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Ca 2+储存耗尽，随后打开CRAC（Ca 2+释放激活的Ca 2+）通道。细胞内Ca 2+浓度的持续增加激活钙调磷酸酶/NFAT（活化T细胞的核因子）途径并开启各种细胞因子的转录程序。最近，Orai 1和STIM 1被确定为CRAC通道的长期寻找的孔组件和内质网（ER）Ca 2+传感器，分别。刺激T细胞受体后，STIM 1感知ER中的Ca 2+耗竭，易位至ER近端的质膜（PM），结合并激活Orai 1。缺乏Orai 1或STIM 1的人类患者具有严重的联合免疫缺陷。然而，这种缺陷已通过造血干细胞移植得到补救，这意味着Orai 1特别在免疫细胞中具有不可撤销的作用。因此，Orai 1的特定阻滞剂可能会改善钙调磷酸酶阻滞剂观察到的副作用。在这里，我们建议开发化学库筛选用于鉴定CRAC通道的小分子阻断剂。为了使用高通量筛选来鉴定Orai 1的特异性阻断剂，我们在初步筛选中测试了超过85，000种化合物。强有力的候选人在二次筛选中得到验证。近期具体目标为：（1）验证Orai 1阻滞剂的特异性和有效性。为了确定阻断剂的特异性和功效，将在T细胞中确定候选化合物的有效剂量。三级筛选工作将利用全细胞膜片钳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中检测候选阻断剂的免疫抑制疗效。为了测试候选分子的治疗效力，我们计划使用动物模型确定候选化合物的注射是否改善自身免疫性疾病。开发CRAC通道的小分子阻断剂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应答。这种靶向Orai 1活性的小分子阻断剂可能比免疫抑制药物如环孢菌素A和他克莫司具有少得多的副作用，因为CRAC通道在免疫细胞中特别占主导地位。 公共卫生相关性：该提案的长期目标是开发CRAC通道的特异性小分子阻断剂，以抑制免疫反应。这种靶向Orai 1活性的小分子阻断剂可能比免疫抑制药物如环孢菌素A和他克莫司具有更少的副作用，因为CRAC通道主要在免疫细胞中表达。开发CRAC通道的小分子阻断剂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应答。

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. PUBLIC HEALTH RELEVANCE: The long-term goal of this proposal is to develop a specific small molecule blocker for CRAC channels to suppress immune responses. 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 predominantly expressed in immune cells. 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.