摘要
自组装是生物大分子结构形成的基础方式之一。酶促自组装(enzyme-instructed self-assembly,EISA)借助工具酶,在特定的部位实现小分子化合物向超分子纳米结构的转换,成为药物开发的全新策略。近年来,EISA在恶性肿瘤的治疗和成像领域取得了长足的进步,实现了纳米结构的精确调控和肿瘤靶向。本文综述了EISA在肿瘤诊疗领域的最新进展,工具酶如碱性磷酸酶、去乙酰化酶、酪氨酸酶、γ-谷氨酰转肽酶和胱天蛋白酶3等的作用与特点,总结了在肿瘤治疗中EISA靶向多种细胞器的研究现状,并介绍了EISA在肿瘤成像中的运用,为EISA策略在肿瘤诊疗中的应用研究提供参考。
自组装是单体在无干预的情况下,从游离的、无规律分布的状态,借助分子间的非共价相互作用(如范德华力、静电力、疏水作用力、氢键和π-π堆积),自发形成具有特定结构的有序状态的过
近年来,采用酶促自组装(enzyme-instructed self-assembly,EISA)策略,在特异性酶的催化下,小分子化合物在肿瘤中形成超分子纳米结构的研究取得了重要进
EISA在肿瘤的治疗和分子成像等领域存在巨大的研究和临床价

图1 酶促自组装(EISA)在肿瘤治疗和成像中的应用
ALP:碱性磷酸酶;SIRT5:去乙酰化酶;GGT:γ-谷氨酰转肽酶
EISA采用具有适度水溶性的小分子化合物,在到达肿瘤部位后,经肿瘤细胞高表达的酶识别,催化小分子转化为亲脂亲水两亲性分子,从而改变分子间的非共价相互作用,激发自组装过程形成纳米结
自从2004年,Yang
作为多肽折叠和蛋白质活性的常用调节开关,磷酸化与去磷酸化已被用来控制超分子纳米结构的转
磷酸化位点影响ALP的水解活性,Chen

图2 ALP酶促自组装的底物结构
除了小分子直接作为自组装的模块,ALP也可以催化纳米结构的去磷酸化,调节其自组装形成更复杂的纳米结构。He
尽管对ALP的底物研究主要集中在含有磷酸酪氨酸的小分子上,但是磷酸丝氨酸、磷酸苏氨酸的潜在应用也不可忽
蛋白质中赖氨酸残基可以发生多种翻译后修饰,包括琥珀酰化、乙酰化和甲基化,而赖氨酸在蛋白质折叠过程中十分重要,所以赖氨酸的修饰会影响蛋白质的结构和功
Yang

图3 SIRT5酶促自组装的底物结构
酪氨酸酶是一种含铜氧化酶,可以使单酚(酪氨酸)氧化生成邻二酚(多巴),后者可进一步氧化生成多巴醌,这是黑色素的前体,广泛存在于动植物
依据酪氨酸酶催化的底物结构,Sun

图4 酪氨酸酶催化底物氧化生成二聚体的结构和反应示意图
γ-谷氨酰转肽酶(γ-glutamyl transpeptidase,GGT)是一种细胞膜结合酶,可以催化γ-谷氨酰基的裂
Ye

图5 GGT与谷胱甘肽参与的点击缩合反应示意图
胱天蛋白酶(caspase)家族被发现与细胞凋亡进程的启动和执行密切相关,caspase-3是细胞凋亡执行酶(executioner caspases)的一种。通过特异性地切割天冬氨酸和半胱氨酸之间的肽键,破坏不同结构和功能的蛋白质而导致不可逆转的细胞凋
Wang

图6 Caspase-3和谷胱甘肽(GSH)参与的点击缩合反应
在EISA中使用的酶种还包括弗林蛋白
细胞器在细胞的各项生命活动中起主导作用,靶向性破坏肿瘤细胞器的功能,可以提高对肿瘤尤其是耐药肿瘤的治疗效
三苯基膦(triphenylphosphinium,TPP)阳离子可以在较高的线粒体膜电位驱动下,实现线粒体积累,是一种靶向线粒体的功能基

图7 线粒体靶向的化合物结构
肠激酶(enterokinase,ENTK)存在于HeLa细胞线粒
破坏细胞高尔基体的功能将严重影响蛋白质的合成,翻译后修饰和运输。Tan

图8 高尔基体靶向的化合物结构
尽管HepG2细胞也过表达ALP,但是胞内过量的GSH会干扰高尔基体中CRPs的捕获。Tan
细胞核是重要的细胞器,许多化疗药物的作用靶点位于细胞核,如喜树碱类抗肿瘤药物的靶标拓扑异构酶Ⅰ。由于进入细胞核的药量少,削弱了其抗肿瘤活
Liu

图9 细胞核靶向的化合物结构
Zhan
溶酶体是细胞内外物质降解与循环的重要细胞器,其酸性的内部环境为多种水解酶参与内吞、自噬和细胞凋亡等过程提供了支

图10 其他细胞器靶向的化合物结构
内质网参与生物体脂质的合成,大多数蛋白质的折叠和修饰,干扰内质网稳态可能会引起程序性细胞死
由于非侵入性和可视化的特点,分子成像技术已经成为研究生物分子变化过程的通用工具,同时也是肿瘤早期诊断的重要手

图11 分子成像探针结构
近年来,EISA技术在分子成像领域的应用引起了越来越多的关注。Wu
最近,Ye课题组发表了一系列将EISA应用在肿瘤多模态成像领域的创新性研究工作,为肿瘤的早期诊断带来了新的思路与方

图12 多模态成像探针结构以及逆电子需求的狄尔斯-阿尔德(IEDDA)反应示意图
随着逆电子需求的狄尔斯-阿尔德(inverse electron demand Diels-Alder,IEDDA)反应在分子成像领域的应
化合物 | 特异性酶 | 临界聚集/ 胶束浓度 | 酶促前纳米性质 | 酶促后纳米性质 | EISA作用 |
---|---|---|---|---|---|
1 | ALP | 2 585.4 μmol/L | 无定形 | α-螺旋的直径7 nm纳米纤维 | - |
2 | ALP | 1 538.3 μmol/L | 无定形 | β-折叠的直径11 ~ 23 nm纳米纤维 | - |
3 | ALP | 1 381.6 μmol/L | 短纤维 | β-折叠的直径6 nm纳米纤维 | - |
5 | ALP | 159 μmol/L | 直径(22.4 ± 7.2) nm纳米颗粒 | 直径(21.9 ± 3.7) nm纳米纤维 | 促进内吞与内涵体逃逸 |
6 | ALP | - | 无定形 | 直径24 nm纳米纤维 | - |
7 | SIRT5 | - | 无定形 | 直径10 ~ 50 nm纳米纤维 | 实现SIRT5的活性荧光成像 |
9 | 酪氨酸酶 | - | - | 纳米颗粒 | 抑制微管蛋白组装 |
10 | GGT | - | - | 直径142 nm纳米颗粒 | 增加肿瘤蓄积,提高PET信号 |
11 | Caspase-3 | - | - | 直径(135 ± 28) nm纳米颗粒 | 增强PA信号 |
12 | ALP | - | 直径(8 ± 2) nm纤维 | 直径约100 nm囊泡 | 定位肿瘤细胞 |
13 | ENTK | 165 μmol/L | 胶束 | 纳米颗粒逐渐转化为纳米纤维 | 实现线粒体靶向 |
14 | ALP | 6.0 μmol/L | 胶束 | 纳米纤维 | 实现高尔基体靶向 |
16 | ALP | 83.3 μmol/L | 纳米颗粒 | 直径(85 ± 13) nm纳米带 | 实现细胞核靶向 |
17 | ALP | 91.7 μmol/L | 无定形 | 直径50 ~ 100 nm纳米颗粒 | 定位肿瘤细胞 |
18 | ALP | 19.0 μmol/L | - | 直径(74.6 ± 11.6) nm纳米纤维 | 定位肿瘤细胞 |
19 | ACP | - | - | 直径6.7 nm纳米纤维 | 靶向溶酶体 |
20 | 胰蛋白酶-1 | 194 μmol/L | 直径(15 ± 2) nm胶束 | 直径(8 ± 1) nm纳米纤维 | 靶向内质网 |
21 | ALP | 16.0 μmol/L | - | 直径(39.4 ± 5.0) nm纳米颗粒 | 增强PA信号 |
22 | ALP | - | - | 直径约66 nm纳米颗粒 | 激活NIRF,增加肿瘤蓄积 |
23 | ALP | - | - | 直径约52 nm纳米颗粒 | 激活NIRF,增加肿瘤蓄积 |
24 | ALP | - | - | 直径约250 nm纳米颗粒 | 激活NIRF,增加肿瘤蓄积 |
ENTK:肠激酶;PET:正电子发射断层扫描;PA:光声;NIRF:近红外荧光成像
虽然近几年来EISA取得了长足的进步,但是仍然有些难题亟待解决。(1)可供选择的酶种类较少。借助肿瘤中高表达的酶,小分子化合物可以自组装形成纳米结构,增大在肿瘤中的积累,但是一种酶促策略难以适用于多种肿瘤细胞,而且正常组织中的酶活性会干扰小分子的原位自组装,因此寻找肿瘤,甚至其他疾病特异性的酶将极大促进EISA的发展。(2)给药剂量大,抗癌效率较低。多数研究中的给药剂量仍然处于微摩尔级别,抗癌效率较低,这与小分子较高的CAC相关,未来对小分子自组装能力的优化,将会减少带给人体生命健康造成的隐患。伴随着对EISA的深入研究,其在肿瘤治疗和成像方面的前景广阔。EISA靶向细胞器,走进亚细胞单位,对自组装实现精确调控,引发细胞器功能障碍,减少耐药性的发生,为肿瘤的治疗带来新的策略。EISA可以实现成像信号“关-开”的控制,延长成像时间,为肿瘤的成像提供新的思路。期待EISA技术能够为肿瘤的诊断和治疗发展带来更多可能。
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