A new class of immunomodulator, CRAC channel blockers

描述（由申请人提供）：刺激T细胞受体导致细胞内Ca2+储存的消耗和随后的CRAC （Ca2+释放激活的Ca2+）通道的开放。细胞内Ca2+浓度的持续增加激活了钙调神经磷酸酶/NFAT（活化T细胞的核因子）途径，并开启了各种细胞因子的转录程序。最近，Orai1和STIM1分别被鉴定为CRAC通道的长期寻找的孔隙成分和内质网（ER） Ca2+传感器。STIM1在T细胞受体刺激后感知内质网中Ca2+的耗尽，易位到质膜（PM）近端内质网，结合并激活Orai1。人类患者缺乏Orai1或STIM1有严重的联合免疫缺陷。然而，这一缺陷已通过造血干细胞移植得到修复，这意味着Orai1在免疫细胞中具有不可逆转的作用。因此，Orai1的特异性阻滞剂可能会改善钙调磷酸酶阻滞剂所观察到的副作用。在这里，我们建议开发化学文库筛选来鉴定CRAC通道的小分子阻滞剂。为了使用高通量筛选确定Orai1的特异性阻滞剂，我们在主筛选中测试了超过85,000种化合物。强有力的候选人在二次筛选中得到验证。短期特异性目的是：(1)验证Orai1阻滞剂的特异性和有效性。为了确定阻滞剂的特异性和有效性，候选化合物的有效剂量将在T细胞中确定。第三阶段的筛选工作将利用全细胞膜片夹持和全内反射荧光显微镜来了解候选药物抑制的分子机制。(2)在临床相关动物模型上检验候选阻滞剂的免疫抑制效果。为了测试候选分子的治疗效力，我们计划用动物模型确定注射候选化合物是否能改善自身免疫性疾病。CRAC通道小分子阻滞剂的开发将代表一种新的和改进的治疗方法，以抑制器官移植和自身免疫性疾病期间的免疫反应。这种靶向Orai1活性的小分子阻滞剂可能比环孢素A和他克莫司等免疫抑制药物副作用小得多，因为CRAC通道在免疫细胞中特别占主导地位。

DESCRIPTION (provided by applicant): Stimulation of T cell receptors causes depletion of intracellular Ca2+ stores and subsequent opening of the CRAC (Ca2+-release-activated Ca2+) channels. A sustained increase in intracellular Ca2+ concentration activates the calcineurin/NFAT (nuclear factor of activated T cells) pathway and turns on transcriptional programs of various cytokines. Recently, Orai1 and STIM1 were identified as a long-sought pore component of CRAC channels and as an endoplasmic reticulum (ER) Ca2+ sensor, respectively. STIM1 senses Ca2+ depletion in ER after stimulation of T cell receptors, translocates to plasma membrane (PM) proximal ER, binds to and activates Orai1. Human patients deficient in Orai1 or STIM1 have severe combined immune deficiency. However, this deficiency has been rescued by hematopoietic stem cell transplantation, implying an irrevocable role of Orai1 specifically in the immune cells. Thus, a specific blocker of Orai1 is likely to ameliorate the side effects observed with calcineurin blockers. Here, we propose to develop chemical library screens for identification of small molecule blockers for CRAC channels. To identify specific blockers for Orai1 using high throughput screening, we have tested more than 85,000 chemical compounds in a primary screen. Strong candidates were validated in secondary screens. The short-term specific aims are: (1) To validate the specificity and efficacy of Orai1 blockers. To determine specificity and efficacy of the blockers, the efficacious dose of the candidate compounds will be determined in T cells. Tertiary screening efforts will utilize whole-cell patch clamping and total internal reflection fluorescence microscopy to understand the molecular mechanism of inhibition by the candidate drugs. (2) To test the efficacy of immunosuppression by the candidate blockers in clinically relevant animal models. To test the candidate molecule's therapeutic potency, we plan to determine whether the injection of the candidate compounds ameliorate autoimmune diseases using animal models. Development of small molecule blockers of CRAC channels would represent a novel and improved therapeutic approach to suppress immune response during organ transplant and autoimmune diseases. Such small molecule blockers targeting Orai1 activity is likely to have much less side effects than immunosuppressive drugs such as cyclosporin A and tacrolimus because the CRAC channel is specifically predominant in immune cells.