CN111560013A - 一种自噬抑制剂及其应用 - Google Patents
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Abstract
Description
技术领域
本发明属于生物医药领域。具体地说,本发明涉及一种具有抑制ATG4B活性及溶酶体功能的双功能自噬抑制剂及其应用。
背景技术
细胞自噬是真核细胞普遍存在的一种生物学现象,是在多种自噬相关蛋白的参与下,对细胞内受损的细胞器等物质通过溶酶体途径进行降解的生物学过程。该过程对于真核细胞维持内环境的稳定和应对环境的变化具有重要意义。自噬异常与多种疾病相关,包括肿瘤、衰老、神经退行性疾病、心血管疾病及微生物感染等,对疾病的发生发展具有重要的调控意义。特别在肿瘤治疗领域,大量研究表明,抑制自噬可成为恶性肿瘤治疗的新途径。自噬是一个动态的不断变化的过程,主要包括以下几个过程:自噬的诱导与成核、自噬体双层膜的延伸、自噬体的形成、自噬体与溶酶体融合及自噬溶酶体的降解。其中,两条泛素化通路在自噬的过程中有着重要的作用,分别为ATG12-ATG5-ATG16L通路和LC3B-PE通路。ATG4B是半胱氨酸组织蛋白酶家族的一个成员,在自噬通路中同时参与LC3B的脂化与去脂化处理过程,通过调控ATG4B介导的LC3B处理过程能有效地调控整个自噬过程。研究表明,在慢性粒细胞性白血病细胞、结肠癌细胞及骨肉瘤细胞中,ATG4B被认为是一个致癌基因,敲低ATG4B的表达量能降低肿瘤细胞的存活率。因此,抑制ATG4B可能成为肿瘤治疗的新手段。自噬途径中的几乎所有步骤都可能成为潜在的药物靶标,目前,溶酶体抑制剂类是应用最为广泛的自噬抑制剂。然而,这些溶酶体抑制剂的抗癌作用可能独立于对自噬的抑制,从而影响其他信号通路来改变细胞的代谢,产生一些不利的副作用。目前已有多篇关于ATG4B抑制剂的报道,但这些报道的ATG4B小分子抑制剂存在特异性差、结构各异、抗肿瘤效价低等缺点,限制了其临床应用。鉴于溶酶体类抑制剂和ATG4B抑制剂单独使用的抗肿瘤效果都不佳,因此,开发同时作用于ATG4B和溶酶体的双功能自噬抑制剂对各类肿瘤治疗具有重要意义。
发明内容
本发明的目的是提供一类嘧啶二胺类化合物用作同时作用于ATG4B和溶酶体的双功能自噬抑制剂,可以用于自噬抑制剂类抗肿瘤药物的制备。
本发明的目的通过以下技术方案实现:
一种取代嘧啶二胺类双功能自噬抑制剂,具有如下所示的结构:
其中,R1为单取代,R2为单取代、双取代或多取代,X是O,S,Se,NH,NR3,P(O)R3,CH2,CHR,CR3R4中的一种。R1,R2,R3,R4取代基独立选自H、卤素、-CF3、-CN、-NO2、-OH、-NH2、-L-C1-C6的烷基、-L-C1-C6的烯基、-L-取代或非取代的杂芳基、或-L-取代或非取代的芳基,其中L是键、O、S、-S(=O)、-S(=O)2、NH、C(O)、CH2、-NHC(O)O、-HC(O)或-C(O)NH中的一种或多种。
优选地,所述的R1,R2为氢,X为O,命名为163N,其化学结构式为:
所述的抑制剂在抑制ATG4B酶活性上的应用。
所述的抑制剂在抑制溶酶体功能上的应用。
所述的抑制剂在制备治疗肿瘤药物中的应用。
所述的抑制剂在制备治疗结肠癌药物中应用。
本发明通过原核蛋白表达与纯化技术获得高纯度的ATG4B,以及底物GABARAPL2和LC3B-GST蛋白,通过荧光共振能量转移和SDS-PAGE的方法来判断化合物对ATG4B酶切活性的影响,并采用半胱氨酸蛋白酶家族成员的通用型抑制剂N-乙基马来酰亚胺(NEM)作为ATG4B抑制剂的阳性对照。
本发明利用LysoTracker Red(LTR)染色和DQ-BSA Red染色的方法来判断化合物对溶酶体pH值和降解能力的影响。
本发明利用CCK-8的方法检测了化合物对多种肿瘤细胞存活率的影响,以判断化合物是否具有抗肿瘤细胞增殖的活性。
本发明通过建立结肠癌荷瘤裸鼠模型来考察化合物在体内是否具有抗肿瘤细胞增殖的活性。
与现有技术相比,本发明具有如下优点:
(1)本发明中的抑制剂可以通过同时作用于ATG4B和溶酶体来发挥自噬阻断作用。与单功能溶酶体抑制剂相比,该抑制剂对自噬通路具有更高的选择性。
(2)本发明的双功能自噬抑制剂抗瘤谱广,对多种肿瘤细胞的增殖均具有良好的抑制活性。
(3)本发明中的双功能自噬抑制剂在体外和体内都具有良好的抑制结肠癌细胞生长的作用,可以用于制备自噬抑制剂类结肠癌治疗药物。
附图说明
图1(a)(b)分别为采用荧光共振能量转移的方法检测阳性化合物NEM及化合物163N抑制ATG4B酶切活性的IC50曲线。
图2为化合物163N可抑制ATG4B对底物LC3B-GST的酶切活性的电泳图。
图3(a)(b)分别为利用LysoTracker Red(LTR)染色和DQ-BSA Red染色的方法来检测化合物对溶酶体pH值和降解能力影响的荧光图;(1)为完全培养基(Complete Medium,CM)组,(2)为163N给药组,(3)为巴弗洛霉素A1给药组。
图4为细胞水平检测化合物163N对HeLa、HCT116、A549、MGC803、SGC7901、U87、KYSE150、MDA-MB-231、T98G和HL60等十种肿瘤细胞存活率影响的抑制作用图。
图5(a)(b)分别为化合物163N腹腔注射给药4周后对结肠癌荷瘤裸鼠模型中肿瘤大小和体积的抑制作用图。
图6(a)(b)分别为化合物163N的氢谱图和碳谱图。
具体实施方式
下面结合具体实例,进一步阐述本发明。应理解,这些实施仅用于说明本发明而不用于限制本发明的范围。凡是依照本发明公开内容所做出的等同替换,均属于本发明的保护范围。
实施例1:化合物163N的合成
化合物163N的合成方法,如路线(1)所示:
具体地,所述制备方法包含以下步骤:
1)称取叔丁醇钾(3equiv)于100ml圆底烧瓶中,依次加入DMF(15ml)、水杨醛(10mmol,1equiv)再缓慢加入对硝基苄溴(10mmol,1equiv)(注意硝基苯化合物的易爆性),插入冷凝管,逐渐升温回流。反应适当的时间,TLC点板检测,待反应完成后,用乙酸乙酯和水萃取,有机相用无水硫酸钠干燥,在旋转蒸发仪上除去有机溶剂,直接快速柱层析分离纯化(石油醚:乙酸乙酯=10:1),得到黄色固体,产率为75%。黄色固体产物溶解于甲醇(20ml),在冰水浴情况下缓慢加入Raney-Ni(w/w=10%),之后再缓慢滴加80%水合肼(2.5equiv)。插入一个干瘪的气球作为保护装置,室温反应,TLC跟踪反应,待反应完成后,使用磁子吸去Raney-Ni催化剂,泡于水中,剩余的反应使用硅藻土过滤,滤液旋干,所得参与有机物柱层析纯化,收集目标组分并浓缩得到浅黄色中间体1,产率为98%。
2)称取4,6-二氯-2-甲基嘧啶(10mmol,1equiv)溶解于EtOH(20ml),在冰水浴情况下,加入Et3N(1.2equiv),缓慢加入对甲氧基苯胺(10mmol,1equiv),移至室温反应,TLC检测反应,待反应完成后,旋干EtOH,加入DCM与H2O萃取,有机相用无水硫酸钠干燥,浓缩,柱层析分离纯化(石油醚:乙酸乙酯=10:1),收集目标组分并浓缩得到白色中间体2,转化率为75%。
3)称取中间体1(1mmol,1equiv)和中间体2(1.2mmol,1.2equiv)于20ml厚壁耐压瓶中,加入3ml叔丁醇和催化量的HCl,升温至150度反应约16h。待反应结束后,冷却抽滤,所得白色固体用DCM和5%的氨水溶液萃取,有机相用无水硫酸钠干燥,浓缩,柱层析分离纯化(二氯甲烷:甲醇=20:1),收集目标组分并浓缩得到目标产物163-N,白色固体,转化率为85%。结构确证:如图6(a)所示,1H NMR(400MHz,DMSO-d6)δ9.36(s,1H),9.09(s,1H),7.91(s,2H),7.76(d,J=8.8Hz,2H),7.63–7.56(m,2H),7.56–7.46(m,2H),7.24(s,3H),6.94(d,J=9.0Hz,2H),6.04(s,1H),3.77(s,3H),2.22(s,3H).如图6(b)所示,13C NMR(101MHz,DMSO)δ164.83,161.35,159.23,155.92,154.99,153.97,142.02,132.92,129.22,125.03,123.81,123.06,122.60,121.85,120.62,118.62,113.93,110.87,99.73,55.23,23.58.
实施例2:ATG4B蛋白、GABARAPL2蛋白和LC3B-GST的表达纯化
将1μL的重组质粒His-ATG4B和His-LC3-GST分别转化至大肠杆菌BL21(DE3)感受态细胞中。将1μL的重组质粒His-GABARAPL2转化至大肠杆菌BL21(DE3)CodonPlus感受态细胞中。分别在LB平板中挑取单克隆接种到LB液体培养基中进行培养,待OD600达到0.5-0.8时加入终浓度为0.5mM的IPTG进行重组蛋白诱导表达。收菌后离心并加入湿菌重量10倍的含5mM咪唑的结合缓冲液后超声破碎菌体。12,000g离心30min后收集上清,与Ni-NTA填料充分结合使蛋白挂柱。分别用10mM、20mM和50mM的咪唑缓冲液梯度洗脱杂蛋白,最后用200mM咪唑洗脱并收集目的蛋白并脱盐保存。
实施例3:FRET方法检测化合物对ATG4B酶活性的影响
384孔黑板中分别加入指定浓度的NEM或163N与0.75mg·L-1的ATG4B在PBS缓冲液中恒温37℃共孵育30min,取出板并加入终浓度为50mg·L-1的底物GABARAPL2使总体系为50μL,37℃继续共孵育30min。反应结束后,测定527/477nm的RFUs比值。ATG4B酶活性抑制率计算公式为:抑制率(%)=(RFUmax-RFUX)/(RFUmax-RFUmin))*100%。其中RFUmax、RFUmin、RFUX分别指指没发生酶切反应时、酶切反应进行到最彻底时及特定化合物处理下的527/477nm的比值。如图1(a)所示,利用该检测体系测得阳性化合物NEM对ATG4B抑制作用的IC50为83.44μM,如图1(b)所示,163N对ATG4B抑制作用的IC50为17.82μM。
实施例4:SDS-PAGE方法检测AG-690抑制ATG4B酶活性
将ATG4B(3ng)单独或与不同浓度的163N在PBS缓冲液中37℃恒温共孵育30min,然后加入底物蛋白LC3B-GST(4μg),反应总体系为20μL,继续反应30min。在反应体系中加入上样缓冲液(Loading Buffer)终止反应,离心后煮沸使蛋白变性,并进行SDS-PAGE电泳,结束后使用考马斯亮蓝染色方法对条带进行着色,之后使用脱色液脱色分析。如图2所示,163N能剂量依赖性地抑制ATG4B对底物蛋白LC3B-GST的酶切活性。
实施例5:LysoTracker Red(LTR)染色和DQ-BSA Red染色法检测化合物163N对溶酶体功能的抑制作用
LTR染色:取0.5μL LTR母液加入到8mL的灭菌HBSS缓冲液中,混匀即为LTR工作液。将消化好的细胞种于96孔板中过夜培养,第二天待细胞完全贴壁后给予不同的处理条件。处理结束后,弃去旧培养基,每孔加入100μL37℃预孵育的LTR工作液,然后放入细胞培养箱中继续培养30min。弃去LTR染色工作液,然后加入新鲜的细胞培养液并在荧光显微镜下拍照。如图3(a)所示,如巴弗洛霉素A1(Baf)处理组类似,163N可以有效地猝灭溶酶体中红色荧光,说明163N可以有效的升高溶酶体的pH值。
DQ-BSA Red染色:取10μL的DQ-BSA Red母液(1mg/mL)于990μL的EBSS缓冲液中,终浓度为10μg/mL。将消化好的细胞种于96孔板中过夜培养,第二天待细胞完全贴壁后去除细胞培养液,加入37℃预温的DQ-BSA Red工作液,放入培养箱中孵育2h。去除DQ-BSA Red工作液,用PBS洗细胞2次,之后加入新鲜的细胞培养基(含药或不含药)继续放入细胞培养箱中培养4-6h。弃去培养基,用PBS洗2次,之后用4%多聚甲醛固定后拍照。如图3(b)所示,如巴弗洛霉素A1(Baf)处理组类似,163N可以有效地猝灭溶酶体中红色荧光,说明163N可以有效地抑制溶酶体的降解功能。
实施例6:CCK-8法检测163N对肿瘤细胞增殖的抑制活性
本实施例采用HeLa、HCT116、A549、MGC803、SGC7901、U87、KYSE150、MDA-MB-231、T98G和HL60等十种肿瘤细胞检测不同浓度163N处理下细胞存活率变化。
将消化好的细胞悬液计数,之后接种于96孔板中(5000个/孔),至少3个复孔,并放置于细胞培养箱中过夜培养。弃去旧培养基,将含有系列浓度梯度163N的新鲜完全培养基加入96孔板中,放置于细胞培养箱中继续培养48h。之后在96孔板中加入10μL的CCK-8溶液,轻晃混匀,继续放置于细胞培养箱中60min。检测在450nm处的吸光度值(约在0.8-1.5之间)。参照CCK-8说明书计算细胞存活率值。细胞存活率(%)=(OD加药组-OD空白组)/(OD对照组-OD空白组)×100%。如图4所示,163N可以剂量依赖性地抑制多种肿瘤细胞的生长且高浓度的163N可完全抑制肿瘤细胞的存活,对HeLa、HCT116、A549、MGC803、SGC7901、U87、KYSE150、MDA-MB-231、T98G和HL60的IC50值分别为7.06μM、5.76μM、7.68μM、9.33μM、21.16μM、10.59μM、13.03μM、9.99μM、7.23μM和12.63μM。实验结果发现,163N对HCT116细胞最为敏感。
实施例7:163N可抑制裸鼠移植瘤的生长
使用4-5周龄的雌性裸鼠皮下注射3×106个HCT116细胞建立结肠癌荷瘤裸鼠模型。待注射1周后,即肿瘤体积约为50mm3时开始分组给药。荷瘤小鼠按照肿瘤体积随机分为2组,溶剂对照组(玉米油)和163N给药组(剂量为50mg/kg体重)。每组5只,各组肿瘤大小无显著性差异(P>0.05)。药物用玉米油溶解后,腹腔注射给药,每周3次,连续给药4周,溶剂对照组给予同等体积的玉米油。每3天检测一次肿瘤体积。按照以下公式计算肿瘤体积:Volume(mm3)=长(mm)×宽2(mm)/2。给药4周后,小鼠称重,1%戊巴比妥钠麻醉后处死取材。如图5(a)和(b)所示,溶剂对照组的肿瘤体积随时间变化显著升高,而163N给药组的肿瘤生长相对缓慢,两组的相对肿瘤体积存在显著性差异,说明163N可抑制裸鼠移植瘤的生长。
Claims (4)
3.权利要求1或2所述的自噬抑制剂在制备抗肿瘤药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述自噬抑制剂在制备治疗结肠癌药物中应用。
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