Store-operated CRAC channel inhibitors: opportunities and challenges

Abstract

Aberrant Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity has been implicated in a number of human disorders, including immunodeficiency, autoimmunity, occlusive vascular diseases and cancer, thus placing CRAC channels among the important targets for the treatment of these disorders. We briefly summarize herein the molecular basis and activation mechanism of CRAC channel and focus on discussing several pharmacological inhibitors of CRAC channels with respect to their biological activity, mechanisms of action and selectivity over other types of Ca²⁺ channel in different types of cells.

As a universal and remarkably versatile second messenger, cytoplasmic Ca²⁺ is important in mediating fundamental biological processes including gene expression, cell proliferation, differentiation and apoptosis [1,2]. There are two major sources contributing to the increase of cytoplasmic Ca²⁺ concentration, in other words, the release of stored Ca²⁺ within the endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) and the influx of extracellular Ca²⁺ across the plasma membrane. Store-operated Ca²⁺ entry (SOCE) is a unique mechanism to generate cytoplasmic Ca²⁺ signals that combines these two processes, which is triggered by depletion of intracellular Ca²⁺ stores (mainly ER), and subsequently followed by Ca²⁺ influx across the plasma membrane by the opening of Ca²⁺ channels [3–5].

The prototypical store-operated Ca²⁺ channel is the Ca²⁺ release-activated Ca²⁺ (CRAC) channel [6–9], which is widely distributed and involved in the regulation of a myriad of cellular activities in different cell types, including various subsets of T cells [10], B cells [11], mast cells [12], endothelial cells [13], platelets [14], vascular smooth muscle cells [15] and skeletal muscle cells [16]. It has an extremely low conductance (in the range of fS compared with pS of most Ca²⁺ channels) but is a highly Ca²⁺-selective channel (P_Ca/P_Na: >1000) that opens in response to Ca²⁺ depletion in intracellular Ca²⁺ stores [17]. The opening of CRAC channel leads to the activation of diverse downstream signaling pathways that regulate cytokine production, gene expression, cell growth, proliferation, differentiation and even cell death.

In recent years, aberrant CRAC channel activity has been noted in several human diseases, including severe combined immunodeficiency (SCID) disorders [18], allergy [19], thrombosis [20], acute pancreatitis [21], inflammatory bowel disease [22] and cancer [23–25], which leads to an increasing interest in developing small molecule compounds that suppress aberrant CRAC channel function.

Molecular basis & activation of CRAC channels

CRAC channel is composed of two key components, STIM (STIM1 and STIM2) [26,27] and ORAI (ORAI1, ORAI2 and ORAI3) [18,28–29], with the combination of STIM1/ORAI1 prevails in most cells and thus best characterized.

STIM1, originally identified as a tumor suppressor protein [30], is one of the two elementary components of SOCE, and functions as an ER Ca²⁺ sensor by detecting the fluctuation of Ca²⁺ concentration in the ER stores [26,27]. STIM1 is a single-pass transmembrane protein located in the ER membrane. The amino-terminal portion of STIM1 is located within the ER lumen [17], composed of an ER retention sequence, a canonical Ca²⁺-binding EF-hand domain, a ‘hidden’ EF-hand domain [31] that does not bind Ca²⁺ and a sterile α-motif (SAM) domain that mediates STIM1 dimerization/oligomerization [32]. The cytosolic domain of STIM1 includes three putative coiled-coiled domains (CC1-3), the CRAC activation domain (SOAR/CAD) that is essential for the gating of ORAI1 [33,34], a serine/proline-rich domain, a TxIP motif associated with microtubule plus-end-tracking protein EB1, and a polybasic C-tail that facilitates efficient targeting of STIM1 toward the plasma membrane through physical association with phosphoinositides embedded in the inner leaflet of the plasma membrane [7,35]. As a homolog of STIM1, STIM2 has also been found to act as ER Ca²⁺ sensor but has a lower affinity to luminal Ca²⁺ and also associates with ORAI proteins to regulate Ca²⁺ influx [36]. However, its physiological functions are less well understood [37].

ORAI1 protein has been recognized as the ion pore-forming subunit of CRAC channels [7,29]. ORAI2 and ORAI3, human homologs of ORAI1, also form Ca²⁺-selective store-operated channels when co-expressed with STIM1 [38]. All three ORAI proteins have four putative transmembrane-spanning domains (TM1–4), one intracellular and two extracellular loop regions, and they are localized to the plasma membrane with their N- and C-termini facing the cytoplasm (Figure 1) [39].

Figure 1. . Domain architecture of STIM1 and ORAI1.

(A) STIM1 is a single-pass transmembrane protein located in the ER membrane. The N terminus is located within the ER lumen and contains an ER-retention sequence, a canonical Ca²⁺-binding EF-hand domain, a ‘hidden’ EF-hand domain and a sterile α-motif domain. The C terminus contains three putative coiled-coiled domains (CC1–3), a serine/proline-rich domain, and a polybasic C-tail. (B) ORAI1 bears four putative transmembrane-spanning domains (TM1-4), one intracellular and two extracellular loop regions. Residues at position E106 and E190 determine the channel selectivity and the dominant-negative mutant R91W has been found to be related to severe combined immunodeficiency.

ER: Endoplasmic reticulum; SAM: Sterile α-motif.

Upon binding of their cognate ligands or antigens, the cell-surface receptors such as receptor tyrosine kinases (RTKs) or G-protein-coupled receptors (GPCRs) activate phospholipase C (PLC) to hydrolyze the membrane phospholipid phosphatidyl-4,5-bisphosphate to generate inositol-1,4,5-trisphosphate (IP₃) [40], followed by release of Ca²⁺ from the Ca²⁺ stores. So far, the IP₃-sensitive ER is the major store that is coupled to CRAC channel activation. The loss of Ca²⁺ from the ER results in Ca²⁺ dissociation from the luminal Ca²⁺-binding EF hand of STIM1 [26,27], leading to the unfolding of EF-SAM domain, followed by STIM1 oligomerization through a mechanism that involves both the luminal and cytosolic domains [32,41–42], which is the essential step responsible for STIM1 conformational switch [43–45], subsequent accumulation at endoplasmic reticulum–plasma membrane (ER–PM) junctions and ultimate activation of ORAI channels [46]. The STIM1 oligomers then migrate from the bulk ER to specialized ER–PM junctions [47,48], during which STIM1 captures diffusing ORAI1 channels, and interaction between the amino and carboxyl termini of ORAI1 with SOAR/CAD on STIM1 leads to the opening of CRAC channel (Figure 2) [49–54].

Figure 2. . Activation of release-activated Ca²⁺ channel.

The increase of IP₃ concentration induced by activation of PLC activates endoplasmic reticulum (ER)-resident IP₃ receptors (IP₃R) and causes the release of Ca²⁺ from ER, which leads to oligomerization and conformational switch of STIM1. The activated STIM1 oligomers then move toward the ER–plasma membrane junctions and trigger Ca²⁺ influx through direct interaction with an opening of ORAI1 Ca²⁺ channels in the plasma membrane.

CRAC: Release-activated Ca²⁺; PLC: Phospholipase C.

Pharmacological inhibitors of CRAC channels

The identification of the molecular identities of CRAC channel kindled an intense interest in the search of small molecule modulators of CRAC channel. CRAC channel modulators may work by targeting either ORAI or STIM to regulate the overall level of CRAC channel activity. The compounds may either modulate channel activity by targeting STIM1 or acting directly at the pore of the ORAI channel by blocking the pore or interfering with the STIM–ORAI interaction.

Although both STIM1 and ORAI1 are widely expressed in a variety of tissues, the major clinical manifestations of patient with CRAC channelopathies are surprisingly limited to the immune system, skeletal muscle and ectodermally derived tissues [7], which agrees well with phenotypes observed in mice with targeted disruptions of the murine Orai1, Stim1 and/or Stim2 [55,56]. These findings indicated that therapies specifically targeting CRAC channels may serve as improved immunomodulators with high selectivity and low toxicity compared with currently US FDA approved immunosuppressive agents, such as cyclosporin A and FK506 that often cause undesired off-target toxicity in patients [57].

Although a number of agents that inhibit CRAC channels have been developed [58–61], most of them by far have not reached clinical trials, primarily owing to their poor selectivity and high toxicity. Nonetheless, a member of the CalciMedica series has reached Phase I clinical trials and it is highly anticipated to reach the milestone of FDA approval in drug development [62]. Apart from this, some CRAC modulators may provide promising lead structures for developing CRAC channel inhibitors with improved specificity and higher potency in the near future. Here we discuss a number of pharmacological agents that are most commonly used to inhibit CRAC channel activity, which are also helpful for understanding the physiological roles and dissecting the structure–function relation of the CRAC channel.

Lanthanides

Similar to other Ca²⁺ entry pathways, store-operated Ca²⁺ channels could also be inhibited by divalent and trivalent cations. Particularly, CRAC channels show high sensitivity to complete blockade by the trivalent ion La³⁺ (lanthanum) and Gd³⁺ (gadolinium) at submicromolar concentration range [63]. This unique feature has been often used to distinguish CRAC channels from other types of less Ca²⁺ selective channels (e.g., TRP channels) [64–66]. The concentrations of Gd³⁺ used to effectively block the endogenous CRAC channel exert no significant inhibitory effect on TRP channels.

Mutation of several key acidic residues in the TM1–TM2 loop of ORAI1 (D110, D112 and D114) reduced the CRAC channel's selectivity for Ca²⁺ and decreased the inhibitory potency of the lanthanides, implying that the binding site of the trivalent ion La³⁺ and Gd³⁺ is located at or nearby that region of ORAI1 [67,68]. However, in the recent determined x-ray crystal structure of Drosophila Orai, Gd³⁺ situates at the same site (E106 in human ORAI1), rather than the acidic region in the first extracellular loop that is proposed to coordinate Ca²⁺ [69].

Lanthanides also showed inhibitory activity against other cationic ion channels, for example, voltage-gated calcium channels and TRP channels [70,71], which limited their potential use in developing CRAC channel inhibitors. Moreover, because the lanthanide salts of other multivalent anions and proteins are insoluble, their utility is also limited in many other applications.

Imidazole compounds

Imidazole antimycotic SKF-96365 (1) was one of the first identified CRAC channel inhibitors for experimental use [58,72], and the structurally related imidazole compounds econazole (2) and miconazole (3), which are primarily used as antimycotics [58], also suppress CRAC channel activity (Figure 3).

Figure 3. . Chemical structures of typical imidazole release-activated Ca²⁺ channel inhibitors.

SKF-96365 (1); econazole (2); miconazole (3).

SKF-96365 inhibited thapsigargin-induced SOCE in Jurkat T cells with an IC₅₀ value (measured by I _CRAC, the current generated by the opening of CRAC channels) of 12 μM in a dose-dependent manner (different groups reported varying IC₅₀ values, which might be mainly due to the methods used to measure CRAC channel activity and the potencies are also cell-type dependent) [72]. Although this compound inhibits agonist-mediated Ca²⁺ influx in many cell types, it also suppresses the activity of other ion channels, such as voltage-gated calcium channels, nonselective cation channels and cyclic AMP-gated Cl⁻ channels with comparable potencies [72,73]. Econazole and miconazole also exhibit a lack of specificity to CRAC channel, thereby limiting its further clinical use as specific CRAC channel modulators.

Diphenylboronate compounds

2-Aminoethyldiphenyl borate (2-APB, 4) has been widely used to characterize the activity of CRAC channel [74]. Its pharmacology is complex with an intriguing biphasic effect on CRAC channel activation. At low concentrations (1–10 μM), 2-APB potentiates CRAC channel activity; while at higher concentrations (20–100 μM), it often causes a transient activation of CRAC channel followed by strong inhibition [75,76]. In recent years, two 2-APB analogs DPB162-AE (5) and DPB163-AE (6), which were identified by Mikoshiba's group, have drawn attention for their higher potencies and greater specificity than 2-APB in terms of suppressing SOCE (Figure 4) [77].

Figure 4. . Chemical structures of 2-APB and its analogs.

2-APB (4); DPB162-AE (5); DPB163-AE (6).

2-APB was initially speculated to inhibit SOCE through its inhibitory effect for IP₃ receptors [78–81]. However, it has been clarified that the inhibition of SOCs has no direct relation with IP₃ [75,82–83].

In recent years, it has been found that formation of STIM1 puncta (clusters of STIM1 at ER–PM regions just below the PM) could be prevented by high concentrations (50 μM) of 2-APB, suggesting that 2-APB could inhibit SOCE through affecting movement of STIM1 [84,85]. However, the inhibition of STIM1-puncta formation by 2-APB could be overcome by coexpression with ORAI1, even though the inhibition of SOCE is still effective [85,86]. These findings imply that the inhibitory effect of 2-APB on SOCE might be due to its effects on one or more steps in the following molecular events: STIM1 multimerization, STIM1 conformational switch, STIM1–ORAI1 interaction or the ORAI channel itself.

Interestingly, the effects of 2-APB on ORAI vary between different ORAI isoforms [85,87]. For example, at high concentrations (50 μM) of 2-APB, ORAI1-mediated Ca²⁺ entry is initially activated, which is quickly followed by a complete inhibition. 2-APB only partially inhibits ORAI2-mediated SOCE. Surprisingly, 2-APB at high concentrations activate, rather than inhibit, ORAI3 channel [88,89]. The different properties of these three ORAI proteins give hope to develop small molecules which could selectively target one specific ORAI protein.

Although 2-APB is widely used to modulate CRAC channel activity, it could also affect the activities of potassium channels [90], SERCA pumps [91] and mitochondrial Ca²⁺ efflux [75]. Notably, 2-APB has been reported to activate the heat-gated recombinant TRPV1, TRPV2 and TRPV3 channels [92].

2-APB derivatives DPB162-AE and DPB163-AE are isomers in chemical structure, which only differ in the linker chain between their two diphenyl groups. In STIM1–ORAI1 overexpressing cells, DPB163-AE had a biphasic effect on SOCE, which is similar to 2-APB but exhibits a higher potency with an IC₅₀ of about 600 nM. DPB162-AE, on the other hand, solely inhibited SCOE with an IC₅₀ of approximately 200 nM, which is two orders of magnitude more potent than 2-APB [76,93]. Similar to 2-APB, both DPB162-AE and DPB163-AE suppressed ORAI1 currents and partially inhibited ORAI2 currents. However, unlike 2-APB, they failed to activate ORAI3 channels at higher concentrations in the absence of STIM1, an observation that could be attributed to their larger size, relative to 2-APB, that likely prohibits access to the ORAI3 pore [77].

The interaction between the STIM1–ORAI1 activating region (SOAR) of STIM1 and a combination of the C- and N-termini of ORAI1 leads to the coupling of STIM1 and ORAI1. The single binding pocket formed by C- and N-termini of ORAI1 plays an important role in both SOAR-binding and gating of the channel [94]. Recent researches have shown that DPB162-AE, not acting as an ORAI1 channel pore blocker, does not prevent the STIM1–ORAI1 interaction but potently inhibits the activation of STIM1-mediated ORAI1 channel [93,94]. Using a unique point mutation in the SOAR of STIM1 (F394H), which prevents both physical binding between SOAR and ORAI1 as well as functional coupling to activate the ORAI1 channel [94], DPB162-AE was found to restore SOAR–ORAI1 binding rapidly but restore ORAI1-mediated Ca²⁺ entry slowly. These findings reveal that DPB162-AE seems to be a potent and relatively specific STIM1–ORAI1 functional uncoupler, and probably acts directly on the coupling interface between SOAR and ORAI1.

In contrast to 2-APB, DPB162-AE has little effect on TRPC channels, L-type Ca²⁺ channels or Ca²⁺ pumps at 2 μM, the maximal CRAC channel-mediated SOCE inhibitory level of DPB162-AE [93,95–97].

Pyrazole compounds: the BTPs

A series of bis(trifluoromethyl)pyrazoles compounds, known as BTP1 (7), BTP2 (8) and BTP3 (9), were initially identified as inhibitors of NFAT activation and T-cell cytokine production by Abbott Laboratories [98–100]. Interestingly, unlike other well-known NFAT inhibitors such as FK506 and cyclosporin A, the BTPs inhibited NFAT nuclear translocation without direct effect on the phosphatase activity of calcineurin, implying a probable effect on the upstream Ca²⁺ signal [98,99]. Indeed, the BTPs were later found to be capable of inhibiting SOCE in many cells at low micromolar to nanomolar concentrations with considerable selectivity over voltage-gated Ca²⁺ entry [61]. In particular, BTP2 (also known as YM-58483) inhibited thapsigargin-induced Ca²⁺ influx in Jurkat T cells with an IC₅₀ value of 100 nM and did not inhibit phosphorylation of PLCγ1 and TCR-mediated Ca²⁺ release from the stores (Figure 5) [101].

Figure 5. . Chemical structures of the BTPs.

BTP1 (7); BTP2 (8); BTP3 (9).

BTP2 has been reported to inhibit CRAC channels in human T cells with an IC₅₀ of about 10 nM [102], which is one order of magnitude higher than the IC₅₀ value described above. The most probable reason for the discrepancy in IC₅₀ values is that the former experiment was performed via preincubation of cells with BTP2 for 18–24 h to reach full inhibition [102], while cytoplasmic Ca²⁺ concentrations in the later study were measured shortly (only a few minutes) after BTP2 treatment [101]. Moreover, it was also found that CRAC channel inhibition mediated by BTP2 was affected by the external Ca²⁺ concentration: higher external Ca²⁺ concentrations are correlated with reduced inhibitory effect on the CRAC channel [102].

A recent study has shown that drebrin, an actin reorganizing protein, is identified as a potential binding site for BTP2 [103]. Knockdown of drebrin by siRNA inhibited SOCE to the same extent as inhibition by BTP2. Thus, the authors of the report concluded that drebrin may play a role in regulating SOCs by affecting the actin cytoskeleton, and that BTP2 may act by inhibiting drebrin. However, previous studies have shown that the actin cytoskeleton is not considered to play a major role in SOCE [104].

Although BTP2 does not inhibit voltage-gated Ca²⁺ channels or K⁺ channels [101,102], it activates TRPM4 channels and inhibits the activities of TRPC3 and TRPC5 channels [105,106].

Pyrazole compounds: the Pyrs

As discussed above, BTP2 (also known as Pyr2, 8) could also inhibit TRPC3 channel activity while it acts on CRAC channels. Recently, the ability of three pyrazole derivative compounds Pyr3 (10), Pyr6 (11) and Pyr10 (12) in inhibiting Ca²⁺ entry have been examined in terms of TRPC/CRAC selectivity in HEK293 cells overexpressing TRPC3 channels and RBL-2H3 cells expressing CRAC channels (Figure 6) [107].

Figure 6. . Chemical structures of the Pyrs.

Pyr2 (8); Pyr3 (10); Pyr6 (11); Pyr10 (12).

Structurally, Pyr6 and Pyr10 have similar structures to the BTPs bearing two trifluoromethyl groups in the C3 and C5 position of the pyrazole ring, which are important in CRAC channel inhibitory activity of BTP2 [108]. Interestingly, Pyr3 shares a carboxylate group in the C4 position of the pyrazole ring instead of the trifluoromethyl group in the C3 position, which appears to contribute to maintaining its inhibitory activity, and the trichloroacryl group of the side chain also seems to be necessary for its inhibitory activity toward both CRAC and TRPC3 channel [107].

Interestingly, Pyr6 displays higher potency to inhibit Ca²⁺ entry mediated by CRAC channel than TRPC3, while Pyr10 exhibits significant selectivity for TRPC3-mediated Ca²⁺ entry. By comparison, Pyr2 and Pyr3 do not show any appreciable selectivity for them [107].

Pyrazole compounds: the GSKs

Recently, several novel pyrazole compounds, GSK-5498A (13), GSK-5503A (14) and GSK-7975A (15), which were developed by GlaxoSmithKline, have been identified as selective CRAC channel inhibitors (Figure 7) [109–111].

Figure 7. . Chemical structures of the GSKs.

GSK-5498A (13); GSK-5503A (14); GSK-7975A (15).

Electrophysiological experiment showed that GSK-5498A inhibits I _CRAC with an IC₅₀ value of about 1 μM in human embryonic kidney cells stably expressing STIM1 and ORAI1 [110]. GSK-5503A and GSK-7975A inhibited STIM1 mediated ORAI1 and ORAI3 currents with an IC₅₀ value of about 4 μM in HEK293 cells [111].

FRET experiments implied that the GSK compounds did not affect STIM1 oligomerization or STIM1–ORAI1 interaction. Compared with wild-type ORAI1, the less Ca²⁺-selective mutant E106D ORAI1 pore requires at least tenfold higher concentrations of GSKs for inhibition, thus pointing to the possibility that these compounds may act by altering the ORAI pore geometry [111].

A recent study has found that blockade of CRAC channels by GSK-7975A effectively precludes palmitoleic acid ethyl ester (POAEE), an important mediator of alcohol-related pancreatitis, from evoking sustained elevation of the Ca²⁺ concentration in the pancreatic acinar cells, activation of protease and necrosis of pancreatic acinar cell [21]. Given these findings, the authors indicated that pharmacological CRAC channel blockade could be applied as a potentially rational therapy against severe acute pancreatitis, which is life-threatening but lacks effective treatment thus far [21,112].

Interestingly, although these GSK compounds were found to have little or no effect on many other ion channels, they potently block TRPV6 channels [111,113].

Synta 66

Synta 66 (16), a selective CRAC channel inhibitor developed by Synta pharmaceuticals, has drawn extensive attentions in recent years [114]. The structure of Synta 66 is similar to Pyr6 (11), whose 3,5-bistrifluoromethyl pyrazole ring is replaced with 2,5-dimethoxy benzene ring (Figure 8).

Figure 8. . Comparison of the chemical structures of Synta 66 and Pyr6.

Pyr6 (11); Synta 66 (16).

This compound inhibits I _CRAC with an IC₅₀ value of 1.4 μM in RBL cells and has no effect on plasma membrane Ca²⁺ ATPase pump and inwardly rectifying K⁺ channels [114,115]. It has also been found that the compound inhibits expression of T-bet and production of IL-2, IL-17 and IFN-γ in biopsy specimens isolated from inflamed areas of IBD patients [115]. The results suggested that Synta 66 could be applied to further investigation of the CRAC channels functions in T-cell signaling and IBD.

A scrutiny of the selectivity of Synta 66 assessed by a panel of 50 specific radioligand-binding assays suggests that, at a concentration of 10 μM, it exerts no significant effect on a series of receptors, enzymes and ion channel targets [115]. Although there are more and more studies employing Synta 66 for probing the physiological role of CRAC channels, the mechanism of action for this compound has not yet been fully clarified.

ML-9

ML-9 (17), an inhibitor of myosin light chain kinase (MLCK), has been found to reversibly inhibit SOCE with an IC₅₀ of approximately 10 μM [116,117]. In HEK293 cells, ML-9 was found to similarly inhibit SOCE and I _CRAC [117].

ML-9 inhibits SOCE at least partially by reversing the formation of STIM1 puncta and blocking its movement to ER–PM junctions. Interestingly, the inhibitory effect of ML-9 does not seem to be related to its well-known inhibition of MLCK [117]. Although STIM1 appears to be the molecular target of ML-9-mediated inhibition on SOCE, it is still unclear on the specific site in STIM1 that ML-9 may act on.

Diethylstilbestrol

Diethylstilbestrol (DES; 18), a synthetic estrogen agonist, inhibits SOCE in a range of cell types including mast cells, vascular smooth muscle cells and rat microglia [118,119]. DES inhibits I _CRAC in RBL cells with an IC₅₀ of approximately 0.6 μM and does not affect TRPM7 channels at a similar concentration range. Interestingly, if it is applied intracellularly, its inhibitory effect on I _CRAC disappears, thus raising the speculation that it might act on the extracellular regions on CRAC channel. Although it might be applied to investigate the function of CRAC channels in vitro, it could not be used in clinical setting due to its activation on estrogen receptors.

Carboxyamidotriazole

Carboxyamidotriazole (CAI; 19) is a potential anticancer drug which has been tested in Phase I and Phase II clinical trials for its activity of inhibiting angiogenesis, tumor growth, invasion and metastasis [120,121]. CAI was initially identified as an inhibitor of SOCE in nonexcitable cells [122]. CAI inhibits I _CRAC with an IC₅₀ value of approximately 0.5 μM in HEK293 cells [120,121]. Carboxyamidotriazole suppresses I _CRAC by reducing the production of IP₃ and depolarizing mitochondria, which induces Ca²⁺-dependent inactivation of the CRAC channels [123–125]. Therefore, although CAI does inhibit I _CRAC, it seems to act in an indirect manner.

RO2959

RO2959 (20), synthesized by Synta Pharmaceutical Corp., has been identified as a novel, potent and selective I _CRAC inhibitor by Roche [126]. RO2959 inhibits I _CRAC with an IC₅₀ value of about 400 nM in RBL-2H3 cells, which has been preincubated with RO2959 [126]. Moreover, due to its ability of inhibiting I _CRAC, RO2959 could potently inhibit human TCR-mediated SOCE, T-cell proliferation, cytokine production and gene expression [126]. In T-REx-CHO cells which could stably express STIM1/ORAI1, STIM1/ORAI2 or STIM1/ORAI3, RO2959 inhibits ORAI1 to a greater extent than ORAI2 and ORAI3 [126], which implies that the compound could be applied as a selective ORAI1 inhibitor. RO2959 had no significant inhibitory effect on a variety of cellular receptors, transporters and ion channels, such as GABA receptors, dopamine transporter, 5-HT transporter, K_v channels, Cl^- channels, TRPC1, TRPM2, TRPM4 and Cav1.2 channels, which showed the high action selectivity of the compound [126]. Notably, TRPC1 channel has some similarities with CRAC channel, a number of drug candidates developed as CRAC channel inhibitors also act on TRPC1 channel. Although TRPC1 can contribute to SOCE along with ORAI1 and STIM1, it also participates in other signaling events that are independent on store depletion. If the CRAC channel inhibitors could potently act on TRPC1, one would therefore expect undesired off-target side effects. Thus, for CRAC channel inhibitors, it is necessary to examine the effect on TRPC1 channels.

Although RO2959 has been shown to be a potent and selective CRAC channel inhibitor, the in vivo efficacy and the exact mechanism of action warrants further investigation.

Linoleic acid

More recently, linoleic acid (21), an 18-C polyunsaturated fatty acid (PUFA), has been reported to effectively inhibit antigen- or thapsigargin-mediated SOCE in mast cells by acute addition at micromolar concentrations [127]. Interestingly, stearic acid, the 18-C saturated fatty acid, does not inhibit SOCE. The authors found that linoleic acid inhibited SOCE by affecting STIM1 oligomerization and subsequent STIM1/ORAI1 coupling. The authors further argue that linoleic acid inhibited STIM1/ORAI1 coupling by disrupting potential electrostatic interactions between STIM1 and ORAI1 [127]. Further studies are needed to delineate its mechanism of action and examine its selectivity over other types of ion channels (Figure 9).

Figure 9. . Chemical structures of several pharmacological inhibitors of release-activated Ca²⁺ channels.

ML-9 (17); Diethylstilbestrol (18); Carboxyamidotriazole (19); RO2959 (20); linoleic acid (21).

1-Phenyl-3-(1-phenylethyl)urea derivatives

A series of 1-phenyl-3-(1-phenylethyl)urea derivatives has been recently identified as CRAC channel inhibitors. As the lead compound, compound 22 could inhibit Ca²⁺ influx with IC₅₀ of 3.25 ± 0.17 μM in HEK293 cells stably co-expressing ORAI1 and STIM1 [128]. The Ca²⁺ influx assay and electrophysiological experiments showed that compound 22 could partially inhibit Ca²⁺ entry in constitutively opened CRAC channels which were formed by ORAI1-SS (monomer ORAI1 covalently linked with two S336–485 domains) and completely inhibit the Ca²⁺ entry and the current mediated by the opened STIM1-free V102A channel (a mutant of ORAI1), which is a constitutively opened CRAC channel, even in the absence of STIM1. Furthermore, this compound could specifically reduce ORAI1/STIM1-mediated Ca²⁺ entry, while exhibited no inhibitory effect on other ORAI channels. These results indicated that compound 22 inhibits CRAC channel by specifically targeting ORAI1 [128].

A total of 40 derivatives have been synthesized, and their primary structure–activity relationships (SARs) study showed that the alkyl substituent on the α-position of the N-phenylethyl group is vital for their inhibition on Ca²⁺ influx [128]. Notably, among these derivatives, compound 23 exhibited low cytotoxicity and relatively improved inhibition of IL-2 production in the Jurkat cell line [128].

It is encouraging that compound 22 could inhibit CRAC channel by specifically targeting ORAI1, however, its mechanism of action and selectivity over other types of ion channels still warrants further studies (Figure 10).

Figure 10. . Chemical structures of two 1-phenyl-3-(1-phenylethyl)urea derivatives.

Compound 22 and compound 23.

The CalciMedica series

CalciMedica has been actively developing novel, potent and specific CRAC channel inhibitors in the past decade. It is worth noting that CM2489, developed by this biotech company, is the only CRAC channel inhibitor tested in human and has completed Phase I clinical trials for treating moderate-to-severe plaque psoriasis [129]. Although there is no chemical structure information disclosed for this compound, through the patent published by CalciMedica, we could find that most compounds bear phenyl or heterocyclic groups linked by carboxyamide, sulfoxamide or alkyl chain (take compound 24 for example) [62], which may help us to estimate the skeleton structure of CM2489 for designing novel CRAC channel inhibitors (Figure 11).

Figure 11. . An example of release-activated Ca²⁺ channel modulators developed by CalciMedica.

Compound 24.

Conclusion

CRAC channels, fundamental to human immune cell function, have shown the physiological importance in many cell types. With the discoveries of STIM1 and ORAI1 proteins, the molecular components of CRAC channel have been identified, which facilitates the functional studies of CRAC channels in a wide range of cellular systems. In the last three decades, several classes of CRAC channel inhibitors have been developed. Although most of them have not reached clinical trials due to their poor selectivity and high toxicity, there are some selective CRAC channel inhibitors that might hold promise for further drug development. Most notably, CM2489 [129], developed by CalciMedica, has reached clinical trials, which is the first CRAC channel inhibitor that has completed the Phase I clinical trials. Unfortunately, there is no sufficient public information available for us to have a thorough study of this compound.

In addition to CM2489, RO2959, recently developed by Synta, has also been shown to act as a selective CRAC channel inhibitor by targeting ORAI1. Thus, developing drugs that target particular component(s) of CRAC channel might be the most efficient and effective way to improve the selectivity and specificity.

Future perspective

Given that CRAC channels have emerged as an attractive target for developing new therapies for autoimmune disorders, allergy, thrombosis and cancer, more and more pharmaceutical companies including Hoffmann-La Roche and GSK are joining the efforts to develop CRAC channel inhibitors. As summarized by Pevarello and colleagues [62], the publication of related patents has been kept growing. Although no CRAC channel inhibitors have reached the milestone of FDA approval and clinical use, the increasing attention paid by pharmaceutical companies, together with our deeper understanding of the activation and regulatory mechanisms of CRAC channel and the advent of novel optogenetic tools to manipulate CRAC channel activity [130–132], would certainly expedite the quest for new drugs that specifically target CRAC channels to treat human disorders associated with dysregulated Ca²⁺ influx (Table 1).

Table 1. . The potencies of leads from each series and their proposed mechanism of action.

Species	CRAC channel inhibitory activity	Selectivity	Proposed mechanism of action
Lanthanides	Complete blockade at submicromolar concentration range [63]	Block other cationic ion channels, such as voltage-gated calcium channels and TRP channels [70,71]	Directly block ORAI1 [69]
SKF-96365	IC50: 12 μM (ICRAC) [72]	Suppresses voltage-gated calcium channels, nonselective cation channels and cyclic AMP-gated Cl− channels [72,73]	Has not yet been fully clarified
2-APB	Activates at low micromolar and inhibits at high micromolar [75,76]	Affect the activities of potassium channels, SERCA pumps, heat-gated recombinant TRPV1, TRPV2 and TRPV3 channels [90–92]	Might act on STIM1 multimerization, STIM1–ORAI1 interaction or the ORAI channel itself [85,86]
DPB162-AE	IC50: 200 nM [93]	Relatively selective [93,95–97]	Probably acts directly on the coupling interface between SOAR and ORAI1 [93,94]
BTP2	Inhibited thapsigargin-induced Ca2+ influx in Jurkat T cells with an IC50 of 100 nM [101]	Activates TRPM4 channels and inhibits the activities of TRPC3 and TRPC5 channels [105,106]	Has not yet been fully clarified
GSK-7975A	IC50: 4 μM (ICRAC in HEK293 cells) [111]	Potently blocks TRPV6 channels [111,113]	May act by altering the ORAI pore geometry [111]
Synta 66	IC50: 1.4 μM (ICRAC in RBL cells) [114,115]	Relatively selective [115]	Has not yet been fully clarified
ML-9	Reversibly inhibit SOCE with an IC50 of approximately 10 μM [116,117]	Inhibits MLCK [116]	Might target STIM1 [117]
DES	IC50: 0.6 μM (ICRAC in RBL cells) [118,119]	Activates estrogen receptors [118]	Might act on the extracellular regions on CRAC channel [118,119]
CAI	IC50: ˜0.5 μM (ICRAC in HEK293 cells) [120,121]	Not very selective	Reduces the production of IP3 and depolarizes mitochondria [123–125]
RO2959	IC50: 400 nM (ICRAC in RBL-2H3 cells) [126]	Relatively selective [126]	Has not yet been fully clarified
Linoleic acid	Inhibit antigen- or thapsigargin-mediated SOCE in mast cells by acute addition at micromolar concentrations [127]	Has not yet been examined	Inhibits SOCE by affecting STIM1 oligomerization and subsequent STIM1/ORAI1 coupling [127]
1-phenyl-3-(1-phenylethyl)urea	Inhibits Ca2+ influx with IC50 of ˜3 μM in HEK293 cells [128]	Has not yet been examined	Targets ORAI1 [128]

Executive summary.

• Store-operated Ca²⁺ entry (SOCE) constitutes one of the major Ca²⁺ entry routes in nonexcitable cells and is implicated in a variety of fundamental biological processes.

• Ca²⁺ release-activated Ca²⁺ (CRAC) channel, which is widely distributed and involved in the regulation of many cellular functions in different cell types, is one of the most well-studied prototypical form of store-operated Ca²⁺ channels.

• Aberrant CRAC channel activity is associated with human disorders involving the immune system, as well as tumor growth and cancer metastasis.

• CRAC channel is composed of stromal interaction molecule (STIM) and ORAI, with the combination of STIM1/ORAI1 most well characterized.

• STIM1 functions as an ER Ca²⁺ sensor and ORAI1 is the ion pore-forming subunit of CRAC channel. The dynamic coupling between STIM1 and ORAI1 is the key step for the opening of CRAC channel.

• Major clinical manifestations of CRAC channelopathies in human are limited to the immune system, skeletal muscle and ectodermally derived tissues. CRAC channels can thus serve as an ideal target for developing novel immunomodulators with improved biosafety profiles.

• Although most agents developed as CRAC channel inhibitors have not reached clinical trials owing to their poor selectivity and high toxicity, the clarification of molecular basis of CRAC channel is anticipated to expedite the development of drug candidates that specially target STIM and/or ORAI. Such compounds might hold great promise to serve as selective CRAC channel inhibitors to treat human disorders arising from imbalanced Ca²⁺ homeostasis.