Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2

Zitt, Christof; Strauß, Bettina; Schwarz, Eva C.; Spaeth, Nicola; Rast, Georg; Hatzelmann, Armin; Hoth, Markus

doi:10.1074/jbc.m309297200

Cited by 266 publications

(272 citation statements)

References 31 publications

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“…With the typical Ca 2+ re-addition protocol (1 lM thapsigargin in Ca 2+ -free (0-Ca 2+ ) Ringer's solution followed by re-addition of 1 mM Ca 2+ Ringer's solution), we measured free Ca 2+ peaks in the range of 2 lM (data not shown). These values are comparable to data published by many different laboratories including our own work [12,24]. Measurements of the [Ca 2+ ] i are also influenced by dye sequestration of fura-2/AM in organelles.…”

Section: Single-cell Imagingsupporting

confidence: 90%

“…Proliferation rates were reduced by about 55% when adding the specific CRAC channel blocker 3,5-bistrifluoromethyl pyrazole BTP2 (Supporting Information Fig. 1), which has been shown to reduce either Ca 2+ influx through CRAC channels [24] and/or to decrease the driving force for Ca 2+ by activating TRPM4 channels [25]. The beads were very efficient in stimulating both PBL and highly purified CD4 + T cells, whereas the commonly used lectin PHA alone was not sufficient to induce proliferation and IL-2 secretion in pure CD4 + T cells, but could do so in PBL (Fig.…”

Section: Bead Stimulation Of Cd4 + T Cells Activates Almost All T Celmentioning

confidence: 99%

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Calcium dependence of T cell proliferation following focal stimulation

et al. 2007

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Clonal T cell expansion through proliferation is a central process of the adaptive immune response. Apoptosis of activated T cells is required to avoid chronic inflammation. T cell proliferation and apoptosis are often analyzed with stimuli that do not induce formation of a functional immunological synapse. Here we analyze the Ca 2+ dependence of proliferation and apoptosis in primary human CD4 + T cells following stimulation with anti-CD3/anti-CD28-coated beads, which induce a tight interaction similar to the immunological synapse. We found this focal stimulation to be much more efficient for stimulating IL-2 production and proliferation than non-focal TCR stimuli. Surprising little Ca 2+ entry through Ca 2+ channels was required for T cell proliferation. Transient free intracellular calcium concentration ([Ca 2+ ] i ) elevations of up to 220 nM from a baseline level of around 40 nM were sufficient for maximal proliferation in primary human CD4 + T cells. We also show that proliferation was very Ca 2+ sensitive in the range 90-120 nM, whereas apoptosis was basically constant for [Ca 2+ ] i levels of 90-120 nM. We conclude that very small changes in [Ca 2+ ] i can dramatically change the ratio between proliferation and apoptosis, thus keeping the balance between overshooting and inefficient immune responses.

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Section: Single-cell Imagingsupporting

confidence: 90%

Section: Bead Stimulation Of Cd4 + T Cells Activates Almost All T Celmentioning

confidence: 99%

Calcium dependence of T cell proliferation following focal stimulation

et al. 2007

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“…Although a majority of these compounds inhibit calcium mobilization dependent on store depletion similar to the recently reported compound 3,5-bistrifluoromethyl pyrazole, BTP2 (20), they are structurally different from the BTP compounds and hence are likely to act by different mechanisms.…”

Section: Discussionmentioning

confidence: 99%

“…The bulk of our mechanistic studies indicated that the NFAT inhibitory compounds acted upstream of NFAT dephosphorylation and calcineurin activation, leaving calcium mobilization as a likely site of action. In most nonexcitable cells, ligand-induced phospholipase C activation leads to inositol trisphosphate (IP 3 ) generation and concomitant IP 3 -triggered release of calcium from intracellular stores (20). This emptying of intracellular stores is thought to trigger the opening of ''capacitative'' or SOC channels in the plasma membrane, which results in sustained elevation of intracellular calcium.…”

Section: Resultsmentioning

confidence: 99%

Chemical genetics to identify NFAT inhibitors: Potential of targeting calcium mobilization in immunosuppression

Natarajan

Feng

DeDecker

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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The development of more selective immunosuppressive agents to mitigate transplant rejection and autoimmune diseases requires effective strategies of blocking signaling pathways in T cells. Current immunosuppressive strategies use cyclosporin A (CsA) or FK506 to inhibit calcineurin, which dephosphorylates and promotes the nuclear import of nuclear factor of activated T cells (NFAT) transcription factors. These nuclear NFATs then transactivate cytokine genes that regulate proliferative responses of T cells. Both CsA and FK506 have debilitating side effects, including nephrotoxicity, hypertension, diabetes, and seizures, that argue for the development of alternative or complementary agents. To this end, we developed cell-based assays for monitoring NFAT dynamics in nonlymphoid cells to identify small molecules that inhibit NFAT nuclear import. Interestingly, we found that the majority of these small molecules suppress NFAT signaling by interfering with ''capacitative'' or ''store-operated'' calcium mobilization, thus raising the possibility that such mobilization processes are relevant targets in immunosuppression therapy. Further, these small molecules also show dose-dependent suppression of cytokine gene expression in T cells. Significantly, the IC50 of CsA in primary T cells was reduced by the addition of suboptimal concentrations of these compounds, suggesting the possibility that such small molecules, in combination with CsA, offer safer means of immunosuppression.capacitative calcium entry ͉ store-operated calcium channels ͉ cyclosporin A D rugs that destroy T cells or block their antigen-dependent activation have been the mainstays of treatment of organ transplant rejection (1). The latter group is represented chief ly by the natural products cyclosporin A (CsA) and FK506, both of which act by suppressing the protein phosphatase calcineurin (2-4). Calcineurin's activity depends on calcium signaling and plays an essential role in T cell signal transduction by controlling the nuclear import of the nuclear factor of activated T cells (NFAT) family of transcription factors. The NFATs are Rel-related transcription factors encoded by four genes (NFATc1-4) that are widely expressed in cells of the immune, cardiovascular, and nervous systems (4 -6). In resting cells, NFATs are localized to the cytoplasm due to a phosphorylation-dependent intramolecular masking of their nuclear location signals (NLSs) (7). During calcium signaling, calcineurin unmasks these NLSs, resulting in a rapid nuclear import of the dephosphorylated NFATs and transcriptional activation of cytokine genes (8). Besides unmasking the NLSs on the NFATs, calcineurin also masks their nuclear export signals, thereby preventing the futile cycling of these transcription factors across the nuclear envelope, thus ensuring their transcriptional functions in the nucleus (9, 10). NFAT proteins are essential for T cell activation and therefore represent potential targets for new immunosuppressive therapies. Interestingly, recent data from mouse knockout studie...

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“…Specific www.nature.com/aps Zhang HZ et al Acta Pharmacologica Sinica npg CRAC channel inhibitors may be developed into new safe and potent immune suppressors, which would benefit patients with these diseases. Thus far, a variety of small molecules blocking the CRAC channel have been identified, such as the imidazole derivative, SKF96365 (IC 50 of approximately 4 μmol/L) [15,16] , 2-APB [at low concentrations of 1-5 μmol/L, 2-APB potentiates CRAC currents, whereas high concentrations (>10 μmol/L) cause a transient enhancement of CRAC currents followed by a complete block in S2 for mammalian cells] [17,18] , YM58483 (IC 50 of approximately 150 nmol/L after 24 h of preincubation; IC 50 of approximately 10 μmol/L immediately after addi tion) [19,20] , and Synta 66 (IC 50 of approximately 3 μmol/L) [21] ( Figure 1); however, these compounds are not specific because they interfere with a variety of other transport processes. The only specific CRAC channel inhibitor tested in human is CM2489 [22] , the structure of which has not yet been disclosed.…”

Section: Introductionmentioning

confidence: 99%

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

Zhang

Chen

et al. 2015

Acta Pharmacol Sin

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Aim: Ca 2+ -release-activated Ca 2+ (CRAC) channel, a subfamily of store-operated channels, is formed by calcium release-activated calcium modulator 1 (ORAI1), and gated by stromal interaction molecule 1 (STIM1). CRAC channel may be a novel target for the treatment of immune disorders and allergy. The aim of this study was to identify novel small molecule CRAC channel inhibitors. Methods: HEK293 cells stably co-expressing both ORAI1 and STIM1 were used for high-throughput screening. A hit, 1-phenyl-3-(1-phenylethyl)urea, was identified that inhibited CRAC channels by targeting ORAI1. Five series of its derivatives were designed and synthesized, and their primary structure-activity relationships (SARs) were analyzed. All derivatives were assessed for their effects on Ca 2+ influx through CRAC channels on HEK293 cells, cytotoxicity in Jurkat cells, and IL-2 production in Jurkat cells expressing ORAI1-SS-eGFP. Results: A total of 19 hits were discovered in libraries containing 32 000 compounds using the high-throughput screening. 1-Phenyl-3-(1-phenylethyl)urea inhibited Ca 2+ influx with IC 50 of 3.25±0.17 μmol/L. SAR study on its derivatives showed that the alkyl substituent on the α-position of the left-side benzylic amine (R1) was essential for Ca 2+ influx inhibition and that the S-configuration was better than the R-configuration. The derivatives in which the right-side R3 was substituted by an electron-donating group showed more potent inhibitory activity than those that were substituted by electron-withdrawing groups. Furthermore, the free N-H of urea was not necessary to maintain the high potency of Ca 2+ influx inhibition. The N,N′-disubstituted or N′-substituted derivatives showed relatively low cytotoxicity but maintained the ability to inhibit IL-2 production. Among them, compound 5b showed an improved inhibition of IL-2 production and low cytotoxicity. Conclusion: 1-Phenyl-3-(1-phenylethyl)urea is a novel CRAC channel inhibitor that specifically targets ORAI1. This study provides a new chemical scaffold for design and development of CRAC channel inhibitors with improved Ca 2+ influx inhibition, immune inhibition and low cytotoxicity.

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Potent Inhibition of Ca2+ Release-activated Ca2+ Channels and T-lymphocyte Activation by the Pyrazole Derivative BTP2

Cited by 266 publications

References 31 publications

Calcium dependence of T cell proliferation following focal stimulation

Calcium dependence of T cell proliferation following focal stimulation

Chemical genetics to identify NFAT inhibitors: Potential of targeting calcium mobilization in immunosuppression

Discovery and structural optimization of 1-phenyl-3-(1-phenylethyl)urea derivatives as novel inhibitors of CRAC channel

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