Project: Identification of ICRAC antagonists

The aim of the project is to identify small molecule antagonists of store-operated calcium entry via the calcium-release-activated calcium (CRAC) channel to treat a range of human inflammatory and autoimmune disorders. Calcium ions are used for intracellular signalling and the pathways involved require tight control of cytoplasmic calcium ion concentrations as well as mechanisms for COtaining the net calcium ion balance. In cells of the immune system, calcium signals are essential for a variety of functions including differentiation, proliferation, effector function and gene transcription. The store-operated calcium current, termed ICRAC, mediated by CRAC channels, is the predominant route of calcium flux into non-excitable cells, including immune cells such as lymphocytes and mast cells, as well as blood platelets. Although the presence of store-operated signalling in immune cells has been known for many years, key proteins in the pathway have only recently been identified. The regulatory protein, Stim1 (stromal interaction molecule), which senses depletion of calcium from the endoplasmic reticulum and Orai1, which forms the pore of the plasma membrane calcium channel, are now known together to form the CRAC channel. In lymphocytes and mast cells, antigen or Fc receptor activation causes release of calcium ions from intracellular stores leading to influx of calcium ions through CRAC channels in the plasma membrane. The resulting increase in intracellular calcium levels activates calcineurin, a phosphatase that regulates the transcription factor, NFAT (nuclear factor of activated T-cells). In response to infection, and during transplant rejection, NFAT translocates to the nucleus and initiates a cascade of events leading to T-cell proliferation and an active immune response, including release of pro-inflammatory cytokines such as interleukin-2, interleukin-4 and interferon-gamma. Individuals who are homozygous for a mutation (Arg91Trp) in the gene encoding Orai1 have one form of severe combined immune deficiency syndrome (SCID) in which T-cells fail to respond to pathogens. When wild-type Orai1 was expressed in T-cells from SCID patients, normal CRAC channel activity and immune function were restored. The therapeutic potential of inhibiting ICRAC has been further established by the clinical use of calcineurin inhibitors such as cyclosporine A and tacrolimus to prevent rejection of organ transplants. Orai1 deficient mice have been shown to be resistant to pulmonary thromboembolism, arterial thrombosis, and ischemic brain infarction, suggesting that Orai1 is also a key mediator of ischemic cardiovascular and cerebrovascular events._x000D__x000D_Molecules that modulate the activity of ICRAC are expected to be useful for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis and also to prevent transplant rejection. Literature reports suggest that such molecules may also have application in the treatment of metastatic breast cancer, diabetes, inflammatory bowel disease, psoriasis, mast cell-related disorders, cardiovascular and cerebrovascular diseases, and certain viral infections. _x000D__x000D_Axxam is a science-driven biotechnology company formed in 2001 which now employs a team of almost 50 qualified personnel. Axxam is privately owned and based in the San Raffaele Biomedical Science Park, Milan. The company specialises in assay development, high throughput screening and compound profiling and offers early stage discovery research services for the life science industry as well as carrying out its own internal discovery research. Innovative state-of-the-art technologies that have been developed to improve assay performance include Photina®, a new and improved calcium activated photoprotein, which has been optimised for high throughput screening assays. The company, which is recognised as a world leader in assay development, also develops stem cells and primary cells for use in screening._x000D__x000D_Xention is a Cambridge, UK-based biopharmaceutical company specialising in the discovery and development of ion channel-modulating drugs. The company has potential breakthrough drugs for atrial fibrillation (AF) and overactive bladder (OAB) in clinical development and also has preclinical research programmes focused on other ion channels involved in atrial fibrillation and also in autoimmune disease. The company has an extensive ion channel knowledge base and uses electrophysiology (EP), the gold standard method for studying ion channels, at all stages of medicinal chemistry. Xention uses several proprietary and non-proprietary ion channel screening platforms to enable high quality EP data generation at all stages in the drug discovery process, allowing medicinal chemists unprecedented access to high-content data.

Acronym ICRAC (Reference Number: 5125)
Duration 01/06/2010 - 31/10/2013
Project Topic The project targets antagonists of calcium influx in T-cells mediated by the CRAC channel as novel therapeutic agents for treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or in the prevention of transplant rejection.
Project Results
(after finalisation)
Axxam has developed two recombinant cell lines, which express functional CRAC (Calcium Release Activated Channels): CHO mito-i-Photina® -CRAC e HEK- mito-I-Photina® -CRAC. They were obtained upon stable transfection of the two channel subunits ORA1 e STIM1 into CHO mito-i-Photina® and HEK- mito-I-Photina reporter cell lines, which stably express the Ca2+ sensing photoprotein mito-I-Photina and which were previously generated at Axxam. Influx of Ca2+ through activated CRAC leads to the activation of the Photoprotein and the consequent emission of a Luminescence signal. In parallel Axxam also developed an assay to study CRAC functionality by a fluorescence detection system in the human Jurkat T cells, which endogenously express the CRAC channel. All these assays were deeply optimized and characterized and they proofed to be suitable for HTS. In the end it was decided to use the Jurkat cells for screening. The recombinant cell lines, used for retsting, were also transferred to Xention, for their use in electrophysiology analysis. _x000D_By mean of HTS, a total of 161.300 compounds, belonging to Axxam and Xention libraries; 137.289 from Axxam and 24.011 from Xention, respectively, were screened on the CRAC-Jurkat T assay. Out of the primary screening a total of 2685 Hits were identified, displaying more than 53% inhibition of the signal. _x000D_These compounds were further tested at different concentrations, also in the recombinant assays, in order to confirm their activity. Finally, 11 hits, belonging to different chemical clusters and showing an IC50 ranging from 2 to 20 microM, were selected for further investigation on secondary assays. In particular, they were tested on Jurkat T cells, which endogenously express CRAC and are able to secrete IL2. The effects of the hits on the nuclear factor NFAT translocation and on the IL2 secretion was analyzed by means of a luminescence based and an ELISA assay, respectively. Most of the tested Hits were active on both the assays, even if with different potency. However, when tested in electrophysiology, which is the gold standard technology for ion channels, those compounds showed no significant block of ICRAC Therefore, due to the low potency displayed in electrophysiology, these compounds were judged to be not the optimal starting point for a medicinal chemistry program in search of selective ICRAC inhibitors. Based on the results obtained during the previous phase, in search for more promising hits, an accurate in silico cheminformatic analysis was performed. Starting from the structure of the validated hits, a preliminary structure activity relationships (SAR), was conducted. Through this approach, 1280 compounds, belonging to Axxam compound collection and analogues to the validated hits were fished out. All these molecules were tested in both CHO mito-i-photina®CRAC and Jurkat T cells. The most promising ones, about 50, were tested in dose/response and 21, out of these 50, showed an IC50 in the range of 4 to 20 microM. The best performers were also tested in electrophysiology, through automated patch clamp, and in secondary assays. In the end 4 compounds showed a good current inhibition, with an IC50 in the low microM range; while only two of them were effective on IL2 secretion, at the highest concentrations. _x000D_To sum up, this program allowed to achieve the following results:_x000D_- development of a functional and HTS suitable CRAC cell-based assay_x000D_- identification, by means of HTS, of 4 molecular classes of compounds acting as CRAC antagonists (hits)_x000D_- confirmation of the specificity of action of the identified hits _x000D_- confirmation of the capacity of these hits to interfere with intracellular pathways such as interleukin 2 (IL2) secretion _x000D_- preliminary toxicity evaluation on hERG channel _x000D__x000D_The program was successfully concluded: indeed the identified hits represent an important starting point for the development of novel and more potent CRAC blocker leads, though medicinal chemist._x000D_
Network Eurostars
Call Eurostars Cut-Off 3

Project partner

Number Name Role Country
2 Xention Limited Coordinator United Kingdom
2 Axxam SpA Partner Italy