CN116903587A - 一种角鲨烯环氧酶抑制剂及其新用途 - Google Patents
一种角鲨烯环氧酶抑制剂及其新用途 Download PDFInfo
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Abstract
本发明利用计算机辅助药物设计软件，高通量筛选得到SQLE的新型抑制剂，研究其对肝癌细胞增殖、转移的抑制作用，以及对胆固醇合成的影响。利用生物信息学方法，分析GEPIA、UALCAN等公共数据库中SQLE在肝癌中的表达情况以及与患者预后的关系，结果显示，SQLE与肝癌的发展密切相关，其高表达与肝癌患者的临床分期和不良预后密切相关。利用药物筛选软件Discovery Studio，从商用化合物数据库Chemdiv中筛选靶向SQLE的335位酪氨酸残基的小分子抑制剂，使用MTT法检测候选化合物对肝癌细胞系Huh7增殖的抑制作用，筛选得到的1个候选SQLE新型抑制剂可抑制肝癌细胞Huh7的增殖。25mM的31#化合物能够抑制Huh7细胞增殖与迁移能力，抑制细胞胆固醇的生成，增加抑癌基因PTEN的表达，抑制PI3K、AKT的蛋白表达。
Description
技术领域
本发明涉及一种角鲨烯环氧酶新型抑制剂及其在制备治疗抗肝癌的药物上的应用，属于制药技术领域。
背景技术
肝癌是我国最常见及致死率最高的恶性肿瘤之一。由于人口老龄化的加剧以及不良的生活习惯，肝癌的发生率及癌症相关死亡率逐年上升。目前，癌症的治疗方法主要是手术治疗或手术结合放化疗，但治疗效果不佳的同时会对人体产生严重的副作用。胆固醇作为一种新的信号分子，在癌症中发挥重要作用。角鲨烯环氧化酶(squalene epoxidase，SQLE)是调节胆固醇含量的关键酶之一。越来越多的研究发现SQLE与癌症关系密切，SQLE在促进肿瘤细胞增殖、迁移，维持肿瘤细胞干性等方面都发挥重要作用，可能是癌症治疗的潜在分子靶点。研究其在肝癌发生、发展中的作用及分子机制，筛选SQLE抑制剂对肝癌治疗及预后有着重要的理论意义和应用价值。
SQLE是一种依赖黄素腺嘌呤二核苷酸(flavin adenine dinucleotide，FAD)的环氧化酶，它催化非甾醇中间体角鲨烯转化为2,3(S)-环氧鲨烯，是胆固醇合成的关键酶之一，其在体内表达受到精密调控以维持胆固醇稳态。癌细胞往往表现出大量胆固醇积累，以增强膜受体信号转导、改变线粒体膜活性、影响肿瘤免疫等，高表达的SQLE会导致胆固醇稳态失衡从而促进癌症发生。现有的SQLE抑制剂，特比萘芬除了有着良好的抑制真菌作用，同样具有显著的肿瘤抑制作用。为了提高特比萘芬对肿瘤的抑制能力，研究人员开发对哺乳动物亲和力更高的特比萘芬衍生物，NB-598。但在动物实验中发现，NB-598有明显的胃肠道和皮肤毒性，这限制了其在肿瘤治疗中的应用。Padyana的团队在2019年解析了SQLE蛋白的晶体结构，并证实了SQLE抑制剂特比萘芬、NB-598均是针对SQLE蛋白第195位酪氨酸残基(Y195)，Y195能与SQLE蛋白168位谷氨酰胺残基(Q168)形成氢键，从而促进SQLE的活化，特比萘芬等抑制剂阻止Y195与Q168间的氢键形成，使SQLE失活。
发明内容
本发明筛选针对SQLE的Y335位点的小分子抑制剂，SQLE的Y335位酪氨酸残基为SQLE与FAD结合所必须，基于该位点的抑制剂可抑制SQLE-FAD复合物形成，仅抑制角鲨烯反应中间体形成。本发明利用Discovery Studio软件，结合SQLE晶体结构，对SQLE蛋白进行分子建模和环境模拟，筛选针对SQLE蛋白Y335位点的小分子化合物。具体技术方案如下：
角鲨烯环氧酶新型抑制剂，所述的抑制剂选自化合物库Chemdiv中S723-2271(31#)。
一种角鲨烯环氧酶新型抑制剂在制备治疗抗肝癌的药物上的应用，所述的抑制剂选自化合物库Chemdiv中S723-2271(31#)。
所述的角鲨烯环氧酶新型抑制剂在制备抑制肝癌细胞系Huh7增殖的药物上的应用。
所述的角鲨烯环氧酶新型抑制剂在制备增加抑癌基因PTEN的表达，以及抑制PI3K、AKT的蛋白表达的药物上的应用。
所述的角鲨烯环氧酶新型抑制剂在制备降低Huh7细胞内的胆固醇含量的药物上的应用，所述的抑制剂选自化合物库Chemdiv中S723-2271(31#)。
本发明的技术方案，利用计算机辅助药物设计软件，高通量筛选得到SQLE的新型抑制剂，研究其对肝癌细胞增殖、转移的抑制作用，以及对胆固醇合成的影响，并初步探究其分子机制。利用药物筛选软件Discovery Studio，从商用化合物数据库Chemdiv中筛选靶向SQLE的335位酪氨酸残基的小分子抑制剂，使用MTT法检测候选化合物对肝癌细胞系Huh7增殖的抑制作用，筛选得到的1个候选SQLE新型抑制剂(即31#化合物)。进一步实验发现25mM的31#化合物能够抑制Huh7细胞增殖与迁移能力，减少细胞内的胆固醇含量，增加抑癌基因PTEN的表达，抑制PI3K、AKT的蛋白表达。
本发明的技术方案首先利用Discovery Studio、CLC Drug Discovery Workbench等药物开发软件，结合SQLE的晶体结构，通过多层级的去重打分，筛选出了35个针对SQLEY335位点的抑制剂。并在肝癌细胞系Huh7中验证其抑制肝癌增殖的作用，得到了1个能显著够抑制Huh7细胞增殖的化合物。随后，我们在Huh7细胞系中验证了所筛选化合物对Huh7细胞迁移能力的抑制。
胆固醇是类固醇激素的前体，也是质膜的重要组成部分，维持细胞膜的完整性和流动性，并且在细胞内信号转导中发挥关键作用。细胞内的胆固醇水平受到精密调控，通过合成、摄取、转运等调节维持稳态。癌细胞往往表现出大量胆固醇积累，以增强膜受体信号转导、改变线粒体膜活性、影响肿瘤免疫等。胆固醇代谢在癌症中发挥重要功能，SOAT1、SQLE和NPC1等新的胆固醇代谢分子，已成为癌症治疗潜在的靶点。高胆固醇水平可以促进包括前列腺癌、食管癌、乳腺癌、结直肠癌在内的多种癌症的发生或发展。在HCC中，SQLE可以通过激活PI3K/AKT信号通路，促进胆固醇的合成，从而加速细胞的增殖与迁移。筛选到的候选化合物小分子对Huh7细胞的增殖有明显的抑制作用，也可以降低胞内胆固醇的含量，这表明候选小分子化合物能有效抑制SQLE的活性，并可能发挥抑制HCC发生发展的作用。
抑癌基因PTEN以及PI3K/AKT信号通路是体内参与细胞增殖、能量代谢等调控的信号通路之一。该通路在HCC中激活可以导致癌细胞增殖和胆固醇的积蓄。为进一步研究候选化合物分子抑制胆固醇合成的机制，我们检测了化合物处理后Huh7细胞PTEN、PI3K、AKT的蛋白表达情况，结果发现，候选的31#化合物能够提高Huh7细胞的PTEN蛋白水平，抑制PI3K、AKT的蛋白水平。因此我们推测所筛选的31#化合物能够通过增加PTEN表达，抑制PI3K/AKT信号通路，抑制细胞内胆固醇的含量。
本发明利用SQLE晶体结构结合药物开发软件，针对SQLE的Y335位点筛选出35个小分子化合物，再通过MTT实验，进一步筛选出1个具有明显Huh7细胞增殖抑制作用的小分子化合物，并验证了其抑制Huh7细胞迁移的能力、抑制胆固醇积累以及增加PTEN表达，抑制PI3K/AKT信号通路激活的能力。
附图说明
图1为小分子化合物的计算机虚拟筛选过程。图2为MTT实验用于检测31#化合物对Huh7细胞增殖的影响。*P<0.05，**P<0.01，***P<0.001VS空白对照组。图3为划痕实验用于检测31#化合物对Huh7细胞迁移的影响。*P<0.05，**P<0.01，***P<0.001VS空白对照组。
图4为Transwell实验用于检测31#化合物对Huh7细胞迁移能力的影响，第一排放大倍数为100×，第二排放大倍数为200×。*P<0.05，**P<0.01，***P<0.001VS空白对照组。
图5为31#化合物对Huh7细胞胆固醇合成的影响。*P<0.05，**P<0.01，***P<0.001VS空白对照组。
图6为31#化合物对Huh7细胞中PTEN、PI3K和AKT蛋白表达的影响。*P<0.05，**P<0.01，***P<0.001VS空白对照组。
具体实施方式
材料与方法仪器：倒置显微镜(上海光学仪器五厂有限公司)；二氧化碳培养箱MCO-175(日本三洋公司)；5417R台式冷冻高速离心机(Eppendorf公司)；Western blot电泳仪、电转膜系统(美国Bio-Rad公司)；凝胶成像仪(美国柯达公司)；精密电子天平JJ 100型(美国双杰兄弟集团常熟双杰测试仪器厂)；精密移液器、超微量分光光度计DanoDrop(美国Thermo Fisher scientific)。
药品与试剂：抗体(万类生物公司)；ECL化学发光试剂盒、5×protein LoadingBuffer、TBS粉末(武汉Servicebio公司)；DMEM培养基、胎牛血清(美国Gibco公司)；Protease Inhibitor Cocktail(美国MedChemExpress公司)；DMSO、EB(美国Sigma)；RIPA裂解液(北京Solarbio)；蛋白定量分析试剂盒、胆固醇检测试剂盒(美国Thermofisher公司)；30％丙烯酰胺-甲叉双丙烯酰胺(武汉博士德生物公司)；TEMED，四甲基乙二胺(美国LifeTechnologies)；PVDF膜(德国Millipore)。
细胞株：人肝癌细胞系(Huh7)：湖北省宜昌市三峡大学，肿瘤微环境与免疫治疗湖北省重点实验室提供。
细胞培养：Huh7细胞用DMEM培养基(含10％胎牛血清，1％的青霉素/链霉素)置于37℃、5％CO2细胞恒温培养箱内培养。当细胞密度达到90％时进行传代，每3天传代1次，取对数生长期细胞用于实验。
MTT：10％DMEM(+)稀释小分子化合物至所需浓度，处理24h，48h和72h。处理结束后，加入MTT工作液，4h后弃上清，每孔加入DMSO溶液150mL。室温振荡240s后，酶标仪检测570nm处的吸光度值。
细胞划痕实验：6孔板背面做横向直线用以标记，接种细胞，确保细胞贴壁后，枪头垂直于标记线均匀划线，吸去培养基，用PBS润洗，加入小分子化合物(0,6.25,25mM)处理，根据划痕与标记线定位拍照。拍照后放入细胞培养箱中继续培养，于24、48、72h拍照，拍照部位一致。利用ImageJ计算各组划痕愈合面积，对进行结果分析，绘制统计图。
Transwell迁移实验：提前一天换无血清培养基饥饿细胞24h，处理完成后，胰酶消化，无血清培养基重悬，取500μl的5×105个细胞，加入Transwell小室，下室加入1mL含20％FBS的培养基。按所需浓度将药物加入小室内。培养24h，24h后清洁小室，染色，风干。第二天拍照。
细胞胆固醇检测：取对数期生长Huh7细胞，接种于6cm皿，药物处理48h，消化后PBS重悬，取部分进行BCA检测蛋白浓度，其余加入配好的脂质萃取液，超声破碎，14000r/min，离心10min。取下层有机相冷冻挥干。使用500mL胆固醇试剂盒中的1×Buffer复溶。配置胆固醇检测工作液、标曲、试卤灵以及阳性对照。37℃反应1h后放入全波长酶标仪，设置激发光530-560nm，560-590的激发光检测，减去无样品孔校正背景荧光。根据标曲计算样品中胆固醇含量，除去BCA所测样品蛋白浓度排除细胞数量对结果的影响。
Western Blot：取对数期生长Huh7细胞，接种于6cm皿，药物处理48h，处理完成后收集并裂解细胞，进行BCA检测蛋白浓度，调整蛋白浓度后加入1×loading buffer金属浴变性。取变性蛋白经SDS-PAGE电泳，湿转转膜，5％牛奶封闭，一抗孵育过夜，TBST洗涤3次，二抗孵育1h，TBST洗涤三次，ECL试剂盒发光显色，ImageJ对条带进行灰度分析。
统计学分析：本课题数据均用SPSS22.0分析并处理，每组数据均进行3次独立重复试验，用means±SD表示；采用t检验比较两样本均数，P<0.05则认为结果有统计学意义。
高通量筛选抑制SQLE的化合物
通过Discovery Studio软件分析，利用3种分子对接评分软件LibDock、CDOCKER、CLC Drug Discovery Workbench，从商用化合物数据库Chemdiv中筛选得到潜在的具有SQLE抑制作用的候选小分子化合物。结合SQLE的晶体结构，在商业化合物库中对化合物进行立体结构、去重及不适合成药结构的筛选，并利用3种分子对接评分软件进行模块打分，初步挑选出了针对SQLE Y335位点、具有SQLE抑制作用的35个小分子化合物进行接下来的研究，流程如图1所示。
小分子化合物对肝癌细胞生物学行为影响
多种肝癌细胞系中筛查小分子化合物对其增殖的影响
将小分子化合物依次编号为1#至35#，用MTT法检测35种化合物50mM浓度下，作用于Huh7细胞48h后细胞的存活率。结果显示50mM的31#化合物能够抑制50％的Huh7细胞存活。因此选择31#化合物为候选小分子化合物，其分子信息见表1。
表1化合物的分子量、分子式及分子结构图
注，上述中，1#、31#号化合物购买自上海陶术生物公司
MTT法检测候选化合物对Huh7细胞增殖能力的影响
MTT法检测候选的31#化合物对Huh7细胞增殖能力的影响，结果如图2所示。随着31#化合物给药浓度和处理时间的增加，对Huh7细胞增殖的抑制越来越显著。100μM的31#化合物处理Huh7细胞24h对Huh7细胞的增殖抑制达不到50％，48h、72h后IC50分别为25.7mM、5.1mM。31#化合物在25mM浓度处理Huh7细胞48h后抑制Huh7细胞50％左右的增殖，在6.25mM浓度时，31#化合物处理Huh7细胞48h后对其增殖无明显影响。因此后续实验选择处理浓度为25μM和6.25μM。
细胞划痕实验检测化合物对Huh7迁移能力的影响
用6.25μM和25μM的31#化合物处理Huh7细胞24h、48h和72h后，发现划痕的面积随着处理浓度增加和处理时间延长而增加，说明化合物可抑制细胞的迁移能力，如图3所示。
实验检测候选化合物分子对Huh7迁移能力的影响
用25μM的31#化合物处理Huh7细胞24h后，观察穿过小室的细胞数量，发现药物处理组穿过的细胞数量明显少于对照组，说明化合物可抑制细胞的迁移，其中25μM的31#的抑制效果比100μM的特比萘芬要更明显，如图4所示。
候选化合物对Huh7细胞中胆固醇水平的影响
用3.125μM、6.25μM、12.5mM、25μM的31#化合物处理Huh7细胞48h后，检测细胞内总胆固醇及游离胆固醇水平，结果如图5所示。25μM 31#化合物处理后，胞内游离胆固醇和总胆固醇水平分别下降至对照组的60％(P<0.05)和58％(P<0.05)。
候选化合物分子对Huh7细胞中PI3K/AKT信号通路的影响为了研究化合物能否通过PTEN调节PI3K/AKT相关通路，我们使用6.25μM和25μM的化合物31#处理Huh7细胞48h，检测细胞内PTEN、PI3K、AKT蛋白表达水平，结果如图6所示。化合物31#能够增加抑癌基因PTEN的表达水平并降低PI3K和AKT的蛋白水平。
Claims (5)
1.角鲨烯环氧酶新型抑制剂，所述的抑制剂选自化合物库Chemdiv中S723-2271，结构式为
2.一种角鲨烯环氧酶新型抑制剂在制备治疗抗肝癌的药物上的应用，其特征在于，所述的抑制剂选自化合物库Chemdiv中S723-2271，结构式为
3.根据权利要求2所述的应用，其特征在于，所述的角鲨烯环氧酶新型抑制剂在制备抑制肝癌细胞系Huh7增殖的药物上的应用。
4.根据权利要求2所述的应用，其特征在于，所述的角鲨烯环氧酶新型抑制剂在制备增加抑癌基因PTEN的表达，以及抑制PI3K、AKT的蛋白表达的药物上的应用。
5.根据权利要求2所述的应用，其特征在于，所述的角鲨烯环氧酶新型抑制剂在制备抑制Huh7细胞的胆固醇的药物上的应用，所述的抑制剂选自化合物库Chemdiv中S723-2271，结构式为
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