Discovery of selective Orai channel blockers bearing an indazole or a pyrazole scaffold.

The calcium release activated calcium (CRAC) channel is highly expressed in T lymphocytes and plays a critical role in regulating T cell proliferation and functions including activation of the transcription factor nuclear factor of activated T cells (NFAT), cytokine production and cytotoxicity. The CRAC channel consists of the Orai pore subunit and STIM (stromal interacting molecule) endoplasmic reticulum calcium sensor. Loss of CRAC channel mediated calcium signaling has been identified as an underlying cause of severe combined immunodeficiency (SCID), leading to drastically weakened immunity against infections. Gain-of-function mutations in Orai and STIM have been associated with tubular aggregated myopathy (TAM), a skeletal muscle disease. While a number of small molecules have shown activity in inhibiting the CRAC signaling pathway, the usefulness of those tool compounds is limited by their off-target activity against TRPM4 and TRPM7 ion channels, high lipophilicity, and a lack of understanding of their mechanism of action. We report structure-activity relationship (SAR) studies that resulted in the characterization of compound 4k [1-(cyclopropylmethyl)-N-(3-fluoropyridin-4-yl)-1H-indazole-3-carboxamie] as a fast onset, reversible, and selective CRAC channel blocker. 4k fully blocked the CRAC current (IC50: 4.9 µM) and the nuclear translocation of NFAT at 30 and 10 µM, respectively, without affecting the electrophysiological function of TRPM4 and TRPM7 channels. Computational modeling appears to support its direction binding to Orai proteins that form the transmembrane CRACchannel.

Agrimonia coreana Extract Exerts Its Therapeutic Effect through CRAC Channel Inhibition for Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (ACext) can be used in treating AD-related dermatologic symptoms. ACext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon ACext treatment. In a mouse model of AD, ACext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD.

ORAI Ca2+ Channels in Cancers and Therapeutic Interventions.

The ORAI proteins serve as crucial pore-forming subunits of calcium-release-activated calcium (CRAC) channels, pivotal in regulating downstream calcium-related signaling pathways. Dysregulated calcium homeostasis arising from mutations and post-translational modifications in ORAI can lead to immune disorders, myopathy, cardiovascular diseases, and even cancers. Small molecules targeting ORAI present an approach for calcium signaling modulation. Moreover, emerging techniques like optogenetics and optochemistry aim to offer more precise regulation of ORAI. This review focuses on the role of ORAI in cancers, providing a concise overview of their significance in the initiation and progression of cancers. Additionally, it highlights state-of-the-art techniques for ORAI channel modulation, including advanced optical tools, potent pharmacological inhibitors, and antibodies. These novel strategies offer promising avenues for the functional regulation of ORAI in research and may inspire innovative approaches to cancer therapy targeting ORAI.

Auxora vs. placebo for the treatment of patients with severe COVID-19 pneumonia: a randomized-controlled clinical trial.

BACKGROUND: Calcium release-activated calcium (CRAC) channel inhibitors block proinflammatory cytokine release, preserve endothelial integrity and may effectively treat patients with severe COVID-19 pneumonia. METHODS: CARDEA was a phase 2, randomized, double-blind, placebo-controlled trial evaluating the addition of Auxora, a CRAC channel inhibitor, to corticosteroids and standard of care in adults with severe COVID-19 pneumonia. Eligible patients were adults with ≥ 1 symptom consistent with COVID-19 infection, a diagnosis of COVID-19 confirmed by laboratory testing using polymerase chain reaction or other assay, and pneumonia documented by chest imaging. Patients were also required to be receiving oxygen therapy using either a high flow or low flow nasal cannula at the time of enrolment and have at the time of enrollment a baseline imputed PaO2/FiO2 ratio > 75 and ≤ 300. The PaO2/FiO2 was imputed from a SpO2/FiO2 determine by pulse oximetry using a non-linear equation. Patients could not be receiving either non-invasive or invasive mechanical ventilation at the time of enrolment. The primary endpoint was time to recovery through Day 60, with secondary endpoints of all-cause mortality at Day 60 and Day 30. Due to declining rates of COVID-19 hospitalizations and utilization of standard of care medications prohibited by regulatory guidance, the trial was stopped early. RESULTS: The pre-specified efficacy set consisted of the 261 patients with a baseline imputed PaO2/FiO2≤ 200 with 130 and 131 in the Auxora and placebo groups, respectively. Time to recovery was 7 vs. 10 days (P = 0.0979) for patients who received Auxora vs. placebo, respectively. The all-cause mortality rate at Day 60 was 13.8% with Auxora vs. 20.6% with placebo (P = 0.1449); Day 30 all-cause mortality was 7.7% and 17.6%, respectively (P = 0.0165). Similar trends were noted in all randomized patients, patients on high flow nasal cannula at baseline or those with a baseline imputed PaO2/FiO2 ≤ 100. Serious adverse events (SAEs) were less frequent in patients treated with Auxora vs. placebo and occurred in 34 patients (24.1%) receiving Auxora and 49 (35.0%) receiving placebo (P = 0.0616). The most common SAEs were respiratory failure, acute respiratory distress syndrome, and pneumonia. CONCLUSIONS: Auxora was safe and well tolerated with strong signals in both time to recovery and all-cause mortality through Day 60 in patients with severe COVID-19 pneumonia. Further studies of Auxora in patients with severe COVID-19 pneumonia are warranted. Trial registration NCT04345614.

Discovery of a Novel Potent and Selective Calcium Release-Activated Calcium Channel Inhibitor: 2,6-Difluoro-N-(2'-methyl-3'-(4-methyl-5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-[1,1'-biphenyl]-4-yl)benzamide. Structure-Activity Relationship and Preclinical Characterization.

The role of calcium release-activated calcium (CRAC) channels is well characterized and is of particular importance in T-cell function. CRAC channels are involved in the pathogenesis of several autoimmune diseases, making it an attractive therapeutic target for treating inflammatory diseases, like rheumatoid arthritis (RA). A systematic structure-activity relationship study with the goal of optimizing lipophilicity successfully yielded two lead compounds, 36 and 37. Both compounds showed decent potency and selectivity and a remarkable pharmacokinetic profile. Further characterization in in vivo RA models and subsequent histopathological evaluation of tissues led to the identification of 36 as a clinical candidate. Compound 36 displayed an excellent safety profile and had a sufficient safety margin to qualify it for use in human testing. Oral administration of 36 in Phase 1 clinical study in healthy volunteers established favorable safety, tolerability, and good target engagement as measured by levels of IL-2 and TNF-α.

GSK-7975A, an inhibitor of Ca2+ release-activated calcium channels, depresses isometric contraction of mouse aorta.

GSK-7975A is described to inhibit stromal interaction molecule 1(STIM1)-mediated Ca2+ release-activated Ca2+ channels ORAI 1, ORAI 2 and ORAI 3 in different cell types. The present study investigated whether isometric contractions of mouse aortic segments were affected by this selective store-operated calcium channel inhibitor. Depending on the way by which Ca2+ influx pathways were activated during contraction, GSK-7975A inhibited contractility of mouse aortic segments with different affinity. When contractile effects were induced by depolarization as with elevated extracellular K+ and opening of voltage-gated calcium channels, the affinity was approximately 10 times lower than when contraction was elicited with Ca2+ influx via non-selective cation channels. GSK-7975A may repolarize the aortic smooth muscle cells by inhibiting non-selective cation channels, has no effect on IP3-mediated phenylephrine-induced phasic contractions or on refilling of the contractile sarcoplasmic reticulum Ca2+ store, but has significant effects on non-contractile store-operated Ca2+ influx.

Auxora for the Treatment of Patients With Acute Pancreatitis and Accompanying Systemic Inflammatory Response Syndrome: Clinical Development of a Calcium Release-Activated Calcium Channel Inhibitor.

OBJECTIVES: To assess the safety of Auxora in patients with acute pancreatitis (AP), systemic inflammatory response syndrome (SIRS), and hypoxemia, and identify efficacy endpoints to prospectively test in future studies. METHODS: This phase 2, open-label, dose-response study randomized patients with AP, accompanying SIRS, and hypoxemia (n = 21) to receive low-dose or high-dose Auxora plus standard of care (SOC) or SOC alone. All patients received pancreatic contrast-enhanced computed tomography scans at screenings, day 5/discharge, and as clinically required 90 days postrandomization; scans were blinded and centrally read to determine AP severity using computed tomography severity index. Solid food tolerance was assessed at every meal and SIRS every 12 hours. RESULTS: The number of patients experiencing serious adverse events was not increased with Auxora versus SOC alone. Three (36.5%) patients with moderate AP receiving low-dose Auxora improved to mild AP; no computed tomography severity index improvements were observed with SOC. By study end, patients receiving Auxora better tolerated solid foods, had less persistent SIRS, and had reduced hospitalization versus SOC. CONCLUSIONS: The favorable safety profile and patient outcomes suggest Auxora may be an appropriate early treatment for patients with AP and SIRS. Clinical development will continue in a randomized, controlled, blinded, dose-ranging study.

Inhibition of Orai Channel Function Regulates Mas-Related G Protein-Coupled Receptor-Mediated Responses in Mast Cells.

Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders.

A first-in-human study to evaluate the safety, tolerability and pharmacokinetics of RP3128, an oral calcium release-activated calcium (CRAC) channel modulator in healthy volunteers.

WHAT IS KNOWN AND OBJECTIVE: RP3128, a novel, orally available modulator of calcium released activated calcium (CRAC) channel, is being developed for the potential treatment of autoimmune and inflammatory diseases. RP3128 showed nano-molar potency and activity in a range of in vitro and in vivo models of inflammation. We report a first-in-human study investigating the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of RP3128 in healthy subjects. METHODS: A randomized, double-blind, placebo-controlled trial of single (25, 50, 100, 200 and 400 mg) and multiple (7 days: 25, 100 and 400 mg once daily) doses of RP3128 were performed. Thirty-two and 24 subjects were randomized in the single ascending dose (SAD) and multiple ascending dose (MAD) parts, respectively. RESULTS AND DISCUSSION: RP3128 was well tolerated, with no dose-limiting toxicity at single and multiple doses. Incidence of treatment emergent adverse events (TEAEs) did not increase with ascending RP3128 doses. No changes were seen in cognitive function and ECG parameters. RP3128 was rapidly absorbed. Elimination was slow with a half-life of more than 80 h. Exposures increased with increasing doses. Accumulation was seen on repeated dosing. PD response, as evidenced by lower plasma levels of tumour necrosis factor-alfa (TNFα) and interleukin-4 (IL-4), was seen when compared to pre-dose values or placebo. WHAT IS NEW AND CONCLUSION: The safety, tolerability and PK/PD profile of RP3128 demonstrates its potential to be developed in inflammatory disorders and support further clinical development (ClinicalTrials.gov number: NCT02958982).

Auxora versus standard of care for the treatment of severe or critical COVID-19 pneumonia: results from a randomized controlled trial.

BACKGROUND: Calcium release-activated calcium (CRAC) channel inhibitors stabilize the pulmonary endothelium and block proinflammatory cytokine release, potentially mitigating respiratory complications observed in patients with COVID-19. This study aimed to investigate the safety and efficacy of Auxora, a novel, intravenously administered CRAC channel inhibitor, in adults with severe or critical COVID-19 pneumonia. METHODS: A randomized, controlled, open-label study of Auxora was conducted in adults with severe or critical COVID-19 pneumonia. Patients were randomized 2:1 to receive three doses of once-daily Auxora versus standard of care (SOC) alone. The primary objective was to assess the safety and tolerability of Auxora. Following FDA guidance, study enrollment was halted early to allow for transition to a randomized, blinded, placebo-controlled study. RESULTS: In total, 17 patients with severe and three with critical COVID-19 pneumonia were randomized to Auxora and nine with severe and one with critical COVID-19 pneumonia to SOC. Similar proportions of patients receiving Auxora and SOC experienced ≥ 1 adverse event (75% versus 80%, respectively). Fewer patients receiving Auxora experienced serious adverse events versus SOC (30% versus 50%, respectively). Two patients (10%) receiving Auxora and two (20%) receiving SOC died during the 30 days after randomization. Among patients with severe COVID-19 pneumonia, the median time to recovery with Auxora was 5 days versus 12 days with SOC; the recovery rate ratio was 1.87 (95% CI, 0.72, 4.89). Invasive mechanical ventilation was needed in 18% of patients with severe COVID-19 pneumonia receiving Auxora versus 50% receiving SOC (absolute risk reduction = 32%; 95% CI, - 0.07, 0.71). Outcomes measured by an 8-point ordinal scale were significantly improved for patients receiving Auxora, especially for patients with a baseline PaO2/FiO2 = 101-200. CONCLUSIONS: Auxora demonstrated a favorable safety profile in patients with severe or critical COVID-19 pneumonia and improved outcomes in patients with severe COVID-19 pneumonia. These results, however, are limited by the open-label study design and small patient population resulting from the early cessation of enrollment in response to regulatory guidance. The impact of Auxora on respiratory complications in patients with severe COVID-19 pneumonia will be further assessed in a planned randomized, blinded, placebo-controlled study. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04345614 . Submitted on 7 April 2020.

Small molecules under development for psoriasis: on the road to the individualized therapies.

Psoriasis is an incurable cutaneous illness characterized by the presence of well-delimited reddish plaques and silvery-white dry scales. So far, there is a limited understanding of its pathogenesis, though recent discoveries on the immunological, genetic and molecular aspects of this disease have significantly contributed to the identification of new targets and the development of novel drugs. Despite these advances, many patients are still dissatisfied, so to improve patient satisfaction, reliability, and clinical outcomes, the individualization of the treatments for this disease becomes a necessity. This review summarizes recent findings related to psoriasis pathogenesis and describes new small molecules and targets recently identified as promising for treatments. Additionally, the current status, challenges and the future directions for achieving individualized therapy for this disease and the need for more collaborative studies are discussed. The individualization of treatments for psoriasis, rather than a goal, is analyzed as a process where a dynamic integration between the needs and characteristics of the patients, the pharmacological progress, and the clinical decisions takes place.

Contribution of Cav1.2 Ca2+ channels and store-operated Ca2+ entry to pig urethral smooth muscle contraction.

Urethral smooth muscle (USM) generates tone to prevent urine leakage from the bladder during filling. USM tone has been thought to be a voltage-dependent process, relying on Ca2+ influx via voltage-dependent Ca2+ channels in USM cells, modulated by the activation of Ca2+-activated Cl- channels encoded by Ano1. However, recent findings in the mouse have suggested that USM tone is voltage independent, relying on Ca2+ influx through Orai channels via store-operated Ca2+ entry (SOCE). We explored if this pathway also occurred in the pig using isometric tension recordings of USM tone. Pig USM strips generated myogenic tone, which was nearly abolished by the Cav1.2 channel antagonist nifedipine and the ATP-dependent K+ channel agonist pinacidil. Pig USM tone was reduced by the Orai channel blocker GSK-7975A. Electrical field stimulation (EFS) led to phentolamine-sensitive contractions of USM strips. Contractions of pig USM were also induced by phenylephrine. Phenylephrine-evoked and EFS-evoked contractions of pig USM were reduced by ~50-75% by nifedipine and ~30% by GSK-7975A. Inhibition of Ano1 channels had no effect on tone or EFS-evoked contractions of pig USM. In conclusion, unlike the mouse, pig USM exhibited voltage-dependent tone and agonist/EFS-evoked contractions. Whereas SOCE plays a role in generating tone and agonist/neural-evoked contractions in both species, this dominates in the mouse. Tone and agonist/EFS-evoked contractions of pig USM are the result of Ca2+ influx primarily through Cav1.2 channels, and no evidence was found supporting a role of Ano1 channels in modulating these mechanisms.

Structural and Mechanistic Insights of CRAC Channel as a Drug Target in Autoimmune Disorder.

BACKGROUND: Calcium (Ca2+) ion is a major intracellular signaling messenger, controlling a diverse array of cellular functions like gene expression, secretion, cell growth, proliferation, and apoptosis. The major mechanism controlling this Ca2+ homeostasis is store-operated Ca2+ release-activated Ca2+ (CRAC) channels. CRAC channels are integral membrane protein majorly constituted via two proteins, the stromal interaction molecule (STIM) and ORAI. Following Ca2+ depletion in the Endoplasmic reticulum (ER) store, STIM1 interacts with ORAI1 and leads to the opening of the CRAC channel gate and consequently allows the influx of Ca2+ ions. A plethora of studies report that aberrant CRAC channel activity due to Loss- or gain-of-function mutations in ORAI1 and STIM1 disturbs this Ca2+ homeostasis and causes several autoimmune disorders. Hence, it clearly indicates that the therapeutic target of CRAC channels provides the space for a new approach to treat autoimmune disorders. OBJECTIVE: This review aims to provide the key structural and mechanical insights of STIM1, ORAI1 and other molecular modulators involved in CRAC channel regulation. RESULTS AND CONCLUSION: Understanding the structure and function of the protein is the foremost step towards improving the effective target specificity by limiting their potential side effects. Herein, the review mainly focusses on the structural underpinnings of the CRAC channel gating mechanism along with its biophysical properties that would provide the solid foundation to aid the development of novel targeted drugs for an autoimmune disorder. Finally, the immune deficiencies caused due to mutations in CRAC channel and currently used pharmacological blockers with their limitation are briefly summarized.

Store-operated calcium channels: Potential target for the therapy of hypertension.

Effective therapy of hypertension represents a key strategy for reducing the burden of cardiovascular disease and its associated mortality. The significance of voltage dependent L-type Ca²âº channels to Ca²âº influx, and of their regulatory mechanisms in the development of heart disease, is well established. A wide variety of L-type Ca²âº channel inhibitors and Ca²âº antagonists have been found to be beneficial not only in the treatment of hypertension, but also in myocardial infarction and heart failure. Over the past two decades, another class of Ca²âº channel - the voltage independent store-operated Ca²âº channel - has been implicated in the regulation and fine tuning of Ca²âº entry in both cardiac and smooth muscle cells. Store-operated Ca²âº channels are activated by the depletion of Ca²âº stores within the endoplasmic/sarcoplasmic reticulum, or by low levels of cytosolic Ca²âº, thereby facilitating agonist-induced Ca²âº influx. Store-operated Ca²âº entry through this pivotal pathway involves both stromal interaction molecule (STIM) and Orai channels. Different degrees of changes in these proteins are considered to promote Ca²âº entry and hence contribute to the pathogenesis of cardiovascular dysfunction. Several blockers of store-operated Ca²âº channels acting at the level of both STIM and Orai channels have been shown to depress Ca²âº influx and lower blood pressure. However, their specificity, safety, and clinical significance remain to be established. Thus, there is an ongoing challenge in the development of selective inhibitors of store-operated Ca²âº channels that act in vascular smooth muscles for the improved treatment of hypertension.

The regulatory roles of calcium channels in tumors.

Calcium (Ca2+) and its relevant transmembrane and intracellular calcium channels were previously thought to be chiefly associated with the regulation of cardiovascular and neuronal systems. Nowadays, an increasing evidence shows those calcium channels are also responsible for tumorigenesis and progression. However, the general underlying mechanisms and the involving signaling transduction pathways still remain unclear. Therefore, in this mini-review, we are mainly focusing on the linkage between calcium channels and major characteristics of tumors such as multi-drug resistance (MDR), metastasis, apoptosis, proliferation, evasion of immune surveillance, and the alterations of tumor microenvironment. We will shed light on the possible therapeutic approaches to counteract tumors regarding the intervention of calcium channel.

Indirect Measurement of CRAC Channel Activity Using NFAT Nuclear Translocation by Flow Cytometry in Jurkat Cells.

Flow cytometry is a powerful technology to assess the presence of NFAT in the nuclei after CRAC channel activation. Here we described a simplified procedure for the analysis of CRAC channel activity using NFAT nuclear translocation by flow cytometry, based on the isolation of Jurkat E6-1 cell nuclei.

CRAC channels as targets for drug discovery and development.

Calcium release-activated calcium (CRAC) channels have been the target of drug discovery for many years. The identification of STIM and Orai proteins as key components of CRAC channels greatly facilitated this process because their co-expression in cell lines produced electrophysiological currents (ICRAC) much larger than those in native cells, making it easier to confirm and characterize the effects of modulatory compounds. A driving force in the quest for CRAC channel drugs has been the immunocompromised phenotype displayed by humans and mice with null or loss-of-function mutations in STIM1 or Orai1, suggesting that CRAC channel inhibitors could be useful therapeutics for autoimmune or inflammatory conditions. Emerging data also suggests that other therapeutic conditions may benefit from CRAC channel inhibition. However, only recently have CRAC channel inhibitors reached clinical trials. This review discusses the challenges associated with drug discovery and development on CRAC channels and the approaches employed to date, as well as the results, starting from initial high-throughput screens for CRAC channel modulators and progressing through target selection and justification, descriptions of pharmacological, safety and toxicological profiles of compounds, and finally the entry of CRAC channel inhibitors into clinical trials.

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

BACKGROUND: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. METHODS: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. RESULTS: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. CONCLUSIONS: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

Efficiency and Safety of CRAC Inhibitors in Human Rheumatoid Arthritis Xenograft Models.

Store-operated Ca2+ release-activated Ca2+ (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483. Systemically constant, s.c. infused CRAC inhibitors showed anti-inflammatory activity in a human-NOD/SCID xenograft RA model as well as protective effects against the destruction of cartilage and bone. hCRACM1-IgG appeared to be safe for systemic application, whereas YM-58483 showed hepatic and renal toxicity in xenograft mice. Treatment with both CRAC inhibitors also caused hyperglycemia in xenograft mice. These results indicate the potential of hCRACM1-IgG and YM-58483 as anti-immunological agents for the treatment of RA. However, some safety issues should be addressed and application methods should be optimized prior to their clinical use.

A study on blocking store-operated Ca2+ entry in pulmonary arterial smooth muscle cells with xyloketals from marine fungi.

In this study, the effect of four xyloketals 1-4 on store-operated calcium entry (SOCE) was investigated in primary distal pulmonary arterial smooth muscle cells (PASMCs) isolated from mice. The results showed that xyloketal A (1), an unusual ketal with C-3 symmetry, exhibited strong SOCE blocking activity. Secretion of interleukin-8 (IL-8) was also inhibited by xyloketal A. The parallel artificial membrane permeability assay (PAMPA) of 1-4 suggested that these xyloketals penetrated easily through the cell membrane. Moreover, the molecular docking study of xyloketal A with activation region of the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1 (STIM1-ORAI1) protein complex, the key domain of SOCE, revealed that xyloketal A exhibited a noncovalent interaction with the key residue lysine 363 (LYS363) in the identified cytosolic regions in STIM1-C. These findings provided useful information about xyloketal A as a SOCE inhibitor for further evaluation.