CN117567385B - 一种靶向clec5a的小分子抑制剂及其应用 - Google Patents
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Abstract
本发明提供了一种靶向CLEC5A的小分子抑制剂及其应用，小分子化合物技术领域。在本发明中，所述抑制剂为：0104‑0071和1869‑0197。本发明提供的小分子抑制剂对CLEC5A蛋白显示出优异的体外亲和力，对HBV复制的抑制和干扰素的诱导有很强的作用以及低细胞毒性，靶向CLEC5A的小分子抑制剂C4治疗持续降低血清HBsAg和HBV DNA以及肝内HBcAg的水平，同时增加血清HBsAb的阳性率和水平，以及血清Ifnα和Ifnβ的水平。
Description
技术领域
本发明涉及小分子化合物技术领域，特别是涉及一种靶向CLEC5A的小分子抑制剂及其应用。
背景技术
髓系C型凝集素5A(C-type lectin domain containing 5A，CLEC5A)也称为髓系DAP12相关凝集素1(MDL-1)，是一种II型跨膜受体，主要在单核细胞、巨噬细胞和中性粒细胞中表达。CLEC5A的短胞质结构域与含有ITAM的跨膜接头DAP-12相互作用，并导致DAP-12磷酸化，随后通过Syk激酶传递信号。CLEC5A不具有完整的Ca2+配位和碳水化合物结合位点，在细胞表面呈现同二聚体结构。
到目前为止，CLEC5A已被证明能够识别多种病毒病原体，包括登革热病毒(Denguevirus，DV)、日本脑炎病毒(Japanese encephalitis virus，JEV)和流感病毒。CLEC5A与DV结合并激活信号级联，同时释放促炎性细胞因子有助于DV感染患者的病理生理变化。CLEC5A识别DV后，DAP-12被磷酸化，导致Syk活化；随后诱导促炎细胞因子IL-1β和IL-18，激活NLRP3炎症小体和caspase-1，最终触发细胞焦亡。此外，DV可以上调CLEC5A的表达，进而激活小鼠单核吞噬细胞中的核因子Nrf2，导致增强肿瘤坏死因子(TNF)-α的生成。抗体介导的CLEC5A阻滞剂减弱了由DV感染的巨噬细胞产生的促炎性细胞因子，这表明CLEC5A靶向可以改善组织损伤。CLEC5A与骨髓源性破骨细胞中的DAP-12和DAP-10结合后，在破骨细胞生成和骨重塑中也是必不可少的。破骨细胞是多核巨细胞，与巨噬细胞不同，参与骨重塑。破骨细胞的DV感染最近被证明可以上调溶骨活性。
在Clec5a-/-小鼠中观察到DV诱导的骨溶解活性的减弱，并且在野生型小鼠中持续给予Clec5a拮抗剂也抑制了DV激活的骨溶解活性。JEV与CLEC5A结合并在巨噬细胞中诱导DAP-12磷酸化。抗体介导的CLEC5A阻滞剂抑制了JEV诱导的小胶质细胞促炎细胞因子的释放，并防止了周围组织对神经细胞的损伤。此外，CLEC5A抗体治疗可减少含有白细胞的JEV向中枢神经系统的渗透，减轻神经炎症，并降低JEV诱导的小鼠致死率。在最近的一项研究中，证实CLEC5A与流感病毒的血凝素(hemagglutinin，HA)蛋白相互作用。抗体介导的CLEC5A阻断或CLEC5A沉默导致人类巨噬细胞产生的促炎性细胞因子水平降低。与野生型小鼠相比，Clec5a-/-小鼠的促炎性细胞因子水平降低，肺部免疫细胞浸润减少，生存率提高，这表明CLEC5A在炎症反应中起着关键作用，从而有助于流感的致病性。然而，靶向CLEC5A的小分子药物还未见报道。
发明内容
为了解决上述问题，本发明提供了一种靶向CLEC5A的小分子抑制剂及其应用，本发明通过数据库分析发现能与CLEC5A结合的小分子化合物；然后，通过SPR等实验验证小分子化合物与CLEC5A的结合；最后，通过功能验证确认小分子化合物的效果。
为了实现上述目的，本发明提供如下技术方案：
本发明提供了一种靶向CLEC5A的小分子抑制剂，所述小分子抑制剂的结构为如下结构中的任一种：
本发明还提供了上述技术方案所述的小分子抑制剂在制备治疗乙肝药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备抑制乙肝病毒复制药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备诱导干扰素药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备降低乙肝病毒感染血清中HBsAg含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备降低乙肝病毒感染血清中HBV DNA含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备降低乙肝病毒感染肝脏内HBcAg含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备增加乙肝病毒感染血清中HBs Ab含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备增加乙肝病毒感染血清中Ifnɑ含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备增加乙肝病毒感染血清中Ifnβ含量药物中的应用。
本发明的有益效果为：
本发明提供的小分子抑制剂对CLEC5A蛋白显示出优异的体外亲和力，对HBV复制的抑制和干扰素的诱导有很强的作用以及低细胞毒性，靶向CLEC5A的小分子抑制剂C4治疗持续降低血清HBsAg和HBV DNA以及肝内HBcAg的水平，同时增加血清HBsAb的阳性率和水平，以及血清Ifnα和Ifnβ的水平。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为筛选靶向CLEC5A的小分子化学抑制剂的流程；
图2为5个小分子化学的化学式及与与固定化CLEC5A蛋白的多种浓度的结合曲线；
图3为功能筛选靶向CLEC5A的小分子抑制剂；
图4为靶向CLEC5A的小分子抑制剂C2和C4的CC50和IC50值；
图5为靶向CLEC5A的小分子抑制剂C4小鼠体内功能确定；
图6为确定靶向CLEC5A的小分子抑制剂C4与CLEC5A的结合。
具体实施方式
本发明提供了一种靶向CLEC5A的小分子抑制剂，所述小分子抑制剂的结构为如下结构中的任一种：
本发明还提供了上述技术方案所述的小分子抑制剂在制备治疗乙肝药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备抑制乙肝病毒复制药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备诱导干扰素药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备降低乙肝病毒感染血清中HBsAg含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备降低乙肝病毒感染血清中HBV DNA含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备降低乙肝病毒感染肝脏内HBcAg含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备增加乙肝病毒感染血清中HBs Ab含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备增加乙肝病毒感染血清中Ifnɑ含量药物中的应用。
本发明还提供了上述技术方案所述的小分子抑制剂在制备增加乙肝病毒感染血清中Ifnβ含量药物中的应用。
为了进一步说明本发明，下面结合实施例对本发明进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
实施例1
实验方法及结果：
(1)高通量筛选靶向CLEC5A的小分子抑制剂
首先，本发明使用计算机辅助高通量虚拟筛选来筛选靶向CLEC5A的小分子化学抑制剂，根据如图1所示的步骤，CLEC5A(PDB ID:2YHF)的晶体结构从RCSB蛋白质数据库获得(https://www.rcsb.org)。通过Discovery Studio 2020软件(BIOVIA，USA)中的LibDock程序，使用包含1539916种化合物的小分子文库(通过Lipinski的无规则过滤)，包括市售文库(例如ChemDiv和ChemBridge)和自建文库，对CLEC5A蛋白进行分子对接，确定了36582个候选化合物。然后，根据LibDockScore的值对前2000个小分子候选进行排序，并进行灵活的对接程序。然后，通过表面等离子体共振(SPR)分析筛选了根据CDOCKER_INTERACTION_ENERGY值排名的柔性对接中的前200个化合物，以测试它们与CLEC5A蛋白的直接相互作用。最后，五种化合物(C1-C5，分别为0242-0318、0104-0071、0242-0435、1869-0197和1262-0251，分子结构见图2)对CLEC5A蛋白显示出优异的体外亲和力，Kd值<1μM，并进行了随后的功能测试(见图2)。
(2)功能筛选靶向CLEC5A的小分子抑制剂
为了在体外测试五种化合物的功能作用，用PMA将THP-1诱导为巨噬细胞后与五种化合物(每个小分子化合物设置3个浓度浓度梯度)以及HepG2.2.15的上清液分别孵育12小时。然后，将/>与HepG22.115共培养48小时。通过qRT-PCR测定HepG22.125中HBVpgRNA、前S/S RNA和ISG15的水平。通过ELISA(CZKWBIO，中国)测量HBsAg和HBeAg的水平，并通过COBAS Amplifrep/COBAS TaqMan HBV Test v2.0(Roche Diagnostics，USA)测量上清液中的HBV DNA水平。用qRT-PCR法测定/>中IFNA1和IFNB1的水平。最后，两种靶向CLEC5A的化合物(C2：0104-0071和C4：1869-0197)以剂量依赖的方式对HBV复制的抑制和干扰素的诱导有很强的作用以及低细胞毒性。
(3)靶向CLEC5A的小分子抑制剂C2和C4的CC50和IC50值确定
随后，本发明进一步确定靶向CLEC5A的小分子抑制剂C2和C4的CC50(细胞毒性浓度，即致死50％宿主细胞所需要的药物的最小浓度)和IC50(最小抑制浓度，即抑制50％的病原所需要的药物的浓度)值。具体地，用PMA将THP-1诱导为巨噬细胞细胞并接种到96孔板中，然后分别加入0.01μM、0.1μM、1μM、10μM、20μM、100μM、500μM和1000μM的C2或C4小分子抑制剂继续培养72小时，最后采用LuciferaseReporterAssay System检测活细胞的数量。如图4所示，C2小分子抑制的CC50值为630μM，而C4小分子抑制的CC50值为770μM。
为了检测C2和C4的IC50，用PMA将THP-1诱导为巨噬细胞后分别加入0μM、0.01μM、0.1μM、1μM、10μM、20μM的C2，或0μM、0.01μM、0.1μM、1μM、10μM、20μM、100μM的C4，以及HepG2.2.15的上清液孵育12小时。然后，将/>与HepG22.115共培养48小时，收集细胞和共培养的上清，通过qRT-PCR测定HepG22.125中HBVpgRNA和前S/S RNA的水平，通过ELISA(CZKWBIO，中国)测量共培养的上清中HBsAg和HBeAg的水平。通过HBVpgRNA、preS/S RNA、HBeAg和HBsAg四个指标的检测，最终确定了C2小分子抑制的IC50值为10μM，而C4小分子抑制的IC50值为20μM。
(4)靶向CLEC5A的小分子抑制剂C4小鼠体内功能确定
接下来，将C4注射到感染HBV的C57BL/6J小鼠模型中，进一步在体内测试它们的作用。简而言之，C57BL/6J小鼠(18-20g)通过流体动力学注射10ug pAAV/HBV1.2感染HBV。一周后，测定血清HBVDNA、HBsAg和HBeAg水平，并选择30只HBV DNA高(5×106拷贝/mL)的小鼠进行后续检测。C4(400μg/只小鼠)或等效溶剂(40％PEG300、5％Tween 80和55％水)每周腹膜内(i.p.)给药于小鼠，每周收集眼眶血，持续20周。使用ELISA试剂盒(R&D，USA)测定小鼠的血清HBsAg、HBsAb、Ifnα和Ifnβ的水平。通过COBAS Amplifrep/COBAS TaqManHBVTestv2.0(Roche Diagnostics，USA)对血清HBV DNA进行定量。IHC染色观察小鼠肝组织中HBcAg的表达。如图5所示，最终确定了靶向CLEC5A的小分子抑制剂C4治疗持续降低血清HBsAg和HBV DNA以及肝内HBcAg的水平，同时增加血清HBsAb的阳性率和水平，以及血清Ifnα和Ifnβ的水平。
(5)确定靶向CLEC5A的小分子抑制剂C4与CLEC5A的结合
最后，本发明又将C4小分子与CLEC5A的结合进行了独立实验验证。首先，为了检测小分子化合物C4是否与内源性人CLEC5A或小鼠CLEC5A结合，我们通过化学反应在C4小分子化合物上偶联了生物素，在或RAW264.7细胞培养基中加入生物素偶联C4或单独加入生物素后继续培养3小时，然后用PBS洗涤3次。后收集/>或RAW264.7细胞，并在含蛋白酶抑制剂混合物(Roche、1mM PMSF、5mM氟化钠和1mM原钒酸钠)的冷RIPA缓冲液(50mM Tris-HCl[pH 7.5]、150mM NaCl、1mM EDTA、1％Triton X-100、0.1％SDS和10％甘油)中裂解。收集细胞裂解液，与链霉亲和素珠在4℃孵育2小时。用裂解缓冲液洗涤微球三次，然后以适当量重新悬浮以进行免疫印迹，以检查C4与人CLEC5A或小鼠CLEC5A之间的相互作用。进而证实了人CLEC5A或鼠CLEC5A蛋白与生物素缀合的C4的直接结合(图6中A)。
其次，结构对接分析预测也证实了人CLEC5A或鼠CLEC5A蛋白与生物素缀合的C4的直接结合(图6中B)。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (8)
1.小分子抑制剂在制备治疗乙肝药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
2.小分子抑制剂在制备抑制乙肝病毒复制药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
3.小分子抑制剂在制备降低乙肝病毒感染血清中HBsAg含量药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
4.小分子抑制剂在制备降低乙肝病毒感染血清中HBV DNA含量药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
5.小分子抑制剂在制备降低乙肝病毒感染肝脏内HBc Ag含量药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
6.小分子抑制剂在制备增加乙肝病毒感染血清中HBs Ab含量药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
7.小分子抑制剂在制备增加乙肝病毒感染血清中Ifnɑ含量药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
8.小分子抑制剂在制备增加乙肝病毒感染血清中Ifnβ含量药物中的应用；
所述小分子抑制剂的结构为如下结构中的任一种：
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