KEY POINTS:This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. ABSTRACT:Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.

BACKGROUND & AIMS:Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.METHODS:Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.RESULTS:GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.CONCLUSIONS:Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

Engagement of the T cell receptor (TCR) triggers complex signaling cascades that result in various effector T cell responses. A critical component of TCR signaling is store-operated calcium entry (SOCE) that occurs through the activation of calcium release-activated calcium (CRAC) channels. Activated CRAC channels cause an increase in cytoplasmic calcium, which results in the transcription of calcineurin/NFAT-dependent genes involved in T cell proliferation and cytokine production. Both CRAC channel inhibitors and calcineurin inhibitors are known to inhibit T cell function. However, the relative impact of CRAC channel inhibitors and calcineurin inhibitors on Th1, Th2, Th17, and Treg functions are not known. Using anti-CD3/anti-CD28 to stimulate cytokine production by peripheral blood mononuclear cells (PBMCs), we demonstrate that novel CalciMedica CRAC channel inhibitors have a pattern of cytokine inhibition that is different from the calcineurin inhibitor cyclosporine (CsA). CRAC channel inhibitors inhibit Th1 and Th17 cytokines (IFNγ and IL-17) more potently than Treg and Th2 cytokines (IL-10 and IL-4), while CsA inhibits IL-10 more potently than IFNγ, IL-4, and IL-17. These results suggest that CRAC channel inhibitors and CsA have different potencies against different effector T cell subsets, and that CRAC channel inhibitors may achieve a more favorable balance of effects on pro- versus anti-inflammatory pathways in immune disorders.

Abstract Calcium-release activated calcium (CRAC) channel inhibitors represent a new class of oral immunomodulatory agents with a potential safety profile suitable for chronic dosing to treat autoimmune disorders. Calcium entry through CRAC channels is a critical step in the functional responses of T cells and mast cells. Patients with mutations in CRAC channels are known to have a form of severe combined immunodeficiency, wherein the adaptive immune response is suppressed without major impairment of other organ systems. CalciMedica has discovered several novel small molecules that selectively inhibit CRAC channels, and is currently developing the first of these inhibitors, CM2489, for the treatment of psoriasis. We describe here the properties of CM3457, another selective CRAC channel inhibitor that is structurally distinct from CM2489. CM3457 potently inhibits CRAC channels, Th1, Th2 and Th17-derived cytokine production, T cell proliferation, and mast cell degranulation. Once-daily oral dosing of CM3457 significantly inhibits inflammation and joint histopathology in a rat collagen-induced arthritis model. CM3457 also inhibits lung inflammation and eosinophilia, as well as improves lung function, in a rat OVA-induced asthma model. These data provide evidence that CM3457 may be an effective therapeutic in the treatment of autoimmune disorders and asthma.

La presente invention concerne des composes et des compositions pharmaceutiques contenant lesdits composes, qui modulent l’activite des canaux calciques dits capacitifs (store-operated calcium (SOC) channels). L’invention concerne egalement des procedes d’utilisation de tels modulateurs des canaux SOC, seuls ou en association avec d’autres composes, pour le traitement de maladies, troubles ou affections qui tireraient un benefice de l’inhibition de l’activite des canaux SOC.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+ concentration near the interface, indicating Ca2+ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+ signaling during subsequent antigen encounter.

Ca(2+) release-activated Ca(2+) (CRAC) channels, located in the plasma membrane, are opened upon release of Ca(2+) from intracellular stores, permitting Ca(2+) entry and sustained [Ca(2+)](i) signaling that replenishes the store in numerous cell types. This mechanism is particularly important in T lymphocytes of the immune system, providing the missing link in the signal transduction cascade that is initiated by T cell receptor engagement and leads to altered expression of genes that results ultimately in the production of cytokines and cell proliferation. In the past three years, RNA interference screens together with over-expression and site-directed mutagenesis have identified the triggering molecule (Stim) that links store depletion to CRAC channel-mediated Ca(2+) influx and the pore subunit (Orai) of the CRAC channel that allows highly selective entry of Ca(2+) ions into cells.

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca2+ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca2+ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-Fis a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca2+ pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca2+ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

As the sole Ca2+ entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca2+ signalling and many other cellular processes1,2,3. A calcium-release-activated calcium (CRAC) channel in T lymphocytes is the best-characterized SOC influx channel4,5,6 and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation7,8,9,10. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes11. Here we show that the expression of EF-hand mutants of Stim or STIM1 activates CRAC channels constitutively without changing Ca2+ store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulum-like sites to the plasma membrane upon depletion of the Ca2+ store. We propose that STIM1 functions as the missing link between Ca2+ store depletion and SOC influx, serving as a Ca2+ sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels.

As the sole Ca entry mechanism in a variety of non-excitable cells, store-operated calcium (SOC) influx is important in Ca signalling andmany other cellular processes. A calcium-releaseactivated calcium (CRAC) channel in T lymphocytes is the bestcharacterized SOC influx channel and is essential to the immune response, sustained activity of CRAC channels being required for gene expression and proliferation. The molecular identity and the gating mechanism of SOC and CRAC channels have remained elusive. Previously we identified Stim and the mammalian homologue STIM1 as essential components of CRAC channel activation in Drosophila S2 cells and human T lymphocytes. Here we show that the expression of EF-handmutants of Stim or STIM1 activates CRACchannels constitutivelywithout changingCa store content. By immunofluorescence, EM localization and surface biotinylation we show that STIM1 migrates from endoplasmic-reticulumlike sites to the plasma membrane upon depletion of the Ca store. We propose that STIM1 functions as the missing link between Ca store depletion and SOC influx, serving as a Ca sensor that translocates upon store depletion to the plasma membrane to activate CRAC channels. We previously characterized a SOC current in Drosophila S2 cells with biophysical properties similar to CRAC channels in human T cells. More recently, Stim was identified in an RNA-mediated interference (RNAi)-based screen with the SERCA (sarcoplasmic/ endoplasmic reticulum Ca2þATPase) pump inhibitor thapsigargin (TG) to evoke SOC influx in Drosophila S2 cells. Uniquely among the 170 candidate genes that were screened, including all trp-related genes, RNAi-mediated suppression of Stim inhibited the Ca2þ influx evoked by TG. By single-cell imaging of cytosolic Ca2þ concentration ([Ca]i) and patch clamp analysis, we confirmed a functional requirement for Stim, and for the human homologue STIM1, to mediate CRAC channel activity in S2 cells and in Jurkat T cells, respectively. Drosophila Stim and mammalian STIM1 (collectively referred to here as Stim1) are modular type I transmembrane proteins with an EF-hand motif near the amino terminus located in the lumen of the endoplasmic reticulum (ER) or outside the cell (Supplementary Fig. 1). Because Stim1 does not resemble any known ion channel, the presence of the EF-hand motif and its localization indicated that Stim1 might function as a sensor of the ER Ca2þ store. According to this proposal, Ca2þ binding to the EFhand domain of Stim1 within the lumen of the Ca2þ store would keep CRAC channels in the plasma membrane closed. To test this possibility, full-length STIM1 and Stim cDNA were cloned from RBL cells and S2 cells and placed into appropriate expression vectors. Mutants in the EF-hand region were prepared, two for STIM1 and four for Stim, on residues known to be critical for Ca2þ binding. Overexpression of wild-type (WT) or mutant Stim1 after transient transfection was confirmed by western blotting (Supplementary Fig. 2). In single-cell Ca2þ imaging experiments, overexpression of WT Stim1 produced no significant difference in resting [Ca]i, TG-independent Ca 2þ influx or TG-evoked store release compared with control blank-transfected cells (Jurkat cells are shown in Fig. 1, S2 cells in Supplementary Fig. 3). A modest increase in TG-dependent (store-operated) Ca2þ influx was seen in Jurkat T cells (Fig. 1b) but not in S2 cells, consistent with the hypothesis that Stim1 by itself is not a functional CRAC channel. In contrast, Jurkat or S2 cells transfected with the EF-hand mutants bore two severe phenotypes: resting [Ca]i and TG-independent Ca 2þ influx were increased from about 50 nM to more than 200 nM on average. Histograms of resting [Ca]i show that expression of EF-hand mutants increased resting [Ca]i to more than 600 nM in many individual cells (Supplementary Fig. 4). The Ca2þ release transient, obtained by adding TG in zero-Ca2þ solution, was not changed (Supplementary Fig. 5), showing that the Ca2þ store content was not affected. To test whether the high values of resting [Ca]i and enhanced TG-independent Ca2þ influx were caused by constitutively opened CRAC channels, 2-aminoethyldiphenyl borate, SKF96365 and Gd3þ were applied as pharmacological tools that block CRAC channels in Jurkat cells and in S2 cells. Each of these agents inhibited TG-evoked SOC influx in control cells and, at the same concentrations, all three inhibited both the high resting Ca2þ concentration and the enhanced TG-independent Ca2þ influx in Jurkat and S2 cells transfected with EF-hand mutants (Fig. 1g–i, and Supplementary Fig. 3c). These results demonstrate that CRAC channels in Jurkat or S2 cells are constitutively opened by overexpression of Stim1 EF-hand mutants. In addition to affecting resting [Ca]i, expression of Stim EFhandmutants arrested the growth of S2 cells, whereas overexpressing WT Stim had a small effect comparedwith that in S2 cells undergoing blank transfection (Supplementary Fig. 6). Most S2 cells overexpressing Stim EF-hand mutants were stained by annexin V, an early marker for the exposure of phosphatidylserine that occurs during apoptosis. The growth arrest and apoptosis were probably triggered by abnormally high resting [Ca]i (refs 19–21). A similar but milder growth defect in Jurkat cells overexpressing STIM1 EF-handmutants was also observed. How does STIM1 regulate the activity of CRAC channels? The subcellular localization of STIM1 was examined by LETTERS

Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 x g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol. In double-labeled experiments with a combination of -l-14C-isopentenyl diphosphate and a [4B-3H]NADPH-generating system, the ratio of 3H- and 14C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal.