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、移植排斥反应、类风湿性关节炎和 已知肺纤维化是由T细胞的病理性激活引起的。虽然T细胞激活是一个关键 这是许多免疫反应的一部分，当T细胞不准确时，这个过程可能会变得病理性 承认病人自己的组织或在组织移植的背景下。而免疫调节剂 包括皮质类固醇和环孢素在内的药物是FDA批准的，这些药物对许多免疫系统起作用 细胞类型，导致广泛的免疫抑制和严重副作用。过去的高吞吐量 筛选鉴定和验证T细胞激活的小分子选择性抑制剂 (SITCAs)在体外和体内发挥作用，而不影响其他细胞的炎症反应 类型。虽然这些分子暗示了新的T细胞选择性免疫调节的潜力 药物，缺乏对其细胞靶标的了解，阻碍了进一步的药物发现努力。 Exportin-1(XPO1)催化数百种蛋白质从细胞核到细胞质的运输，还具有 在调控着丝粒和转录方面起着丝点作用。剧毒天然产物 用来证实阻断XPO1介导的核输出会导致癌细胞死亡， 后来的努力导致FDA批准Selinexor，一种核出口(SINE)的选择性抑制剂，用于 之前至少四次治疗失败的多发性骨髓瘤患者。我们的数据证实了 T细胞激活的选择性抑制剂也针对XPO1，但具有新的药理作用：这些 拮抗剂抑制XPO1‘S在T细胞活化过程中的新作用但对 核出口，细胞毒性大大降低。这些数据表明，XPO1代表了 新型部分拮抗剂有望成为阻断病理性T细胞活化的新靶点 Profile是理想的，以避免与现有XPO1调节剂相关的靶向细胞毒性。 本建议旨在了解和优化XPO1部分拮抗剂在免疫系统中的应用。 媒介疾病。首先，我们试图使用结构和功能分析来理解 结合XPO1相同位点的小分子对细胞表型的影响是不同的 包括核出口和细胞存活率。在目标2中，我们将建立细胞机制， XPO1调节剂阻断T细胞的激活，假设从XPO1的染色质解离， 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 药物化学优化部分拮抗剂图谱，评价主要部分拮抗剂 在T细胞功能的临床前模型中，包括使用人类原代T细胞和在小鼠模型中 已知T细胞在肺纤维化中起作用。总之，这些研究将把XPO1扩展为 通过优化新型XPO1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.