摘要
病原微生物的入侵和细胞受损导致细胞质中DNA异常聚集,环化核苷酸合成酶(cGAS)通过识别细胞质中的DNA,催化生成第二信使2',3'-cGAMP,将信号传递给下游干扰素基因刺激因子(STING),诱导转录因子IRF3和NF-κB入核,表达和分泌Ⅰ型干扰素等炎症因子,进而激活机体固有免疫和适应性免疫反应。cGAS-STING信号通路调控紊乱将导致病原体感染,以及肿瘤和自身免疫疾病等多种疾病发生和发展,因此靶向cGAS和STING蛋白进行的药物开发具有十分重要的临床价值。本文讨论cGAS-STING信号通路的最新研究进展以及其在不同疾病中发挥的功能,并总结目前已报道的调节cGAS和STING的小分子化合物,为后续相关的药物研发提供理论参考。
固有免疫系统是人体免疫系统的第一道防线,可以迅速识别外源病原体入侵,并且诱导巨噬细胞和自然杀伤细胞对病原体进行吞噬和降解,同时释放大量干扰素和炎症因子,促进树突状细胞对抗原的递呈作用,进而激活适应性免疫,形成长期的免疫记
环化核苷酸合成酶 (cyclic GMP-AMP synthase, cGAS) 就是一种位于细胞质中的dsDNA识别受体,和下游主要调节蛋白干扰素基因刺激因子(stimulator of interferon genes, STING) 介导了细胞质DNA引起的固有免疫反应。DNA-cGAS-STING信号通路的激活促进细胞内Ⅰ型干扰素 (interferons, IFNs) 与肿瘤坏死因子α (tumor necrosis factor, TNF-α) 和白介素6 (interleukin 6, IL-6) 等炎症因子的表达和分
人源cGAS由522个氨基酸组成,相对分子质量大小为60 kD,位于N末端的130-155 aa为非保守序列,功能研究尚不明确,而155-522 aa构成了cGAS的C末端核酸转移酶结构域,含有一个位于中心的催化区域和分散在两侧的阳离子表面区

图1 环化核苷酸合成酶(cGAS)和干扰素基因刺激因子(STING)的蛋白结构
A: cGAS-dsDNA-2',3'-cGAMP复合物结构(PDB code: 4LEZ); B: 左边为静息状态下STING C末端结构域(CTD区域)的蛋白结构(PDB code: 4EMU),右边是STING结合2',3'-cGAMP的蛋白结构(PDB code: 4KSY),结合2',3'-cGAMP后,STING CTD区域发生构象变化,形成特殊的“盖子”结构
2',3'-cGAMP在与下游受体蛋白STING结合后完成信号的传

图2 cGAS-STING信号通路示意图
外源DNA和内源DNA激活cGAS, 生成第二信使2',3'-cGAMP。2',3'-cGAMP与下游受体蛋白STING结合后,引起STING向高尔基体转移,并发生多聚化,招募TBK1,转录因子IRF3和NF-κB,促进IRF3和NF-κB磷酸化和二聚化入核,激活炎症因子相关基因转录。TBK1: TANK结合激酶; IRF3: 干扰素调节因子3; NF-κB: 核因子κB; IFNB: 干扰素β基因; TNFA: 肿瘤坏死因子α基因; IL6: 白介素6基因
在机体的抗病毒、抗细菌方面,cGAS-STING信号通路发挥着重要作用。但是细菌和病毒已经进化出一系列逃逸机制,抑制了固有免疫信号通路的激活。Su
cGAS-STING信号通路通过促进Ⅰ型IFNs,TNF-α和IL-6等炎症因子表达,一方面能诱导细胞产生大量干扰素刺激基因 (interferon-stimulated genes,ISGs),而另一方面能促进树突细胞向T细胞呈递抗原,加强抗肿瘤免
机体内STING信号通路的持续异常激活能导致多种自身免疫疾病。第一类是因基因突变导致细胞内核酸代谢酶功能异常,比如TREX1, RNASEH2A, SAMHD1等基因发生突变,促进DNA在细胞质中的聚集,诱导炎症细胞因子的持续释放,导致自身免疫疾病发
除了上述疾病外,近年来人们发现越来越多的代谢与炎症疾病与cGAS-STING信号通路调控密切相关。在急性胰腺炎模型中,坏死组织泄露的外源DNA会诱导胰腺中的巨噬细胞分泌大量炎症因子,造成组织损伤,而这一现象在cGAS和STING基因敲除小鼠中均有改
KO: 基因敲除; NA: 无法获取; DMBA: 二羟甲基丁酸; SAVI: 诱发于婴儿时期的STING相关血管病变; RPE: 视网膜色素上皮细胞; ALD: 酒精性肝疾病; MCD: 蛋氨酸胆碱缺乏饮食; HFD: 高脂饮食
鉴于cGAS-STING信号通路在疾病的发生发展中发挥的重要作用,而且一些免疫佐剂如铝盐、明矾和壳聚糖等能够通过诱导细胞死亡或线粒体损伤导致细胞质内的DNA聚集进而激活cGAS-STING信号通路,促进机体的免疫功
目前,直接靶向cGAS的激动剂还没有报道,虽然一些金属离子(例如锰离子、锌离子等)可以增加cGAS的酶活,但其所需的有效浓度过高且易在体内产生毒性不利于后续药物研
除了全新发现的小分子抑制剂,一些已经上市的药物也被发现有类似的功能。An
综上,现有针对cGAS酶活区域的结构或者cGAS/DNA复合物的结构进行设计筛选的化合物具有较弱的生物活性,所以建立起一个具有高通量、高灵敏度的检测平台,获得更加全面的蛋白质结构数据,将极大促进靶向cGAS的小分子研究进程。而且,越来越多的数据证明,通过影响蛋白质翻译后修饰过程,可以有效调控蛋白质介导的信号转导,所以开发靶向蛋白翻译后修饰的化合物也是未来的研究方向。
由于STING信号通路的激活促进下游Ⅰ型 IFNs的表达在抗病毒免疫和抗肿瘤免疫中发挥着重要的作用,所以靶向STING蛋白激动剂开发一直是该领域的研究热点。STING激动剂的研究主要集中在环化核苷酸(cyclic dinucleotides, CDNs)类似物的优化和新型小分子激动剂的筛选。CDNs最早在细菌中发现,是由腺嘌呤核糖核苷酸(adenosine monophosphate, AMP)和鸟嘌呤核糖核苷酸 (guanosine monophosphate, GMP)通过经典磷酸二脂键连接形成的环化二鸟嘌呤c-di-GMP和环化二腺嘌呤c-di-AMP或者cGAMP。由cGAS催化生成的2',3'-cGAMP是哺乳动物体内唯一已知的CDNs。相对于3',3'-cGAMP和其他的CDNs,2',3'-cGAMP和STING具有更强的亲和力,在解离常数 (Kd)方面,比c-di-GMP,3',2'-cGAMP和3',3'-cGAMP低99%,比2',2'-cGAMP低98

图3 天然环化核苷酸化学结构式
CDNs可以在肿瘤微环境中促进CD
it:瘤内注射; iv: 静脉注射; ip: 腹腔注射; po:口服;SITC 2018:肿瘤免疫治疗学会2018年度会议; AACR 2017:美国肿瘤研究协会2017年肿瘤免疫和免疫治疗会议; PD-1:程序性死亡蛋白1; ENPP1:核苷酸内焦磷酸酶/磷酸二酯酶1; TREX1:脱氧核糖核酸酶3
近年来研究发现STING的异常激活导致的免疫功能紊乱能诱发AGS、SLE和SAVI等自身免疫疾病,这也使得研究者开始重视STING抑制剂的开发。Merck公司的Siu
除了上述靶向STING蛋白CBD区域的小分子抑制剂外,靶向抑制STING N端跨膜区内半胱氨酸的棕榈酰化修饰的小分子抑制剂也逐渐成为研究热点。Mukai

图4 cGAS-STING信号通路抑制剂
作为固有免疫系统的重要组成部分,cGAS-STING信号通路在维持机体的稳态过程中发挥着重要作用。相应的cGAS-STING功能的紊乱导致机体免疫功能的异常会诱发包括肿瘤、自身免疫疾病在内的多种疾病。迄今为止,研究人员通过多种筛选方法发现了多种新型的靶向调控cGAS或STING蛋白功能的小分子激动剂或抑制剂,且这些化合物在临床前的研究中也表现出很好的生物学活性。但是,研究者也发现了现有小分子中存在的一些问题,例如cGAS的抑制剂抗疟疾药物衍生物的细胞毒性,STING激动剂CDNs类似物的细胞通透性差且易被降解,靶向STING蛋白半胱氨酸的共价抑制剂具有脱靶作用以及在筛选化合物过程中发现的种属特异性问题等。因此,靶向cGAS和STING的新颖激动剂和抑制剂依然有待开发,筛选出具有更好生物活性的先导化合物,为临床研究提供更多的候选分子。
靶向调控cGAS-STING信号通路的小分子化合物在多种疾病的治疗中都具有显著疗效。比如在小鼠多种肿瘤模型中,STING激动剂与PD-1 (programmed cell death protein 1, PD-1) 或者PD-L1 (programmed cell death 1 ligand 1, PD-L1) 抑制剂联用能协同促进机体的抗肿瘤免疫且在体内能形成长期的免疫记忆,有效地控制肿瘤复发;在自身免疫疾病治疗中,AGS和SAVI等罕见病并没有有效的治疗药物,临床上目前以使用Janus激酶抑制剂为主控制疾病进展,与其相比靶向cGAS或STING的抑制剂不仅能控制Ⅰ型IFNs诱导的组织损伤,还能控制TNF-α和IL-6等炎症因子导致的组织炎症。此外靶向STING蛋白的抑制剂也能改善STING异常激活导致的T, B细胞和内皮细胞等多种细胞死
此外,在开发调控cGAS-STING信号通路的激动剂或抑制剂时,其潜在的药物不良反应值得关注。比如在利用STING激动剂进行肿瘤免疫治疗,很可能爆发“免疫风暴”,过度激活的免疫系统导致机体损伤甚至死
越来越多的研究表明,除了发挥固有免疫功能外,cGAS和STING蛋白还能在疾病中独立发挥作用。比如:当DNA发生损伤时,cGAS可以入核干扰DNA同源重组修复过程,促进肿瘤生长,而这一过程不依赖于STING;在白血病中,激活STING将通过IRF3,p53或线粒体介导的细胞凋亡信号通路,促进肿瘤细胞凋
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