Selective Inhibitors of T Cell Activation Target Exportin-1 at Cys528 to Suppress Pathological T Cell Activation

摘要 多种疾病，包括移植物抗宿主病、移植排斥、类风湿性关节炎和 已知肺纤维化是由T细胞病理性活化驱动的。虽然T细胞活化是关键， 作为许多免疫反应的一部分，当T细胞不准确地 识别患者自身的组织或在组织移植的背景下。虽然免疫调节 包括皮质类固醇和环孢素在内的药物是FDA批准的，这些药物作用于许多免疫系统， 细胞类型，导致广泛的免疫抑制和严重的副作用。过去的高通量 筛选工作鉴定并验证了小分子T细胞活化的选择性抑制剂 在体外和体内发挥功能而不影响其它细胞中炎症反应的SITCA 类型虽然这些分子提示了新的T细胞选择性免疫调节的潜力， 由于缺乏对细胞靶点的了解，进一步的药物发现工作受到阻碍。 Exportin-1（XPO 1）催化数百种蛋白质的核质转运， 在调节着丝粒和转录中的作用。剧毒的天然产物 Leptomycin被用于确定阻断XPO 1介导的核输出导致癌细胞死亡， 后来的努力导致FDA批准了selinexor，一种选择性核出口抑制剂（SINE）， 多发性骨髓瘤患者至少有四种既往治疗失败。我们的数据表明 T细胞活化的选择性抑制剂也靶向XPO 1，但具有新的药理学：这些“部分” 拮抗剂抑制XPO 1在T细胞活化过程中的新作用，但对 核输出，并且细胞毒性大大降低。这些数据表明，XPO 1代表了一种 有希望的新靶点，用于阻断病理性T细胞活化，并且新的部分拮抗剂 期望的是，该特性可避免与现有XPO 1调节剂相关的中靶细胞毒性。 该提案旨在了解和优化XPO 1部分拮抗剂在免疫治疗中的应用。 介导的疾病。首先，我们试图使用结构和功能分析来了解不同的 结合XPO 1相同位点的小分子对细胞表型表现出如此不同的影响 包括核输出和细胞活力。在目标2中，我们将建立细胞机制， XPO 1调节剂阻断T细胞活化，假设XPO 1从染色质解离， NFAT转录因子和其他染色质因子起着核心作用。最后，我们将使用 药物化学，以优化部分拮抗剂特征并评价主要部分拮抗剂 在T细胞功能的临床前模型中，包括使用人原代T细胞和在小鼠模型中， 已知T细胞在其中起作用的肺纤维化。这些研究将共同扩展XPO 1作为一种 通过优化XPO 1的新型部分拮抗剂，治疗晚期癌症患者。

ABSTRACT Multiple diseases, including graft-versus-host disease, transplant rejection, rheumatoid arthritis, and lung fibrosis are known to be driven by pathological activation of T cells. While T cell activation is a key part of many immune responses, this process can become pathological when T cells inaccurately recognize a patient’s own tissues or in the context of tissue transplantation. While immunomodulatory drugs including corticosteroids and cyclosporine are FDA-approved, these agents act on many immune cell types, leading to broad immunosuppression and severe side effects. Past high-throughput screening efforts identified and validated small molecule ‘Selective Inhibitors of T Cell Activation (SITCAs)’ that function in vitro and in vivo without influencing inflammatory responses in other cell types. While these molecules suggested the potential for novel T cell-selective immunomodulatory agents, lack of understanding of their cellular targets prevented further drug discovery efforts. Exportin-1 (XPO1) catalyzes nuclear-to-cytoplasmic transport of hundreds of proteins and also has established roles in regulating the centromere and transcription. The highly toxic natural product Leptomycin was used to establish that blocking XPO1-mediated nuclear export led to cancer cell death, and later efforts led to FDA approval of selinexor, a Selective Inhibitor of Nuclear Export (SINE), for multiple myeloma patients who have failed at least four prior therapies. Our data establish that multiple Selective Inhibitors of T Cell Activation also target XPO1, but with novel pharmacology: these ‘partial antagonists’ inhibit XPO1’s novel role in the T cell activation process but have minimal effects on nuclear export and are substantially less cytotoxic. These data suggest that XPO1 represents a promising new target for blocking pathological T cell activation, and that the novel partial antagonist profile is desirable to avoid on-target cytotoxicity associated with existing XPO1 modulators. This proposal seeks to understand and optimize XPO1 partial antagonists for application in immune- mediated diseases. First, we seek to use structural and functional assays to understand how different small molecules that bind the same site of XPO1 show such divergent effects on cellular phenotypes including nuclear export and cell viability. In Aim 2, we will establish the cellular mechanisms by which XPO1 modulators block T cell activation, with the hypothesis that dissociation from chromatin of XPO1, NFAT transcription factors, and other chromatin factors plays a central role. Finally, we will use medicinal chemistry to optimize the partial antagonist profile and evaluate leading partial antagonists in preclinical models of T cell function, including using human primary T cells and in a mouse model of lung fibrosis in which T cells are known to play a role. Together these studies will extend XPO1 as a therapeutic beyond late-stage cancer patients by optimizing novel partial antagonists of XPO1.