非肽类共价蛋白酶体抑制剂的研究文献综述

一、课题背景

蛋白酶体(proteasome)是一个ATP依赖的多亚单位组成的蛋白酶复合物，在泛素蛋白酶体通路(ubiquitin proteasome pathway，UPP)关键步骤中起催化作用，广泛参与细胞周期调控、细胞凋亡、DNA修复、细胞信号转导、蛋白的跨膜定位和细胞表面膜受体的内化等多种生理过程，对细胞生长、个体发育有重要影响。

蛋白酶体的组分通常根据它们的斯维德伯格沉降系数（以S标记）来命名，最普遍的蛋白酶体形式是26S蛋白酶体，分子量2000kDa。26S蛋白酶体包含一个20S核心颗粒（CP）和一个19S调节颗粒（RP）。20S核心颗粒类似一个4个环组成的圆柱状物（2个alpha;环和2个beta;环）。每个alpha;环由七个不同的alpha;亚基（alpha;₁-alpha;₇）组成；同样的，每个beta;环由七个不同的beta;亚基（beta;₁-beta;₇）组成，其中beta;₅，beta;₂，beta;₁为催化亚基，分别具有胰凝乳蛋白酶样（chymotrypsin like，ChT-L）活性，胰蛋白酶样（tryps-in like，T-L）活性和半胱天冬酶样（caspase like，C-L）活性。由于蛋白酶体广泛参与细胞周期调控、细胞凋亡、DNA修复、细胞信号转导、蛋白的跨膜定位和细胞表面膜受体的内化等多种生理过程，抑制蛋白酶体的活性，尤其是抑制ChT-L活性已成为癌症药物治疗的重要策略。

2003年，二肽硼酸类蛋白酶体抑制剂硼替佐米（Bortezomib，商品名万珂，PS-341）经FDA批准成为治疗多发性骨髓瘤（multiplemyeloma，MM）和套细胞淋巴瘤的上市药物，证实蛋白酶体为药用靶标。

尽管这些药物在最初的临床试验中取得成功，但不断出现的耐药现象和对实体瘤无效，都促使研究者寻找新的蛋白酶体抑制剂。同时，已有的上市药物均属于多肽类药物，具有多肽类药物共有的缺点：较差的细胞膜渗透率，较快的血浆清除速度，具有免疫原性，需注射使用等，其较差的体内稳定性和药代动力学性质与其对实体瘤的疗效较差密切相关。而非肽类抑制剂可以克服现有药物的以上缺陷。

二、化合物设计

为了克服现有药物肽类骨架带来的缺陷，我们通过寻找非肽骨架，并对其进行结构优化以期获得活性较好的非肽蛋白酶体抑制剂。

2010年，Harshani Lawrence，et al.报道了非肽类蛋白酶体抑制剂PI-8182及其类似物。PI-8182结构如下：

Figure 1非肽类蛋白酶体抑制剂PI-8182的化学结构

PI-8182具有非肽骨架，生物介质中稳定性好，对实体瘤有活性，作用不可逆等优点，但活性不佳。

报道的作用模式图中羧酸跟beta;5亚基重要的Thr1残基有氢键作用，我们的对接结果也证实了这一点。

分析PI-8182的构效关系，去掉羧酸部分或羧酸成酯活性均下降，用羧酸的生物电子等排体四氮唑替换羧酸，活性得到提高。分析替换为四氮唑后的化合物与蛋白的对接结果：

Figure 2 四氮唑替换后化合物与受体的对接结果

我们认为，四氮唑增强了与Thr1之间的氢键作用，使化合物活性得到了提高。因此我们也可以通过对羧酸部分的改造来提升其对Thr1残基的相互作用，从而提升化合物活性。

硼替佐米通过硼酸药效团与Thr1残基发生共价结合，将硼酸基团替换为醛基或乙烯亚砜共价结合片段，活性大幅下降，说明硼酸基团为活性较好的共价结合片段。利用拼合原理，将PI-8182的非肽骨架与硼替佐米共价结合药效团硼酸进行拼合，获得非肽共价蛋白酶体抑制剂。

设计的化合物除了保持硼酸共价结合片段与受体较好的结合外，还与其它关键残基如Asp114，Thr21有氢键作用。虽然氢键数要少于硼替佐米，但设计的化合物中的萘环与下方的残基有很好的疏水作用，芳香磺酰胺片段与下方的一个疏水空腔有较好的相互作用，可以弥补减少肽键而氢键作用减少所造成的结合力的损失。

如下图：

Figure 3 硼替佐米与蛋白酶体晶体复合物相互作用示意图

Figure4 设计的化合物与蛋白酶体对接示意图

为了考察共价结合片段合适的长度及侧链影响，设计了三种侧链

Figure 5 三种拥有不同共价结合片段的化合物

为了考察不同芳香磺酰胺片段对疏水空腔的结合力，设计了以下三种磺酰胺片段

Figure 6 三种不同的磺酰胺片段

总共得到三种化合物

Figure 7 设计的化合物一览

三、合成路线

Scheme 1 目标化合物的合成路线

四、课题进度，遇到的困难及解决办法

目前，合成路线已打通，并合成出了四个目标化合物，在实验过程中发现中间产物和终产物在硅胶中稳定性不高，易分解或氧化，寻找到了打浆或重结晶的纯化方式进行产品纯化。

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