CN106905217A - 一种自噬关键蛋白atg4b酶抑制剂及其应用 - Google Patents
一种自噬关键蛋白atg4b酶抑制剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种自噬关键蛋白ATG4B酶抑制剂及其应用，属医药技术领域。该抑制剂结构通式如(1)所示，其中，R1，R2为单取代、双取代或多取代，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。该抑制剂可以通过特异性的抑制ATG4B活性，降低细胞自噬水平，进而发挥阻止肿瘤细胞生长的作用，实现其作为抗肿瘤药物的应用。
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酶活性的抑制剂及其应用。
背景技术
细胞自噬是真核细胞所特有的对细胞内受损的细胞器及长寿命蛋白通过溶酶体途径进行降解的细胞生物学过程。自噬对于维持细胞稳态、调节细胞物质能量代谢具有重要意义，因此2016年诺贝尔生理与医学奖就授予细胞自噬的研究专家大隅良典。细胞自噬是一个动态变化的过程，可大致分为以下几个阶段：诱导与成核、延伸、自噬体的成熟、自噬体与溶酶体的融合及其内容物的降解[3]。在这一过程中，有多种自噬相关基因(Autophagy-related genes,ATG)的参与，其中两条泛素样通路ATG12-ATG5-ATG16和ATG8-PE(phosphatidylethanolamine)在自噬体的延伸和成熟过程中起着重要的作用，而ATG12-ATG5-ATG16复合物作为E3样酶有助于ATG8与PE的结合。ATG8必须经过一个蛋白水解过程暴露C末端的甘氨酸才能锚定在自噬泡膜上。ATG4作为C54家族的一种半胱氨酸蛋白酶，在ATG8连接系统中起了非常关键的作用。ATG4可以剪切ATG8的C端精氨酸，使其暴露出C端的甘氨酸残基，以便与PE共价连接形成ATG8-PE锚定在自噬泡膜上。接下来ATG4还能去脂化ATG8-PE，以便使自噬体与溶酶体融合。因此，通过调控ATG4介导的ATG8-PE的去脂化过程能调控整个自噬的过程。
ATG4家族成员在脂化与去脂化ATG8家族成员以形成自噬体的过程中扮演着非常重要的作用。ATG4在酵母中仅有一个成员，其功能的缺失将阻断整个自噬的进程。而在哺乳动物细胞中ATG4有四个家族成员：ATG4A、ATG4B、ATG4C和ATG4D。ATG4B作为ATG4家族中研究最为广泛的成员，对ATG8家族成员(LC3家族及GABARAP家族)均具有酶切活性。
已有文献报道，ATG4B在肿瘤的发生发展过程中起了重要作用。ATG4B被认为是一个致癌基因，能促进结肠癌细胞的生长且独立于其自噬调节作用。除此之外，在慢性髓细胞性白血病及骨肉瘤细胞中，ATG4B也被看作致癌基因。另外使用ATG4B的小分子抑制剂NSC185058能抑制骨肉瘤细胞的生长。不仅如此，ATG4B在博来霉素诱导的心肌纤维化过程中起了重要作用。目前关于ATG4B小分子抑制剂的研究较少，抑制剂效价偏低。因此，开发高特异性和效价的ATG4B抑制剂对各类肿瘤治疗具有重要意义。
发明内容
本发明解决的问题在于通过前期药物筛选，提供一种针对ATG4B酶的抑制剂AG-690及其类似物，可以用于抗肿瘤药物、慢性髓细胞白血病药物、抗心肌纤维化药物的制备。
本发明的技术方案如下：
一种自噬关键蛋白ATG4B酶抑制剂，其化学结构式为：
其中，R1，R2为单取代、双取代或多取代，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。
所述的ATG4B酶抑制剂，优选地，所述的R1为双取代，甲基取代位于羟基的邻位，硝基取代位于羟基的对位，R2为氢，命名为AG-690，其化学结构式为：
所述的抑制剂在抑制ATG4B酶上的应用。
所述的抑制剂在制备治疗肿瘤药物中应用。
所述的抑制剂在制备治疗结肠癌药物中应用。
本发明通过原核表达和亲和纯化技术获得高纯度的酶ATG4B蛋白以及底物FRET-GATE16，通过蛋白电泳检测底物酶切后的条带亮度变化来判断化合物抑制ATG4B酶活的能力。
本发明还利用荧光能量荧光功能转移的方法，通过酶标仪检测底物酶切后荧光的变化来判断化合物抑制ATG4B酶活的能力。
本发明采用非特异性的半胱氨酸蛋白酶抑制剂NEM作为ATG4B抑制剂的阳性对照。
本发明检测了化合物对其他半胱氨酸蛋白酶如Caspase3的酶活抑制能力，以判断抑制能力是否特异。
与现有技术相比，本发明具有如下优点：
(1)本发明抑制剂可以通过特异性的抑制ATG4B活性，降低细胞自噬水平，对肿瘤细胞活力有明显抑制，进而发挥阻止肿瘤细胞生长的作用，实现其作为抗肿瘤药物的应用。
(2)本发明抑制剂比已报道的抑制剂NSC185058化合物效价高。
(3)本发明采用结肠癌细胞HCT116检测不同浓度抑制剂下细胞的活力变化，证明抑制剂对结肠癌细胞具有细胞毒活性。
附图说明
图1为底物FRET-GATE16经过ATG4B酶切后的电泳图。
图2为非特异性半胱氨酸蛋白酶抑制剂作为阳性对照抑制ATG4B的IC50曲线。
图3为不同浓度AG-690抑制ATG4B的酶切能力(电泳图)。
图4为AG-690抑制ATG4B活性的IC50曲线。
图5为不同化合物抑制Caspase3活性的差异效果图。
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：重组蛋白的表达纯化
将重组质粒FRET-GATE-16和ATG4B分别转化至大肠杆菌BL21(DE3)CodonPlus和BL21(DE3)PLYSs中。LB平板挑取单克隆接种到LB液体培养基中，37℃、220rpm过夜培养，1:100进行扩增培养，当OD600达到0.6-0.8时加入0.5mM的IPTG进行诱导，16℃，16h培养后收菌。离心收集菌体，加入湿菌重量5到10倍的结合缓冲液(含5mM的咪唑)稀释菌体后超声破碎菌体。离心收集上清，使用镍NTA填料进行纯化，加入菌液上清使目的蛋白挂柱，之后分别用20mM和50mM的咪唑缓冲液进行梯度洗脱，最后用200mM的咪唑洗脱并收集洗脱液。将收集到的洗脱液过脱盐柱后浓缩并于-80℃冰箱保存。为保证该体系的可靠性和稳定性，用考马斯亮蓝染色的方法对纯化出来的两种蛋白的纯度及活性进行了验证。FRET-GATE16(4μg)与合适量的ATG4B(3ng)在37℃共孵育0min或30min。如图1所示，全长的FRET-GATE16(0min)的纯度(＞90％)可以用于接下来的实验，而30min时全长的FRET-GATE16(0min)几乎可以完全被3ng的ATG4B酶切为CFP-GATE16和CFP两部分，说明ATG4B的活性良好。
实施例2：FRET方法检测AG-690抑制ATG4B的酶活性
384孔黑板中加入终浓度为100μM的抑制剂(AG-690)与0.75mg·L-1的ATG4B在Tris缓冲液中37℃共孵育30min，之后加入50mg·L-1的FRET-GATE16，反应总体系为50μL，反应时间为30min。该体系的中含有0.1％DMSO终浓度。527/477nm的RFUs比值在反应30min时测定。ATG4B相对酶切活性的计算公式为：抑制率(％)＝(RFUmax-RFUX)/(RFUmax-RFUmin))*100％，其中RFUmax指没发生酶切反应时的527/477nm的比值，RFUmin指酶切反应进行到最彻底的527/477nm的比值，RFUX指在特定化合物处理条件下的527/477nm的比值。我们选择半胱氨酸蛋白酶的通用型抑制剂N-乙基马来酰亚胺(NEM)作为本次筛选的阳性对照，利用该检测体系测得NEM的IC50值为134.2μM(图2)。
实施例3：SDS-PAGE方法检测AG-690抑制ATG4B酶活性
将3ng的ATG4B单独或与0-100μM的抑制剂(AG-690)在缓冲液中37℃共孵育30min，之后加入4μg的底物蛋白FRET-GATE16，反应总体系为20μL，反应时间为30min。用5X上样缓冲液终止反应，将蛋白变性，采用SDS-PAGE进行电泳，电泳结束后用考马斯亮蓝染色方法对条带进行着色，之后脱色进行分析。为进一步验证AG-690对ATG4B的体外抑制活性，采用考马斯亮蓝染色的方法检测该化合物对ATG4B的酶切抑制效果。如图3所示，AG-690能剂量依赖性地抑制ATG4B的活性，且在100μM的条件下几乎可以完全抑制住ATG4B的活性。FRET方法测得该化合物的IC50值为36.8μM(图4)。
实施例4：AG-690特异性抑制作用分析
Hela细胞于含有10％胎牛血清的DMEM培养基中，于37℃，5％CO2的孵箱中培养。用星形孢菌素(1μM,5hrs)来诱导Hela细胞凋亡，对照组细胞不做任何处理在同样的条件下培养。非变性提取细胞总蛋白，BCA试剂盒检测细胞蛋白含量，上样量为10μg。384孔黑板中加入终浓度为100μM的指定化合物与10μg的细胞裂解液在Tris缓冲液中37℃共孵育30min，之后加入终浓度为25μM的荧光底物Ac-DEVE-AFC，反应总体系为50μL，立即检测荧光值，测定时间为60min，反应温度为37℃。分别于激发光400nm和发射光505nm处检测荧光AFC的动力学曲线。如图5所示，星形孢菌素能有效的诱导Hela细胞产生凋亡。Caspase-3的特异性抑制剂Z-VAD-FMK在50μM的浓度下能有效抑制Caspase-3的酶切活性。而AG-690在100μM的浓度下对Caspase-3的酶切活性没有影响，说明该化合物不是普遍的半胱氨酸蛋白酶抑制剂。
实施例5：AG-690抑制肿瘤细胞活力的测定
本实施例采用结肠癌细胞HCT116检测不同浓度抑制剂下细胞的活力变化。在96孔板中接种结肠癌细胞HCT116，每孔100μl，细胞于含有10％胎牛血清的DMEM培养基中，于37℃，5％CO2的孵箱中培养16小时。之后更换培养基含有浓度为200μM，100μM，50μM，25μM，12.5μM，6.2μM，3.1μM，0μM的AG-690，继续培养48小时后，加入10μl的CCK8溶液。放置2-4个小时后取出，在450nm下读取OD值，根据不同浓度下的细胞相对活力，计算得到化合物AG-690抑制结肠癌细胞HCT116活力的IC50为25μM。
Claims (5)
1.一种自噬关键蛋白ATG4B酶抑制剂，其特征在于，其化学结构式为：
其中，R1，R2为单取代、双取代或多取代，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。
2.根据权利要求1所述的抑制剂其特征在于，其化学结构式为：
3.权利要求1或2中所述的抑制剂在抑制ATG4B酶上的应用。
4.根据权利要求3所述的应用，其特征在于，所述的抑制剂在制备治疗肿瘤药物中应用。
5.根据权利要求4所述的应用，其特征在于，所述的抑制剂在制备治疗结肠癌药物中应用。
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