CN114948955A - 靶向环鸟苷酸-腺苷酸合成酶的小分子共价抑制剂及其用途 - Google Patents
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Abstract
一种靶向环鸟苷酸‑腺苷酸合成酶的小分子共价抑制剂及其用途，该小分子共价抑制剂具有如式(1)或式(2)所示结构：
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及靶向环鸟苷酸-腺苷酸合成酶的小分子共价抑制剂及其用途。
技术背景
环鸟苷酸-腺苷酸合成酶(简称为cGAS)是一种细胞DNA感受器，主要通过识别双链DNA(简称为dsDNA)激活环鸟苷酸-腺苷酸合成酶-干扰素基因刺激蛋白(简称为cGAS-STING)信号通路。当有细胞出现不稳定因素导致DNA的异常积累或者病原体通过微生物感染的方式将DNA传递到细胞质中被cGAS识别后，就会激活cGAS-STING信号通路，从而引起先天性免疫反应和炎症的发生。因此，开发靶向cGAS的小分子抑制剂在炎症免疫性疾病治疗中具有重要意义，cGAS抑制剂的研发也已成为炎症免疫性药物研究前沿中的热点。
近些年来，在针对cGAS的研究过程中，已经发现了一些老药新用小分子化合物，如羟氯喹和苏拉明。它们是通过干扰cGAS与dsDNA的相互作用来抑制cGAS的激活，而不是直接干扰cGAS酶活性，而且与dsDNA结合的非特异性相互作用容易导致药物脱靶。后续有研究又陆续发现了几个直接靶向cGAS的小分子抑制剂，但是这些小分子抑制剂中部分在细胞上不具有活性，而活性良好的部分小分子抑制剂只在分子和细胞水平上具有活性。因此目前亟需扩展或探索活性好、选择性高的cGAS小分子抑制剂。
发明内容
有鉴于此，本发明的主要目的之一在于提出靶向环鸟苷酸-腺苷酸合成酶的小分子共价抑制剂及其用途，以期至少部分地解决上述技术问题之一。
为了实现上述目的，作为本发明的一个方面，提供了一种靶向环鸟苷酸-腺苷酸合成酶的小分子共价抑制剂，其具有如式(1)或式(2)所示结构：
作为本发明的另一个方面，提供了一种如上所述的小分子共价抑制剂在制备抑制环鸟苷酸-腺苷酸合成酶的试剂中的用途。
作为本发明的再一个方面，提供了一种如上所述的小分子共价抑制剂在制备治疗炎症免疫性疾病的药物中的用途。
基于上述技术方案可知，本发明的靶向环鸟苷酸-腺苷酸合成酶的小分子共价抑制剂及其用途具有以下有益效果其中之一或其中一部分：
1、本发明公开的小分子共价抑制剂，即式(1)所示的噻唑类化合物和式(2)所示的喹啉类化合物对cGAS蛋白表现出良好的抑制活性，对cGAS介导的信号通路具有选择性。
2、本发明的小分子共价抑制剂具备与靶标作用时间长、药效强等优点，为帮助靶向cGAS的药物研发提供新思路、新用途，具有重要的药物研究价值。
附图说明
图1为本发明实施例1中基于酶活实验发现cGSA小分子共价抑制剂的图；其中A-B为化合物1和2的结构；C-D分别为化合物1和2对cGAS酶活性的抑制率。
图2为本发明实施例2中小分子共价抑制剂与cGAS体外直接结合的图；其中A为化合物1和cGAS蛋白的体外结合；B为化合物2和cGAS蛋白的体外结合。
图3为本发明实施例3中荧光偏振实验排除了化合物1和2与酶活体系中DNA的结合的图。
图4为本发明实施例4中小分子共价抑制剂在细胞水平上的活性测定的图；其中A-C为化合物1和2对由dsDNA刺激引起的炎性因子表达的作用；D为化合物1和2对由Pam3CSK4引起的炎性因子表达的作用。
图5为本发明涉及的cGAS-STING信号通路示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
本发明提供了一个噻唑类化合物和一个喹啉类化合物，可与cGAS共价结合并选择性地抑制cGAS的激活。
具体而言，根据本发明的一些实施例，提供了一种靶向cGAS的小分子共价抑制剂，其具有如式(1)或式(2)所示结构：
通过这两个小分子化合物直接靶向cGAS蛋白，通过抑制cGAS进而避免cGAS-STING信号通路被异常激活，具有高效性和高选择性。
根据本发明的一些实施例，还提供了一种如上所述的小分子共价抑制剂在制备抑制cGAS的试剂中的用途。
在本发明的一些实施例中，抑制cGAS为抑制cGAS介导的cGAS-STING信号通路。
其中，异常激活的cGAS-STING信号通路如图5所示，cGAS识别dsDNA产生环鸟腺苷酸(简称为cGAMP)，cGAMP结合STING促进炎症因子例如IFN-β、CXCL10、IL-6、TNF等的产生。本发明提供的小分子共价抑制剂对cGAS蛋白表现出良好的抑制活性，对cGAS介导的信号通路具有选择性抑制的效果，从而有利于抑制炎症免疫性反应的发生。
根据本发明的一些实施例，还提供了一种如上所述的小分子共价抑制剂在制备治疗炎症免疫性疾病的药物中的用途。
其中，所述炎症免疫性疾病为由cGAS-STING信号通路异常激活而引起。
以下通过具体实施例结合附图对本发明的技术方案做进一步阐述说明。需要注意的是，下述的具体实施例仅是作为举例说明，本发明的保护范围并不限于此。下述实施例中使用的药品或试剂均为市售所得或通过公知的制备方法自制得到。
本发明所用到的主要材料如下：
cGAS质粒为cGAS对应的全长核酸序列(UniProt Q8C6L5-1)，购自苏州泓迅生物科技股份有限公司；大肠杆菌焦磷酸酶(Sigma-Aldrich，I5907)；HT-DNA(Sigma-Aldrich，D6898)；ATP和GTP购自MCE上海皓元生物医药科技有限公司；黑色384孔板(Coming，3575)；细胞培养用96孔板、24孔板、6孔板(Coming)；10cm培养皿(Corning，430293)；淬火溶液50mM乙二胺四乙酸(Sigma-Aldrich，03609)；孔雀绿溶液(Sigma-Aldrich，213020)；5×SYPROorange(Invitrogene，S6651)；高糖DMEM培养基(BasalMedia，L110KJ)；胎牛血清(Gibco，10099141C)；双抗(Gibco，2321118)购自上海紫骥生物科技有限公司；Polyjet DNA转染试剂(SignaGen Laoratories，SL100688)；RNA提取试剂(Vazyme，R701-01)；4×gDNA wiperMix(Vazyme，R323-01-AB)；ChamQ SYBR qPCRMaster Mix(Vazyme，Q331-AA)。
本发明涉及到的数据采用Graphpad 8.4.2进行数据分析，利用student’s t-test进行统计分析，各组之间的统计量用(mean±SD)表示，P＜0.05时认为两组之间具有显著性差异。
实施例1通过cGAS酶活实验进行筛选发现了cGAS抑制剂
实验方法：酶活反应总体系的体积为40μL，其中化合物、cGAS、大肠杆菌焦磷酸酶、HT-DNA、ATP和GTP的终浓度分别为200μM、200nM、50nM、5μg/mL、1mM和300μM。将384孔板中的混合物3000r/min离心30s，紧接着室温下孵育90min。用40μL的淬火溶液(50mM乙二胺四乙酸)和20μL孔雀绿溶液停止反应。接下来孔板中的混合物室温孵育10min。测量各孔在620nm处的吸光度，并与对照组进行比较。将2’，3’-cGAMP的生成与DMSO阳性对照和缺少cGAS蛋白的阴性对照进行归一化处理：抑制率％＝1-(Abs样本平均值-Absnegative conntrol平均值)/(Abspositive control平均值-Absnegative control平均值)。
实验结果：如图1C-D所示，酶活筛选得到的小分子化合物1和2可以显著抑制cGAS酶的活性，实验测得抑制率的IC50分别为4.85μM和3.01μM。
实施例2化合物与cGAS体外直接结合，化合物可提高cGAS蛋白热稳定性
实验方法：用实时荧光定量PCR仪检测cGAS蛋白的热稳定性。先用分子筛缓冲液配制终浓度为1.25mM的cGAS蛋白和5×SYPRO orange(Invitrogene，S6651)的混合液，混匀后加入96孔无裙边PCR板(DN Biotech)中，每孔19μL，再分别加入1μL化合物(12.5mM)，封板后1000rpm离心1min，放入PCR仪启动程序，在25～90℃内监测和采集荧光信号，用蛋白质热迁移软件(Bio-Rad)测定cGAS蛋白的熔解温度(Tm)值。
实验结果：如图2A-B所示，小分子化合物1和2增强了cGAS蛋白的热稳定性，使cGAS的Tm值分别提高了3.5℃和5.5℃，该结果证明化合物1和2与cGAS蛋白有直接结合。
实施例3荧光偏振实验排除了化合物与酶活体系中DNA的结合
实验方法：实验在384孔黑板上进行，每孔反应总体积为40μL。每孔含有10μL的HEN缓冲液(10mM HEPES pH＝7.5，1mM EDTA pH＝7.5，100mM NaCl)，10μL的150nM吖啶橙溶液，10μL的HT-DNA溶液(37.5μg/mL)，10μL的50μM化合物。以已知的DNA嵌入剂米托蒽醌(50μM)作为阳性对照，以DMSO代替化合物作为阴性对照。加好后的黑板在室温下避光孵育30min。1000rpm离心甩板1min后在多功能酶标仪上测量所有荧光偏振实验(FP)的荧光偏振值mP，激发波长为485nm，发射波长为535nm。
实验结果：如图3所示，吖啶橙荧光偏振法实验结果显示，与阳性对照米托蒽醌和对照组DMSO的mP值比较得出结论，化合物1和2不能够插入DNA。结果证明小分子化合物1和2抑制cGAS酶活性并非是由于插入dsDNA作用导致的。
实施例4化合物选择性抑制cGAS介导的信号通路
实验方法：把Raw 264.7细胞平均种在六孔板中，过夜培养。第二天先用10μM化合物或者DMSO预处理细胞1h，然后分别用不同配体刺激细胞：①用PolyJet转染1μg/mL dsDNA刺激细胞4小时；②Pam3CSK4是一个模拟细菌分泌物的TLR2/TLR1配体，可以促进转录因子NF-κB的有效激活，我们用1μg/mL Pam3CSK4刺激细胞18小时。之后提取细胞RNA，进行RT-qPCR实验。
实验结果：如图4A-C所示，化合物1和2可以抑制由双链DNA(ISD)刺激引起Ifnb1、Cxcl10和Il6等炎症因子的表达，但是并不能抑制由Pam3CSK4引起的Tnf的表达(如图4D所示)，表明化合物1和2可以特异性抑制由cGAS激活引起的炎症因子的表达。总的来说，即化合物1和2只能够抑制dsDNA引起的刺激，不能抑制其他的免疫原性刺激，具有选择性。
从上述实施例可以看出，本发明提供的化合物1和2直接靶向cGAS，在分子和细胞水平上均表现出对cGAS良好的抑制活性。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种靶向环鸟苷酸-腺苷酸合成酶的小分子共价抑制剂，其具有如式(1)或式(2)所示结构：
2.一种如权利要求1所述的小分子共价抑制剂在制备抑制环鸟苷酸-腺苷酸合成酶的试剂中的用途。
3.根据权利要求2所述的用途，其中，所述抑制环鸟苷酸-腺苷酸合成酶为抑制环鸟苷酸-腺苷酸合成酶-干扰素基因刺激蛋白信号通路。
4.一种如权利要求1所述的小分子共价抑制剂在制备治疗炎症免疫性疾病的药物中的用途。
5.根据权利要求4所述的用途，其中，所述炎症免疫性疾病为由环鸟苷酸-腺苷酸合成酶-干扰素基因刺激蛋白信号通路异常激活而引起。
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