CN104814962B - 4N杂环化合物作为Th17细胞分化抑制剂的应用 - Google Patents
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Abstract
本发明公开了4N杂环化合物作为Th17细胞分化抑制剂的应用，发明人通过基于转录因子活性的荧光素酶活性筛选***，发现通式为
Description
技术领域
本发明涉及化合物的新应用，特别涉及4N杂环化合物作为Th17细胞分化抑制剂的应用。
背景技术
自免疫疾病大约影响了5%的人口，包括牛皮癣，多发性硬发症，风湿性关节炎，哮喘和炎症性肠道病在内的大约70多种人类疾病都和自免疫失调有关(Goodnow et al.,2005)。目前自免疫疾病的治疗主要依赖于一些非选择性的免疫抑制剂，其疗效有限且副作用大，因而在临床中还没有非常特效的治疗自免疫疾病的药物。因此开发新型的高疗效低副作用的自免疫药物成为一种急切的临床需要。最新研究表明一种新的T细胞亚群Th17细胞和人类自免疫疾病和相关动物模型的发生相关(Linden 2006; Kikly et al., 2006)。在风湿性关节炎、牛皮癣、哮喘、炎症性肠炎等自免疫疾病中，Th17细胞的特征细胞因子IL17表达上调，而抑制IL17表达和Th17细胞形成可以减轻自免疫疾病的发生或临床严重程度(Bowman et al., 2006;Kikly et al., 2006)。现有研究表明Th17细胞的分化形成受到转录因子RORγt的控制，而RORγt基因敲除的小鼠中Th17细胞的分化能力下降，数量减少，同时诱导发生的小鼠EAE自免疫疾病概率和临床打分指标均有显著的降低(Ivanov etal., 2006)。这意味着RORγt的功能抑制剂可以做为开发Th17介导的自免疫疾病治疗药物的定向靶标。
RORγt是类固醇类核受体家族成员，蛋白分子中包括一个保守的DNA结合域和有12个螺旋构成的配体结合域，而配体结合域是结合配体、核定位和二聚体形成的重要区域。类固醇类受体共激活分子SRCs能够通过结合配体结构域的AF2区域来解聚转录抑制复合物，招募转录激活分子，启动相关基因转录(Glass and Rosenfeld, 2000)。目前RORγt的天然配体还没有找到，但最近两个研究找到了人工合成分子地高辛及其衍生物(Huh etal., 20011; Fujita-Sato et al., 2011)和SR1001(Solt et al., 2011)能够特异性的结合到RORγt的配体结构域，抑制RORγt的功能并降低Th17细胞的分化能力，减弱小鼠自免疫疾病EAE的临床症状。然而地高辛及其衍生物具有很强的毒性(Paula et al., 2005)，而SR1001分子尽管在体外能够有效抑制Th17细胞分化，但在体内实验中即使在高浓度给药下(40 mg/kg)，也只能略微减轻EAE的临床症状。此外，SR1001能够同其他的RORs作用，选择性较差(Solt et al., 2011)。这些研究一方面表明抑制RORγt的功能来治疗自免疫疾病是一个可行的方案，但目前还没有找到一个理想的靶标药物。因此寻找高效低毒且有较强特异性的RORγt功能抑制剂将是开发Th17介导的自免疫疾病药物的一个重要方向。
参考文献：
Bowman EP, Chackerian AA, Cua DJ. Rationale and safety of anti-interleukin-23 and anti-interleukin-17A therapy. Curr Opin Infect Dis 2006;19: 245-52.
Fujita-Sato S, Ito S, Isobe T, Ohyama T, Wakabayashi K, Morishita K,Ando O, Isono F. Structural basis of digoxin that antagonizes RORγt receptoractivity and suppresses Th17 cell differentiation and interleukin (IL)-17production. J Biol Chem 2011; 286: 31409-17.
Glass, C. K., and Rosenfeld, M. G. (2000). The coregulator exchangein transcriptional functions of nuclear receptors. Genes Dev 14, 121-141
Goodnow CC, Sprent J, Fazekas de St Groth B, Vinuesa CG. Cellular andgenetic mechanisms of self tolerance and autoimmunity. Nature 2005; 435: 590-7.
Huh JR, Leung MWL, Huang PX, Ryan DA, Krout MR, Malapaka RRV, Chow J,Manel N, Ciofani M, Kim SV, Cuesta A, Santori FR, Lafaille JJ, Xu HE, Gin DY,Rastinejad F, Littman DR. Digoxin and its derivatives suppress TH17 celldifferentiation by antagonizing RORγt activity. Nature 2011; 472: 486-90.
Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ,Cua DJ, Littman DR. The orphan nuclear receptor RORgammat directs thedifferentiation program of proinflammatory IL-17+T helper cells. Cell 2006;126: 1121-33.
Kidly K, Liu L, Na S, Sedgwick JD. The IL-23/Th(17) axis: therapeutictargets for autoimmune inflammation. Curr Opin Immunol 2006; 18: 670-5..
Paula S, Tabet MR, Ball WJJ. Interactions between cardiac glycosidesand sodium/potassium-ATPase: three-dimentional structure-activityrelationship models for ligand binding to the E2-Pi form of the enzyme versusactivity inhibition. Biochemistry 2005; 44: 498-510.
Solt LA, Kumar N, Nuhant P, Wang YJ, Lauer JL, Liu J, Istrate MA,Kamenecka M, Roush WR, Vidović R, Schűrer SC, Xu JH, Wagoner G, Drew PD,Griffin PR, Burris TP. Suppression of Th17 differentiation and autoimmunityby a synthetic ROR ligand. Nature 2011; 472: 491-4。
发明内容
本发明的目的在于提供4N杂环化合物作为Th17细胞分化抑制剂的应用。
发明人通过基于转录因子活性的荧光素酶活性筛选***，发现4N杂环连苯化合物具有抑制Th17细胞分化的关键转录因子RORγt的能力，抑制能力EC50在0.2-1.5μM之间，在利用体外Th17细胞分化实验验证这些化合物具有抑制Th17分化的的功能，包括能够显著降低RORγt的转录表达，能够抑制Th17的效应分子IL17A和IL17F的转录表达，IL17A细胞因子在培养物中的分泌水平也得到明显抑制，有望开发成为治疗自免疫疾病，特别是牛皮癣，多发性硬发症，***性红斑狼疮，风湿性关节炎，哮喘或炎症性肠道病的药物。
4N杂环化合物的通式为，式中，R1、R2独立为H或C1～C3的饱和烃基，位于苯环1～5位中的两个上；R3为含有至少一个N的杂苯并二环基团。
特别的，上述通式所示的化合物中，R3选自：
、、、或，
式（2）、式（3）或式（6）中，苯环上的1’～3’中的一个C被N取代，或1’和3’的C同时被N取代；
R4、R5独立选自C～C4的饱和烃基；
R6选自H、C1～C3的饱和烃基；
X为卤素；
Y1和Y2中的至少一个为N。
特别的，上述通式所示的化合物中，R4、R5独立选自C2或C3的饱和烃基。
式（6）中的X为Cl。
式（2）、式（3）中，1’和3’位的C被N取代；
式（4）、式（5）中，Y1和Y2同时为N，R6为甲基，R4、R5同时为乙基；
式（6）所示的基团具体为。
如（化合物7）的EC50活性为1.503μM，（化合物11）的EC50活性为0.257μM，（化合物14）的EC50活性为0.679μM。
附图说明
图1是不同化合物浓度与相对荧光强度之间的关系图；
图2是不同化合物对转录因子RORγt mRNA表达量的影响结果；
图3是不同化合物对IL17A细胞因子分泌量的影响图。
具体实施方式
本申请所述的4N杂环化合物连苯化合物，如化合物7、11和14均可以购自Enamine公司，其他类似的化合物可以购买得到的化合物的基础之上，应用现有技术进行简单的修饰得到或直接委托合成机构合成。
以下实验中，化合物7、化合物11和化合物14分别指、和。
基于转录因子活性的荧光素酶活性筛选***可以按现有方法构建。或按下述的方法构建得到：
材料：
细胞系 Jurkat和293T为本实验室保存；DH5α细菌菌株由北京中科院遗传所马润林教授馈赠；限制性内切酶购自美国fermentas公司；DNA连接酶购自美国的NEB公司；报告基因序列IRES-GFP为发明人自有保存（也可以采用其他现有的报告基因序列）；报告质粒pGL4.31[luc2P/GAL4UAS/Hygro] 质粒和pBIND质粒购自美国promega公司；DMEM培养基和RPMI1640培养基以及培养细胞用的丙酮酸钠、谷氨酰胺、β巯基乙醇、非必须氨基酸、双抗是购自美国life公司；胎牛血清（FBS）购自美国hyclone公司；荧光素酶报告基因检测试剂盒购自美国Promega 公司；Lipo2000 购自美国 life公司；无内毒素质粒提取试剂盒购自Sigma 公司；8-12周龄的C57小鼠购自中山大学实验动物中心（SPF级）；T细胞电转试剂盒和电穿孔仪购自瑞士的lonza公司；潮霉素B购自瑞士Roche公司；逆转录试剂盒购自大连Takara公司；Realtime检测试剂盒购自美国promega公司；细胞因子hTGF-β和mIL-6购自美国RD公司；小鼠CD3和CD28抗体购自美国eBioscience公司；小鼠IL-17A的ELISA检测试剂盒购自武汉华美公司；其他常用化学试剂购自 Sigma公司和上海生工公司。
方法：
质粒构建：
将荧光蛋白基因IRES-GFP目的片段通过Not1单酶切位点***到pBIND载体中完成pBIND-IRES-GFP的质粒构建；用PCR法从PBMC的cDNA中调取人的RORγt（hRORγt）目的基因，hRORγt调取时两端加上BamH1的单酶切位点；再将hRORγt基因克隆到pBIND-IRES-GFP质粒中，完成pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒的构建（Gal4DBD的序列是pBIND质粒自带的）。
细胞培养：
293T细胞培养在含10%FBS（胎牛血清），1%双抗的DMEM完全培养基中；Jurkat细胞培养在含10%FBS（胎牛血清），1%双抗，2mM谷氨酰胺，1mM丙酮酸钠，50μM的β巯基乙醇的RPMI1640完全培养基中。细胞置于5% CO2，37 ℃培养箱中恒温培养，约每两天传代一次。
稳定细胞系的构建：
pGL4.31+ hRORγt+293T稳定细胞系的构建：首先用Not1单酶切质粒pGL4.31[luc2P/GAL4UAS/Hygro]，然后将4μg酶切后的质粒用lipo2000转染至293T（1×106）细胞中，转染后的细胞用100μg/ml的潮霉素B进行抗性筛选；筛选进行2-3周后，再将4ug用限制性内切酶Eam1105酶切的pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒转染（lipo2000）至经潮霉素B筛选后的293T细胞中，再次转染后的细胞继续培养2-3周后，用GFP荧光蛋白标记进行流式分选，并收集GFP+阳性293T细胞。
pGL4.31+ hRORγt+Jurkat稳定细胞系的构建：类似于293T稳定细胞系的构建过程。将20ug经Not1单酶切的质粒pGL4.31[luc2P/GAL4UAS/Hygro]电转入Jurkat细胞（1×107）中，再用200μg/ml的潮霉素B进行抗性筛选；筛选进行2-3周后，再将20ug用限制性内切酶Eam1105酶切的pBIND-Gal4DBD-hRORγt-IRES-GFP重组质粒电转至经潮霉素B筛选后的Jurkat细胞中，再次转染后的细胞继续培养2-3周后，用GFP荧光蛋白标记进行流式分选，并收集GFP+阳性Jurkat细胞。
高通量筛选：
将pGL4.31+ hRORγt+Jurkat细胞铺板至96孔圆底板中，每孔2×104个细胞，5%CO2，37 ℃培养箱中恒温培养过夜，然后将终浓度为50μM化合物（DMSO溶解）加到细胞中，每孔细胞加一种化合物，同时在每块96孔板中的第12竖排加相同量（2μL）的DMSO作为阴性对照，化合物作为加样完成后，将细胞重新放回培养箱中，在5% CO2，37 ℃条件中继续培养6h。6小时后，1500rpm，10min离心后，弃上清，用100ul裂解液裂解细胞后，取20ul上清进行Report assay以检测luciferase活性。
化合物对Th17细胞的体外分化的影响：
实验前一天晚上用5ug/ml的anti-CD3、1ug/ml的anti-CD28的PBS溶液包被六孔板，每孔1ml，4度包被过夜。第二天开展实验，首先用Miltenyi磁珠分选小鼠脾脏的CD4+T细胞，分选后的细胞按1×106个/ml的细胞密度重悬细胞，向包被后的六孔板的每个孔加2ml的细胞悬液，即每孔细胞数为2×106个，激活24h。24h后，收集六孔板中的细胞至50ml离心管中，再向溶液中加入终浓度为5ng/ml的hTGF-β和30ng/ml的mIL-6的细胞因子，混匀，体外诱导CD4+T细胞向Th17细胞分化。混匀后的细胞悬液，按每孔100ul分配至96孔板中，再向每孔加1ul的终浓度为5um化合物（并设置3个孔的DMSO对照组），37度、5%二氧化碳培养48h。48h后，每孔补加100ul RPMI1640完全培养基以及终浓度为5ng/ml的hTGF-β、30ng/ml的mIL-6的细胞因子和1ul的终浓度为5um化合物，补加完成后再培养48小时。至此，CD4+T 细胞体外诱导Th17分化培养了约5天，5天后，分别收集细胞和细胞上清，细胞用于抽提RNA，上清立即保存于-80度冰箱用于ELISA检测IL-17A细胞因子的分泌水平。
cDNA的合成及荧光定量PCR的检测：
Trizol法提取细胞的总RNA，并按逆转录试剂盒的操作说明反转总RNA合成cDNA。然后用合成的cDNA按promega公司的使用说明进行RORγt、IL-17A、IL-17F三个基因的荧光定量PCR检测（以GAPDH基因为内参）。
ELISA检测：
用IL-17A的ELISA试剂盒检测冻存的细胞上清中IL-17A的含量。实验操作严格按照试剂盒操作说明进行。
EC50的测定
EC50的测定基于构建的Gal4/hRORγt的报告体系：将pGL4.31+ hRORγt+Jurkat细胞铺板至96孔圆底板中，每孔2×104个细胞，5% CO2，37 ℃培养箱中恒温培养过夜，然后从5μM到8nM按5倍的浓度梯度设置5个浓度值，在这5个浓度的化合物下作用6h，6h后，收集细胞，进行luciferase活性检测以得到化合物的EC50的值。
实验结果分别如图1～3所示。
图1是不同化合物浓度与相对荧光强度之间的关系图；从图中可以看出，化合物7的EC50值为2.605μM，化合物11的EC50值为0.257μM，化合物14的EC50值为0.679μM。
图2是不同化合物对转录因子RORγt mRNA表达量的影响结果；从图中可以看出，化合物7、化合物11和化合物14均可以有效降低RORγt、IL-17A和IL-17F的表达量。
图3是不同化合物对IL17A细胞因子分泌量的影响图，从图中可以看出，化合物7、化合物11和化合物14均可以有效降低IL-17A细胞因子的分泌量。
Claims (2)
1.4N杂环化合物在制备RORγt抑制剂或自免疫疾病药物的应用，其中，4N杂环化合物选自、或。
2.根据权利要求1所述的应用，其特征在于：自免疫疾病选自牛皮癣，***性红斑狼疮，多发性硬发症，风湿性关节炎，哮喘或炎症性肠道病。
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