P9, 2-(bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine
1.48 g of 2-(bis(4-(tert-butyl)phenyl)boryl)oxy)ethanamine were obtained following the general
1
procedure using 2.34 g of 4-n-butyl-bromobenzene as a white solid. (70% yield). H NMR (400 MHz,
DMSO-d6):
δ(ppm) 7.29 (d, J = 7.7 Hz, 4H), 6.95 (d, J = 7.7 Hz, 4H), 5.96 (bt, 2H), 3.75 (t, J = 6.4 Hz,
2H), 2.88–2.77 (p, J = 6.4 Hz, 2H), 2.58–2.42 (m, 4H), 1.51 (m, 4H), 1.30 (m, 4H), 0.89 (t, J = 7.4 Hz,
6H). 11B NMR (128 MHz, DMSO-d6):
δ δ (ppm) 148.6,
(ppm) 5.8 (s). 13C NMR (100 MHz, DMSO-d6):
138.4, 131.5, 126.6, 62.4, 41.3, 34.8, 33.5, 21.8, 13.8. Melting point: 99.0 ◦C. LC-MS (ESI): C22H32BNO
674.85 [2M+H]+.
P10, 2-((bis(3,5-dimethylphenyl)boryl)oxy)ethanamine
1.02 g of 2-((bis(3,5-dimethylphenyl)boryl)oxy)ethanamine were obtained following the general
1
procedure using 2.04 g of 4-bromo-m-xylene as a white solid (85% yield). H NMR (300 MHz, DMSO-d6):
δ
(ppm) 6.97 (s, 4H), 6.65 (s, 2H), 5.92 (bt, 2H), 3.70 (t, J = 6.25 Hz, 2H), 2.78 (p, J = 6.25 Hz, 2H),
2.18 (s, 12H). δ δ (ppm)
11B NMR (96 MHz, DMSO-d6): (ppm) 5.6 (s). 13C NMR (100 MHz, DMSO-d6):
134.6, 129.4, 126.3, 62.3, 41.3, 21.3. Melting point: 294.5 ◦C. LC-MS (ESI): C18H24BNO 562.75 [2M+H]+.
P11, 2-(dibiphenyl-4-ylboryloxy)ethanamine (102032-41-7)
1.55 g of 2-(dibiphenyl-4-ylboryloxy)ethanamine were obtained following the general procedure
using 2.56 g of 4-bromobiphenyl as a white solid (79 % yield). 1H NMR (300 MHz, DMSO-d6):
δ (ppm) 7.60 (d, J = 7.3 Hz, 4H), 7.55 (d, J = 8.0 Hz, 4H), 7.46 (d, J = 7.3 Hz, 4H), 7.41 (d, J = 8.0 Hz,
2H), 7.30 (t, J = 7.3 Hz, 2H), 6.19 (bt, 2H), 3.83 (t, J = 6.4 Hz, 2H), 2.95–2.83 (p, J = 6.4 Hz, 2H). 11B NMR
(96 MHz, DMSO-d6):
δ δ (ppm) 151.0, 141.3, 137.0,
(ppm) 7.8 (s). 13C NMR (100 MHz, DMSO-d6):

Int. J. Mol. Sci. 2020, 21, 9777
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132.2, 128.8, 126.7, 126.4, 125.1, 62.6, 41.5. Melting point: 232.0 ◦C. LC-MS (ESI): C26H24BNO 754.80
for [2M+H]+.
P12, 2-(dibenzo[b]thiophen-5-ylboryloxy)ethanamine
1.34 g of 2-(dibenzo[b]thiophen-5-ylboryloxy)ethanamine were obtained following the general
procedure using 2.34 g of 5-bromobenzo[b]thiophene as a white solid (68 % yield). 1H NMR (300
MHz, DMSO-d6):
δ (ppm) 7.92 (s, 2H), 7.75 (d, J = 8.1 Hz, 2H), 7.55 (d, J = 5.4 Hz, 2H), 7.47 (d, J = 8.1
Hz, 2H), 7.33 (d, J = 5.4 Hz, 2H), 6.21 (bt, 2H), 3.85 (t, J = 6.4 Hz, 2H), 2.98–2.85 (p, J = 6.4 Hz, 2H).
11B NMR (96 MHz, DMSO-d6):
δ δ (ppm) 147.3, 138.9,
(ppm) 6.7 (s) 13C NMR (100 MHz, DMSO-d6):
136.4, 128.8, 126.4, 125.2, 124.0, 120.5, 62.6, 41.5. Melting point: 228.0 ◦C. LC-MS (ESI): C18H16BNOS2
674.55 [2M+H]+.
P13, 2-(bis(4-chlorophenyl)boryl)oxy)ethanamine (61733-90-2)
1.132g of 2-(bis(4-chlorophenyl)boryl)oxy)ethanamine were obtained as a white solid starting
1
from 575 mg of diisopropylamine borane complex following the general procedure (80%). H NMR
(300 MHz, DMSO-d6):
δ (ppm) 7.38–7.35 (dd, J = 9Hz, 4H), 7.19–7.16 (t, J = 9Hz, 4H), 6.18 (brs, 2H),
3.77–3.73 (t, J = 6Hz, 2H), 2.87–2.79 (p, J = 6Hz, 2H). 11B NMR (100 MHz, DMSO-d6):
13C NMR (75 MHz, DMSO-d6): δ (ppm) 133.8, 130.5, 129.1, 127.0, 62.9, 41.8.
P14, 2-(bis(4-fluorophenyl)boryl)oxy)ethanamine (176913-70-5)
δ (ppm) 4.0.
871 mg of 2-(bis(4-fluorophenyl)boryl)oxy)ethanamine were obtained as a pale yellow solid
starting from 575 mg of diisopropylamine borane complex following the general procedure (80%).
1H NMR (300 MHz, DMSO-d6):
δ (ppm) 7.40-7.36 (t, J = 6Hz, 4H), 6.98–6.92 t, J = 9Hz, 4H), 6.11 (brs,
2H), 3.77 (t, J = 6Hz, 2H), 2.84 (p, J = 6 Hz, 2H). 11B NMR (100 MHz, CDCl3):
(75 MHz, CDCl3): δ (ppm) 163.0, 159.8, 133.6, 113.5, 62.9, 41.8.
δ
(ppm) 3.9. 13C NMR
P16, 2-(((4-methoxyphenyl)(p-tolyl)boryl)oxy)ethanamine (7294-53-3)
To a suspension of magnesium (18.7 mmol, 455 mg) in a solution of diisopropylaminoborane
(7.5 mmol, 1.2 mL) in anhydrous THF (30 mL), at◦ room temperature, was added 855 mg of
4-bromotoluene. The mixture was then heated at 70 C under stirring during 20 h. After cooling
at room temperature, all the volatiles were removed under vacuum and to the resulting oil were
added anhydrous THF (40 mL) and 1.40 g of 4-bromoanisole (7.5 mmol) and the mixture was heated
at 70 ◦C during 20 h. After cooling at 78 ◦C, the reaction was quenched with a 3 M aqueous HCl
−
solution (25 mL) and was stirred for 45 min at room temperature. To this solution was added water
(20 mL), diethyl ether (20 mL), and the organic phase was separated. The aqueous phase was then
extracted with DCM (6
×
30 mL) and the combined organic phases were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The residue was dissolved in Et2O (25 mL) and
1.2 eq of ethanolamine was added. Depending on substituents, precipitation occurs spontaneously or
requires addition of pentane. Crystals were filtered, washed with pentane, and dried under vacuum to
1
yield 2-(((4-methoxyphenyl)(p-tolyl)boryl)oxy)ethanamine as a white solid in 90% yield). H NMR
(300 MHz, DMSO-d6):
δ (ppm) 7.26 (m, 4H), 6.94 (d, J = 7.5 Hz, 2H), 6.71 (d, J = 8.4, 1.8 Hz, 2H),
5.89 (bt, 2H), 3.74 (t, J = 6.1 Hz, 2H), 3.67 (s, 3H), 2.80 (p, J = 6.1 Hz, 2H), 2.21 (s, 3H). 11B NMR (96 MHz,
DMSO-d6):
δ δ (ppm) 157.3, 133.3, 132.6, 131.6, 127.3,
(ppm) 5.19 (s). 13C NMR (100 MHz, DMSO-d6):
112.3, 62.4, 54.6, 41.3, 20.9. Melting point: 154.3◦C LC-MS (ESI): C16H20BNO2 538.70 [2M+H]+: 539.32
4.9. Statistical Analysis
Given values are representative of at least three independent experiments and are given as mean
± SEM. When used, a t-test < 0.05 is considered as significant.
5. Conclusions
This study confirms that from the non-specific SOCE inhibitor, 2-APB, it is possible to create
new analogues with more efficient properties. Thus, we designed, synthetized, and characterized
16 new molecules. Among them, the molecule we called P11 unveils interesting properties: Full SOCE
inhibition for concentration less than 1 µM with an inhibition constant between 30 and 75 nM according

Int. J. Mol. Sci. 2020, 21, 9777
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to the cell lines, absence of effect on the two other main targets of 2-APB, SERCA and IP3R, absence of
cytotoxicity on non-activated cells, but the ability to induce the caspase-3 activity and the apoptosis
of PHA-activated cells. Thus, P11 is able to highjack the Ca2+ signaling of the T Jurkat cells from
proliferation to death.
6. Patents
The 2-APB analogues are patented: FR1907184.
Author Contributions: Formal analysis, C.L.G., T.L., J.B.P., M.P. and O.D.; writing—review and editing, C.L.G. and
O.D. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors want to thank Ch. Chomienne and A. Trautmann for their helping talks.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
2-APB
2-aminoethyl diphenylborinate
Cytosolic Ca2+ concentration
di-aminodo Phenyl Indol
Enzyme Linked Immunosorbent Assay
Endoplasmic Reticulum
Interleukin-2
Inositol 1,4,5-triphosphate
IP3 receptor
Inhibition constant
[Ca2+
DAPI
]
cyt
ELISA
ER
IL-2
IP3
IP3R
Ki
SOCC
SOCE
TG
Store-Operated Calcium Channel
Store-Operated Calcium Entry
Thapsigargin
TUNEL
SOCC
Terminal Uridine Nick-End Labeling
Store-Operated Calcium Channel
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