CN114053414A - 一种黄酮类化合物usp22抑制剂在制备抗肿瘤免疫药物中的应用 - Google Patents
一种黄酮类化合物usp22抑制剂在制备抗肿瘤免疫药物中的应用 Download PDFInfo
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Abstract
本发明属抗肿瘤免疫药物应用领域，尤其涉及一种黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用。黄酮类化合物USP22抑制剂的骨架为：
所述黄酮类化合物USP22抑制剂为宝藿苷I、桑辛素或白杨素。该抑制剂可选择性抑制USP22，具有较好的抗肿瘤免疫作用。
Description
技术领域
本发明属抗肿瘤免疫药物应用领域，尤其涉及一种黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用。
背景技术
泛素化是蛋白质的翻译后修饰的过程，它与多种生物活动相关，而去泛素化酶(deubiquitinases，DUBs)是可以逆转泛素化的一类酶，其中最大且异质性最高的一类是泛素特异性蛋白酶(ubiquitin-specific proteases，USPs)。泛素特异性蛋白酶22(ubiquitin-specific protease 22，USP22)属于USPs，作为11个癌症死亡标签之一，它与多种癌症侵袭性生长、转移和治疗耐药性相关。
USP22的表达上调与多种癌症中的发生发展及不良预后相关。USP22被鉴定为人染色质重组复合体即SAGA(Spt-Ada-Gcn5乙酰转移酶)转录调控复合物的保守组分，通过从组蛋白H2A和H2B上去除泛素部分，来诱导基因启动子区域的变化，从而导致转录激活。此外，USP22通过去泛素化作用稳定脱乙酰酶Sirt1，抑制p53的转录激活，进而抑制细胞凋亡。抑制USP22对抗肿瘤治疗具有巨大的治疗潜力。
调节性T细胞(Treg)是近些年免疫学领域比较重要的发现，其介导的免疫逃逸是肿瘤免疫治疗的主要障碍。遗憾的是，目前尚缺乏特异抑制Treg细胞功能的有效手段。叉头盒P3转录因子(Forkhead Box P3，FoxP3)是Treg细胞谱系的标志物，也是其抑制功能的程序调控员。先前研究发现USP22是稳定Foxp3表达的阳性调节因子，是抗肿瘤免疫治疗的重要靶点。另外，USP22可与免疫检查点PD-L1相互作用并增强其稳定性，促使PD-L1脱去泛素链，并防止其被蛋白酶体降解。综上，抑制USP22功能不仅诱导肿瘤细胞直接死亡，而且还可以通过削弱Treg细胞的功能和降低PD-L1的稳定性来增强抗肿瘤免疫。因此，抑制USP2功能具有抗肿瘤和增强肿瘤免疫″一靶双效″的治疗作用。针对特异抑制USP22靶向药物的开发，将为肿瘤的治疗提供新的途径和方向。
USP22小分子抑制剂的研发，主要难点体现在以下几点：
(1)人源化USP22晶体结构难以获取。USP22蛋白水溶性差，纯化困难，至今没有共结晶结构数据报道，给基于靶点结构的药物设计带来了难度。(2)基于酶促反应的高通量筛选方法无法用于该体系。USP22是转录调控组蛋白乙酰化复合物hSAGA(Human Spt-Ada-Gcn5-acetyltransferase)的去泛素化模块的成员。Atanassov等发现USP22需要结合到hSAGA复合体上才能发挥去泛素酶活性，说明USP22单独无催化活性，而是同其他相互作用的蛋白结合形成复合物，才能表现其生物学功能。因此，通过酶活性方法高通量筛选USP22小分子抑制剂难以实现。
迄今为止，尚未见USP22抑制剂的相关报导及其抗肿瘤免疫中的用途。
发明内容
本发明的目的在于提供一种黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用。该抑制剂可选择性抑制USP22，具有较好的抗肿瘤免疫作用。
为解决上述技术问题，本发明是这样实现的：
黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其中，黄酮类化合物USP22抑制剂结构式如下：
黄酮类化合物骨架结构(fIavonoids)。
进一步地，所述黄酮类化合物USP22抑制剂可选择宝藿苷I、桑辛素或白杨素。
进一步地，所述宝藿苷I结构为：
宝藿苷I(Baohuoside I)；
所述桑辛素结构为：
所述白杨素结构为：
白杨素(chrysin)。
进一步地，所述宝藿苷I、桑辛素或白杨素可用以制备抑制结肠癌细胞USP22功能活性药物。
进一步地，所述宝藿苷I、桑辛素可用以制备特异性抑制USP22功能活性药物。
进一步地，所述宝藿苷I、桑辛素或白杨素可用以制备抑制Treg细胞功能活性药物。
本发明黄酮类化合物USP22抑制剂可选择性抑制USP22，具有较好的抗肿瘤免疫作用，可抑制结肠痘细胞USP22功能活性、特异性抑制USP22功能活性及Treg细胞功能活性。表1为实施例1中分子动力学模拟和MM/PBSA结合自由能计算宝藿苷I、桑辛素、白杨素分别与USP22蛋白结合能。
表1.USP22分别与宝藿苷I、桑辛素、白杨素的结合自由能。
附图说明
图1-A、图1-B、图1-C为实施例1中分子对接和分子动力学模拟分析宝藿苷I、桑辛素、白杨素分别与USP22蛋白结合模式，和结合稳定性。
图2-A、图2-B、图2-C为实施例2中Western Blot方法检测宝藿苷I、桑辛素、白杨素抑制USP22功能活性。
图3-A、图3-B为实施例3中Western Blot方法检测宝藿苷I、桑辛素特异性抑制USP22功能活性。
图4为实施例4中流式细胞术方法检测宝藿苷I、桑辛素、白杨素抑制小鼠中Treg细胞的FoxP3表达。
具体实施方式
本发明中宝藿苷I，分子式：C27H30O10，CAS号：113558-15-9，宝藿苷I为小檗科淫羊藿属植物淫羊藿(Epimedium brevicornum Maxim.)中的一种多羟基黄酮类单体成分，有较好的抗肿瘤和抗骨质疏松活性。桑辛素，分子式C25H24O6，CAS号：62596-29-6，桑科植物桑根皮内的主要酚类成分，主要存在于桑白皮黄棕色粗皮中，具有抗肿瘤，抗HIV，抗菌作用。白杨素，分子式：C15H10O4，CAS号：480-40-0，存在于紫葳科植物木蝴蝶的种子、茎皮，松科植物山白松的心木，芒松的心木等，在蜂胶中含量较高。具有抗氧化、抗肿瘤、抗病毒、抗高血压、抗糖尿病、抗菌、抗过敏等广泛药理生理活性。
实施例1
宝藿苷I、桑辛素、白杨素分别与USP22蛋白的分子间相互作用分析
我们利用同源建模方法，构建了人源USP22蛋白结构，依据此结构模型，利用计算机辅助药物设计方法虚拟筛选了天然化合物文库，发现黄酮类化合物具有潜在抑制USP22功能活性的作用。分子对接结果表明，宝藿苷I、桑辛素、白杨素与USP22蛋白受体催化结构域口袋匹配。如图1-A左侧图显示，棍状结构为宝霍苷I的3D结构，宝霍苷I稳定结合在USP22蛋白受体催化结构域口袋中。为了进一步了解宝霍苷I与USP22蛋白是否稳定性结合，我们使用分子动力学模拟方法，分析了宝霍苷I配体结构及USP22蛋白质结构的各原子位置与初始结构位置的RMSD值(root mean square deviations，均方根偏差)，如图1-A右侧图所示，USP22的RMSD在前300ps左右迅速升高，之后保持相对稳态，宝藿苷I在体系中波动相较平缓，表明宝藿苷I与USP22稳定结合。桑辛素和白杨素计算模拟结果与宝藿苷一致(图1-B，图1-C)。进而，分析了这些化合物与USP22的结合能。如表1所示，宝藿苷I、桑辛素、白杨素与USP22的MM/PBSA结合能分别为-244.497、-161.591、-80.486kJ/mol，表明具有较强的结合能力。
图1-A、图1-B、图1-C为实施例1中分子对接和分子动力学模拟分析宝藿苷I、桑辛素、白杨素分别与USP22蛋白结合模式，和结合稳定性。图1-A为宝藿苷I与USP22稳定结合(左)RMSD值(右)。图1-B为桑辛素与USP22结合(左)RMSD值(右)。图1-C为白杨素与USP22稳定结合(左)RMSD值(右)。
实施例2
宝藿苷I、桑辛素、白杨素抑制USP22功能活性
在hSAGA复合物内DUBm通过组蛋白H2A和H2B与核小体结合，研究表明组蛋白H2A、H2B均可被USP22抑制其单泛素化过程，而USP22被抑制后组蛋白H2A、H2B的单泛素化程度会提高。依据宝藿苷I、桑辛素、白杨素对人结肠癌HCT116细胞的IC50值，取对数生长期的人结肠癌HCT116细胞，分别给药20μM、8μM、40μM共培养12小时，培养结束后利用Western Blot方法，对单泛素化的H2A、H2B进行蛋白水平检测。结果如图2-A所示，USP22蛋白表达与对照组比几乎一致，说明化合物不影响USP22蛋白的表达。而单泛素化的H2A(H2Aub1)和H2B(H2Bub1)蛋白表达均上调(图2-A)。
先前的研究表明，USP22可以去泛素化SIRT1来抑制细胞凋亡，当USP22被抑制后，SIRT1泛素化进程加快进而促使其降解。而作为免疫检查点的PD-L1也与USP22相关，USP22可以稳定PD-L1，防止其被蛋白酶体降解，当USP22被抑制后，PD-L1也会随之减少。我们进一步对给药后的HCT116细胞的PD-L1和SIRT1的蛋白水平进行检测。如图2-B所示，结肠癌HCT116细胞加入宝藿苷I(20μM)、桑辛素(8μM)、白杨素(40μM)培养24小时后，PD-L1和SIRT1蛋白表达明显下调。当加入蛋白酶体抑制剂MG132后，消除了化合物处理对PD-L1和SIRT1蛋白水平的影响，表明宝藿苷I、桑辛素、白杨素通过促进其泛素化抑制了PD-L1和SIRT1蛋白水平。
参见图2-A，HCT116细胞分别加入宝藿苷I(20μM)、桑辛素(8μM)、白杨素(40μM)孵育12小时后，H2Aub1、H2Bub1、USP22的蛋白水平变化。参见图2-B，HCT116细胞分别加入宝藿苷I(20μM)、桑辛素(8μM)、白杨素(40μM)共培养24小时后，PD-L1和SIRT1的蛋白水平变化。参见图2-C，细胞中加入宝藿苷I(20μM)、桑辛素(8μM)、白杨素(40μM)培养20小时，加或不加蛋白酶体抑制剂MG1324小时后，PD-L1和SIRT1的蛋白水平变化。
实施例3
宝藿苷I、桑辛素特异性抑制USP22功能活性
为了验证宝藿苷I、桑辛素的促泛素化作用靶点是否为USP22，我们采用了Si-RNA干扰技术沉默USP22表达。作为对比，同时检测了未被Si-RNA干扰的正常给药组细胞H2Bub1的变化，如图3-A所示，未被干扰的细胞给药后对USP22的表达几乎没有影响，而给药组的H2Bub1相较不加药组都有明显提升。如图3-B所示，当用Si-RNA干扰HCT116细胞后，被干扰细胞的H2Bub1相较对照组大幅提高，而宝藿苷I、桑辛素给药组与Si-RNA组相比基本没有进一步促进H2B的单泛素化，所以宝藿苷I、桑辛素的促泛素化作用的原因是抑制了USP22，当细胞内USP22被沉默时，这种作用也就不复存在。由此，可以判定宝藿苷I、桑辛素为USP22特异性抑制剂。尽管白杨素有抑制USP22功能活性的作用，但是由于其药效较差，未对其特异性进行检测，白杨素可作为黄酮类USP22抑制剂骨架结构，值得进一步优化改造。
参见图3-A，HCT116细胞与宝藿苷I、桑辛素共培养12小时后USP22和H2Bub1变化；参见图3-B，Si-RNA干扰的HCT116细胞与宝藿苷I、桑辛素共培养12小时后USP22和H2Bub1变化。
实施例4
宝藿苷I、桑辛素、白杨素抑制Treg细胞FoxP3的表达
先前的研究表明，抑制USP22可以抑制Treg细胞功能从而增强抗肿瘤免疫。为了验证宝藿苷I、桑辛素、白杨素是否抑制Treg细胞的FoxP3蛋白表达，我们提取了小鼠的Treg细胞，分别给予宝藿苷I(20μM)、桑辛素(8μM)、白杨素(40μM)共培养24小时，利用流式细胞技术对各组细胞的FoxP3平均荧光强度(MFI)进行检测。如图4所示，当我们用3种化合物处理小鼠Treg细胞后，其FoxP3的MFI都有一定程度的降低，总体而言宝藿苷I、桑辛素、白杨素均能降低Treg细胞FoxP3的蛋白表达水平，使Treg细胞稳态失衡。综上所述，黄酮类化合物宝藿苷I、桑辛素、白杨素可作为USP22抑制剂，具有抗肿瘤和增强肿瘤免疫“一靶双效”的治疗作用。
参见图4，小鼠Treg细胞分别与宝藿苷I(20μM)、桑辛素(8μM)、白杨素(40μM)共培养24小时后的FoxP3平均荧光强度(MFI)。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (6)
1.一种黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其特征在于，黄酮类化合物USP22抑制剂的骨架为：
2.如权利要求1所述黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其特征在于：所述黄酮类化合物USP22抑制剂为宝藿苷I、桑辛素或白杨素。
3.如权利要求2所述黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其特征在于：所述宝藿苷I结构为：
所述桑辛素结构为：
所述白杨素结构为：
4.如权利要求3所述黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其特征在于：所述宝藿苷I、桑辛素或白杨素用以制备抑制结肠癌细胞USP22功能活性药物。
5.如权利要求3所述黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其特征在于：所述宝藿苷I、桑辛素用以制备特异性抑制USP22功能活性药物。
6.如权利要求3所述黄酮类化合物USP22抑制剂在制备抗肿瘤免疫药物中的应用，其特征在于：所述宝藿苷I、桑辛素或白杨素用以制备抑制Treg细胞功能活性药物。
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