Malcolm Begg

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca2+ concentration inside pancreatic acinar cells ([Ca2+]i), due to excessive release of Ca2+ stored inside the cells followed by Ca2+ entry from the interstitial fluid. The sustained [Ca2+]i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca2+ release-activated Ca2+ channels (CRAC) would prevent sustained elevation of [Ca2+]i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca2+ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca2+. Ca2+ entry then occurred upon admission of Ca2+ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca2+ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC50 = 3.4 μM), but had little or no effect on the physiological Ca2+ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca2+]i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca2+]i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.

The action of selective CRAC channel blockers is affected by the Orai pore geometry.

As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC50 of approximately 4 μM and exhibited a substantially slower rate of onset than the typical pore blocker La3+, together with almost no current recovery upon wash-out over 4 min. For the less Ca2+-selective Orai1 E106D pore mutant, ICRAC inhibition was significantly reduced. FRET experiments indicated that neither STIM1–STIM1 oligomerization nor STIM1–Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.

CRACM/Orai ion channel expression and function in human lung mast cells

Background Influx of extracellular Ca2+ into human lung mast cells (HLMCs) is essential for the FcεRI-dependent release of preformed granule-derived mediators and newly synthesized autacoids and cytokines. However, the identity of the ion channels underlying this Ca2+ influx is unknown. The recently discovered members of the CRACM/Orai ion channel family that carries the Ca2+ release–activated Ca2+ current are candidates. Objectives To investigate the expression and function of CRACM channels in HLMCs. Methods CRACM mRNA, protein, and functional expression were examined in purified HLMCs and isolated human bronchus. Results CRACM1, -2, and -3 mRNA transcripts and CRACM1 and -2 proteins were detectable in HLMCs. A CRACM-like current was detected following FcεRI-dependent HLMC activation and also in HLMCs dialyzed with 30 μM inositol triphosphate. The Ca2+-selective current obtained under both conditions was blocked by 10 μM La3+ and Gd3+, known blockers of CRACM channels, and 2 distinct and specific CRACM-channel blockers—GSK-7975A and Synta-66. Both blockers reduced FcεRI-dependent Ca2+ influx, and 3 μM GSK-7975A and Synta-66 reduced the release of histamine, leukotriene C4, and cytokines (IL-5/-8/-13 and TNFα) by up to 50%. Synta-66 also inhibited allergen-dependent bronchial smooth muscle contraction in ex vivo tissue. Conclusions The presence of CRACM channels, a CRACM-like current, and functional inhibition of HLMC Ca2+ influx, mediator release, and allergen-induced bronchial smooth muscle contraction by CRACM-channel blockers supports a role for CRACM channels in FcεRI-dependent HLMC secretion. CRACM channels are therefore a potential therapeutic target in the treatment of asthma and related allergic diseases.

“Chemokine receptors as therapeutic targets: Why aren’t there more drugs?”

Chemokines are a family of around 40 small proteins, which are secreted by a variety of cells, including structural cell types and leukocytes of the immune system. Chemokines bind to their specific 7-transmembrane G protein-coupled receptors (GPCRs) and induce a variety of downstream signals which notably modulate polymerization of the actin cytoskeleton and thus drive cellular motility. Excessive or inappropriate release of chemokines is observed in many inflammatory diseases and so there has been a great effort in industry to target chemokine receptors. The large family of GPCRs regulate many physiological cellular processes and they have proved to be highly amenable to pharmacological intervention with small chemicals. Consequently GPCRs make attractive targets for drug discovery and indeed a large number of successful current therapeutics are either agonists or antagonists of GPCRs. The apparent lack of success with chemokine receptors has been frustrating and in this paper we discuss potential reasons for previous failures and also why there is considerable cause for optimism.

Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations

Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders.

High glucose and diabetes enhanced store-operated Ca2+ entry and increased expression of its signaling proteins in mesangial cells

The present study was conducted to determine whether and how store-operated Ca(2+) entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7-day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca(2+) channels. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca(2+) currents were significantly enhanced in the MCs treated with HG for 7 days, and the enhanced response was abolished by both GSK-7975A and La(3+). In contrast, receptor-operated Ca(2+) entry in MCs was significantly reduced by HG treatment. Western blotting showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real-time RT-PCR revealed that a 6-day HG treatment significantly increased the mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription but increases Orai1 protein through a posttranscriptional mechanism.

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.

Synthesis and Structure–Activity Relationships of Indazole Arylsulfonamides as Allosteric CC-Chemokine Receptor 4 (CCR4) Antagonists

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.

The role of Ca2+ influx in endocytic vacuole formation in pancreatic acinar cells.

The inducers of acute pancreatitis trigger a prolonged increase in the cytosolic Ca2+ concentration ([Ca2+]c), which is responsible for the damage to and eventual death of pancreatic acinar cells. Vacuolization is an important indicator of pancreatic acinar cell damage. Furthermore, activation of trypsinogen occurs in the endocytic vacuoles; therefore the vacuoles can be considered as ‘initiating’ organelles in the development of the cell injury. In the present study, we investigated the relationship between the formation of endocytic vacuoles and Ca2+ influx developed in response to the inducers of acute pancreatitis [bile acid taurolithocholic acid 3-sulfate (TLC-S) and supramaximal concentration of cholecystokinin-8 (CCK)]. We found that the inhibitor of STIM (stromal interaction molecule)/Orai channels, GSK-7975A, effectively suppressed both the Ca2+ influx (stimulated by inducers of pancreatitis) and the formation of endocytic vacuoles. Cell death induced by TLC-S or CCK was also inhibited by GSK-7975A. We documented the formation of endocytic vacuoles in response to store-operated Ca2+ entry (SOCE) induced by thapsigargin [TG; inhibitor of sarcoplasmic/endoplasmic reticulum (ER) Ca2+ pumps] and observed strong inhibition of TG-induced vacuole formation by GSK-7975A. Finally, we found that structurally-unrelated inhibitors of calpain suppress formation of endocytic vacuoles, suggesting that this Ca2+-dependent protease is a mediator between Ca2+ elevation and endocytic vacuole formation.

CRAC channel inhibition produces greater anti-inflammatory effects than glucocorticoids in CD8 cells from COPD patients.

There are increased numbers of pulmonary CD8 lymphocytes in COPD (chronic obstructive pulmonary disease). CRAC (calcium release-activation calcium) channels play a central role in lymphocyte activation though the regulation of the transcription factor NFAT (nuclear factor of activated T-cells). We studied the expression of NFAT in lungs from COPD patients compared with controls, and evaluated the effects of CRAC channel inhibition compared with corticosteroids on NFAT activation and cytokine production in CD8 cells from COPD patients. The effects of the corticosteroid dexamethasone, the calcineurin inhibitor cyclosporin and the CRAC channel inhibitor Synta 66 were studied on cytokine production and NFAT activation using peripheral blood and isolated pulmonary CD8 cells. NFAT1 and CD8 co-expression in the lungs was compared in COPD patients and controls using combined immunohistochemistry and immunofluorescence. NFAT inhibition with either cyclosporin or Synta 66 resulted in significantly greater maximal inhibition of cytokines than dexamethasone in both peripheral blood and pulmonary CD8 cells [e.g. >95% inhibition of IFNγ (interferon γ) production from pulmonary CD8 cells using cyclosporin and Synta 66 compared with <50% using dexamethasone]. The absolute number of pulmonary CD8 cells co-expressing NFAT1 was significantly raised in lungs from COPD patients compared with controls, but the percentage of CD8 cells co-expressing NFAT1 was similar between COPD patients and controls (80.7% compared with 78.5% respectively, P=0.3). Inhibition of NFAT using the CRAC channel Synta 66 produces greater anti-inflammatory effects on CD8 cells from COPD patients than corticosteroids. NFAT is expressed at a high level in pulmonary CD8 cells in COPD.