CN114796254B - 多杀菌素衍生物作为精氨酸代琥珀酸合成酶激活剂及其应用 - Google Patents
多杀菌素衍生物作为精氨酸代琥珀酸合成酶激活剂及其应用 Download PDFInfo
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Abstract
本发明公开了多杀菌素A(Spinosyn A)及其衍生物作为精氨酸代琥珀酸合成酶1(ASS1)和突变型精氨酸代琥珀酸合成酶ASS1G362V的激活剂及其应用，所述多杀菌素衍生物具有结构通式(I)：该多杀菌素及其衍生物通过靶向激活精氨酸代琥珀酸合成酶1(ASS1)和突变型精氨酸代琥珀酸合成酶ASS1G362V，从而作为治疗I型瓜氨酸血症的药物和抗肿瘤药物。
Description
本发明是多杀菌素衍生物作为精氨酸代琥珀酸合成酶激活剂及其应用的分案申请，母案的申请日为2020年5月7日，申请号为202010375736.2，申请名称为多杀菌素衍生物作为精氨酸代琥珀酸合成酶激活剂及其应用。
技术领域
本发明涉及多杀菌素A(Spinosyn A)及其衍生物可调节激活精氨酸代琥珀酸合成酶(Argininosuccinate synthetase 1,ASS1)。该类化合物可作为氨酸代琥珀酸合成酶缺陷相关疾病的治疗，如抗肿瘤、瓜氨酸血症，属于医药领域。
背景技术
精氨酸代琥珀酸合成酶(Argininosuccinate synthase，ASS1；EC 6.3.4.5)首先在肝脏中发现，后来在哺乳动物中被认为是普遍存在的酶。ASS1基因位于染色体9q34.11，基因长度为56kb，开放阅读框长度1239bp，带有16个外显子，编码412个氨基酸，分子量为46kDa。ASS1催化瓜氨酸和天冬氨酸在ATP功能的条件下生成精氨酸代琥珀酸，该化合物在精氨酸代琥珀酸裂解酶的作用下进一步分解为精氨酸和延胡索酸，其中，精氨酸进一步进入尿素循环或用于蛋白质合成等代谢过程。其中尿素循环可以将有毒的氨转化为无毒的尿素排出体外，是体内解氨毒的主要途径。如果ASS1表达下调或突变，其酶催化活性下降或者缺乏，将使尿素循环受阻，出现瓜氨酸水平上升。精氨酸代琥珀酸合成酶(是尿素循环关键酶(见图2)。同时，天冬氨酸是ASS1的关键底物之一，ASS1表达量的高低及其活性将决定用于尿循环中的天冬氨酸利用度。ASS1低表达将限制天冬氨酸的利用。
瓜氨酸血症(Citrullinemia，CTLN)是一种常染色体隐性遗传的尿素循环障碍疾病，临床表现上主要为瓜氨酸增加和高氨血症，根据发病机制的不同分为Ⅰ型和Ⅱ型。Ⅰ型瓜氨酸血症(Citrullinemia type 1，CTLN 1)，是由于ASS1基因缺陷导致的，发病率约为 1/250,000，是第三大尿素循环障碍；Ⅱ型瓜氨酸血症(Citrullinemia type 2，CTLN 2)是由于Citrin基因突变所致。CTLN1在临床表现上主要为高氨血症的毒性现象，由发病时间及疾病的严重程度分为经典型和迟发型。经典型瓜氨酸血症于新生儿期发病，表现为高氨血症伴随着神经系统功能衰退，预后差，死亡率高；迟发型瓜氨酸血症则发病较晚，症状较轻，患者可能表现出反复出现的神经症状，如嗜睡、智力障碍等，有部分患者没有症状，只有在新生儿筛查中检测到的生化表型。
导致CTLN1的精氨酸琥珀酸合成酶基因缺陷的突变有很多，目前已被报道的突变有 137种，主要是错义突变，另外也有少数病例出现无义突变、异常剪接及缺失突变。前八种最常出现的突变类型为p.Gly390Arg、p.Trp179Arg、p.Gly362Val、p.Arg363Trp、p.Gly324Ser、p.Arg157His、p.Arg304Trp以及p.Val263Met,分别对应出现在124、27、24、17、16、14、13、以及12个病例中。有研究通过构建体外表达载体的方式体外表达了部分突变型ASS1并检测其酶活性，在突变率最高的前八位突变中，突变p.Gly390Arg、 p.Arg157His、p.Gly324Ser在体外实验中表现为完全失活，而突变p.Trp179Arg、 p.Val263Met、p.Gly362Val在体外仍保留有部分酶活性。这个结果与其他报道携带这些突变的患者的临床表现基本一致。但目前为止还没有明确Ⅰ型瓜氨酸血症基因型与表型之间的相关性。有报道部分患者直到处于高分解代谢状态(手术期间和术后、产后或发烧)时才产生严重的高氨血症。
瓜氨酸血症是一种染色体异常的疾病，目前是没有根治的方法，治疗上主要是低蛋白饮食，降血氨治疗，如果症状比较严重，或者血氨过高，则要依靠血液或是腹腔透析治疗。因此发明治疗瓜氨酸血症的药物有十分重要的临床价值。
肿瘤细胞增殖速度快，需要更多的能量和营养物质，包括核苷酸、蛋白质、脂质等。CAD[氨甲酰磷酸合成酶Ⅱ(Carbamoylphosphate synthetaseⅡ，CPSase)、天冬氨酸转氨甲酰酶 (Aspartate transcarbamoylase,ATCase)和二氢乳清酸脱氢酶(Dihydroorotatehydrogenase， DHOase)]是嘧啶类核苷酸从头合成的关键限速酶。嘧啶类核苷酸从头生物合成如下所述：谷氨酰胺、二氧化碳在胞液中由ATP供能，氨基甲酰磷酸合成酶Ⅱ催化下，生成氨基甲酰磷酸。后者又在天冬氨酸转氨甲酰酶催化下，将氨基甲酰基转移到天冬氨酸的氨基上生成氨甲酰天冬氨酸。氨甲酰天冬氨酸脱水环化，生成二氢乳清酸，再脱氢即成乳清酸。乳清酸是嘧啶类核苷酸的必需前体。由此可知，天冬氨酸也是CAD的关键底物。肿瘤细胞异常增殖，就必须有更多的天冬氨酸参与合成核苷酸。
天冬氨酸含有两个羧基，极性大，食物等外源性天冬氨酸酸很难进入细胞中，细胞内的天冬氨酸的来源依赖于其内源性生物合成。天冬氨酸的分解代谢途径决定其在细胞内作用和功能。天冬氨酸是ASS1和CAD共同的底物，因此，ASS1和CAD二者竞争性利用天冬氨酸。
如果肿瘤细胞ASS1的下调或者缺陷，这导致其底物天冬氨酸的更多地用于嘧啶类核苷酸合成,促进细胞增殖。ASS1与肿瘤生长紧密相关，在一些肿瘤中，ASS1表达下调或存在缺陷，包括乳腺癌、黑素瘤、肝细胞癌、前列腺癌、膀胱癌、间皮瘤，卵巢癌、肾癌、胰腺恶性肿瘤、鼻咽癌、骨肉瘤和粘液纤维肉瘤等。ASS1缺陷与癌症预后不良之间存在明显相关性，被认为是抑癌蛋白，提示ASS1在多种肿瘤中表现为抑癌功能，特别在ASS1缺陷的肿瘤中，激活ASS1可以抑制经天冬氨酸途径合成肿瘤细胞增殖必需的嘧啶核苷酸。因此，我们认为ASS1蛋白可以成为潜在的抗肿瘤药物直接作用靶点。迄今为止，没有任何文献公开报道可以调节ASS1活性的化学小分子。
多杀菌素Spinosyn是土壤刺糖多胞菌(Saccharoplyspora spinosa)经过有氧发酵所产生的胞内次级代谢产物，是大环内酯类抗生素，具有杀虫活性。多杀菌素商品名为Spinosad(SP)，其主要活性成分A(Spinosyn A，SPA,85-90％)和D(Spinosyn D，10-15％)，Spinosyn包含一个独特的四环结构，连接着两个不同的六元糖。SP作为广谱的生物农药，主要防治鳞翅目、缨翅目害虫。哺乳动物的慢性毒性实验表明，SP无致癌、致畸、致突变性或神经毒性，急性毒性试验表明，对哺乳动物的毒性低(老鼠口服(mg/kg)LD50＝3783- 5000；老鼠皮肤(mg/kg)LD50＞2000)。
本发明公开了多杀菌素衍生物作为ASS1激活剂，可以激活ASS1和突变ASS1G362V酶活性，用于治疗ASS1缺陷相关的疾病的药物中，特别是瓜氨酸血症、抗肿瘤。
发明内容
本发明解决的技术问题是，提供一类化合物新用途，该化合物属于多杀菌素衍生物，可作为精氨酸代琥珀酸合成酶(ASS1)激活剂。
本发明的技术方案是，提供一种多杀菌素衍生物及其在医学上可接受的盐作为精氨酸代琥珀酸合成酶(ASS1)激活剂的应用，所述多杀菌素衍生物具有结构通式(I)：
其中，R1选自下列II-Ⅷ基团：
R8、R9均独立地选自氢、1-20个碳原子的烷基(优选2-16个碳原子的烷基，更优选2- 10个碳原子的烷基)、1-20个碳的卤代烷基(优选2-16个碳的卤代烷基，更优选2-10个碳的卤代烷基)、1-6个碳烷基胺基取代的1-10个碳原子烷基(优选2-6个碳原子的烷基)、酰氧基取代的1-10个碳原子的羟烷基(优选2-6个碳原子的羟烷基)、芳甲基、磷酰基、1-10 个碳原子的烷酰基(优选2-6个碳原子的烷酰基)、芳酰基、、 其中，J选自卤原子、R19R20N-、四氢吡咯基、哌啶基、吗啉基、哌嗪基、其中R16选自氢、1-10个碳的烷基(优选1-6个碳原子的烷基)；
R10、R11、R12均独立地选自氢、1-20个碳的烷基(优选2-16个碳原子的烷基)、1-20个碳的烷烯基(优选2-16个碳原子的烷烯基，更优选2-10个碳原子的烷烯基)、芳甲基；
R13选自氢、R14R15N-、含氮杂环、含氧杂环、含硫杂环、含磷杂环；
R14、R15、R19、R20均独立地选自氢、1-6个碳原子的烷基、胺基取代的1-10个碳原子的烷基；
R2选自乙基、丙基、丁基、3-4个碳的烯基；
R3选自氢、甲基；
R4选自氢、羟胺基、-S-R17；其中，R17选自氢、1-6个碳的取代烷基、1-6个碳的烯基、芳甲基、芳基、-(CH2)qCH2YR18；在-(CH2)qCH2YR18中，R18选自H、1-6个碳烷基、芳酰基，取代芳酰基、芳基胺基甲酰基、芳香杂环酰基、1-5碳烷基酰基、芳基烷酰基、N,N-取代氨基甲酰基、烷氧基甲酰基，Y为氧或氮原子，-q＝1,2或3；
R5、R6、R7均独立地选自氢、1-3个碳的烷基、乙酰基、丙酰基；
R21选自A-B选自CH2-CH2、CH＝CH；
M-Q选自CH-CH、C＝CH；
W选自CH2、O、NH、S；
X为阴离子；
X为阴离子，氯、溴、碘、硫酸根、硫酸氢根、磷酸根、甲磺酸根、苯磺酸根、对甲苯磺酸根、氢氧根；
n为0-4的整数，m为0-20的整数。
进一步地，所述R2为乙基。
进一步地，所述R4为氢。
进一步地，所述R5、R6、R7均独立地选自甲基或乙基。
进一步地，所述W选自O、NH、NCH3、S。
进一步地，所述含氮杂环、含氧杂环、含硫杂环、含磷杂环分别是指杂环中的杂原子分别为氮、氧、硫、磷。
进一步地，所述含氮杂环中的杂原子为氮原子，数量为1-3个。
进一步地，所述含氮杂环为四氢吡咯基、哌啶基、吗啉基、哌嗪基、其中R16选自1-10个碳的烷基。
进一步地，所述多杀菌衍生物具有结构：
本发明涉及通式(I)所述化合物，其特征在于专利CN201610355188.0，ZL201010123056.8，CN201610356840.0及US6001981所涉及的多杀菌素及其衍生物。
另外，本发明还提供了包含结构通式(I)所示的小分子组合物作为精氨酸代琥珀酸合成酶的应用，所述小分子组合物除了包含结构通式(I)所示的化合物，还可以包含一种或多种药物上可接受的载体或赋形剂
本发明所述的该药物组合物中包含结构通式(I)所示的多杀菌素及其衍生物，还可以包含一种或多种药物上可接受的载体或赋形剂。
所述载体或赋形剂可以包含甘油、乙醇、缓冲盐水、生理盐水及其组合。所述药物组合物还可以包含渗透促进剂、抗氧化剂等。
另一方面，本发明提供如通式(I)所示多杀菌素及衍生物LM-2I在预防和治疗与ASS1 低表达或突变型ASS1G362V相关疾病的应用。其中所ASS1低表达或突变型ASS1G362V相关疾病包括但不限于I型瓜氨酸血症和相关癌症。
本发明通过利用生物素探针法鉴定小分子靶点，发现了结构通式(I)化合物作用靶点为ASS1。发明人合成和发现多杀菌素A-生物素探针，如Xn-03-17A(简写为17A)，发现其具有良好的抗肿瘤增殖活性，可以替代药物进行靶点实验。我们用Xn-03-17A进行靶点鉴定，结果显示多杀菌素衍生物的作用靶点为蛋白ASS1。并使用ASS1单克隆抗体进行Western blot分析并结合质谱鉴定确证为ASS1。
本发明根据精氨酸代琥珀酸合成酶(ASS1)的活性测定原理和方法测定多杀菌素衍生物对ASS1的激活活性。ASS1活性检测原理是：ASS1催化瓜氨酸与天冬氨酸反应，生成精氨酸代琥珀酸，该反应会消耗ATP生成焦磷酸(PPi)，生成的PPi在焦磷酸酶的催化作用下生产磷酸，磷酸与钼酸铵反应生成磷钼杂多酸，在维生素C的还原下生成蓝色的磷钼蓝，在OD660有特征吸收峰。结果显示，多杀菌素衍生物能够不同程度增加ASS1酶活性，与 ASS1阳性对照相比，酶活性增幅为5.85-203.86％。
本发明采用MTT实验筛选了多杀菌素衍生物对多种人源性肿瘤细胞活性。ASS1低表达与乳腺癌患者的总生存率低、无病生存率低相关，ASS1是乳腺癌总生存率、无病生存率的独立预后因素，可作为乳腺癌的新型候选分子分型标记物。
本发明通过ASS1体外酶活实验发现多杀菌素及其衍生物LM-2I能够不同程度增加ASS1G362V酶活性。
本发明的有益效果：多杀菌素及其衍生物系首次发现的靶向抑癌蛋白精氨酸代琥珀酸合成酶1(ASS1)和I型瓜氨酸血症的突变型精氨酸代琥珀酸合成酶ASS1G362V的一类激活剂，由于ASS1在肿瘤中普遍表达下调或存在缺陷，因而多杀菌素及其衍生物的广泛抗肿瘤疗效显著。同时对于因ASS1基因突变ASS1G362V所导致的难以治愈的先天性遗传病—I型瓜氨酸血症，多杀菌素及其衍生物亦可以作为孤儿药通过逆转并恢复ASS1G362V功能活性发挥明显疗效。多杀菌素及其衍生物主要作为治疗ASS1缺陷相关的疾病的药物，特别是用于I型瓜氨酸血症和抗肿瘤治疗。
附图说明
图1表示多杀菌素衍生物的结构通式；
图2表示精氨酸代琥珀酸合酶1(ASS1)催化反应过程；
图3表示天冬氨酸代谢途径；
图4表示化合物作用靶点的鉴定；图中，4A)细胞裂解液中Pull-down产物银染结果；4B) ASS1抗体Western blot检测图；
图5表示质谱鉴定探针Biotin-SPA结合的蛋白为ASS1；
图6表示不同化合物对ASS1的催化相对活性；
图7表示多杀菌素A(SPA)和LM-2I对突变型ASS1的活性影响；
图8表示ASS1和ASS1G362V的体外酶活实验结果；
图9表示ASS1在乳腺癌多细胞系的表达存在差异。(a)Western blot检测ASS1在多种乳腺癌细胞系中的表达情况，软件Image J对条带进行灰度扫描，以表达量最低的MDA-MB-231 为参照，定义为1，进行相对定量，得到ASS1的相对表达谱(b)；
图10表示乳腺癌多细胞系增殖率与ASS1表达量呈显著负相关。MTT法检测多细胞系48h 的增殖率(a)，(b)细胞增殖率与ASS1表达量的相关性分析；
图11表示ASS1敲低或过表达后对药物敏感性检测。SPA或LM-2I分别处理MDA-MB-231 ASS1过表达细胞系(a)或MCF-7ASS1敲低细胞系(b)48h，MTT检测细胞存活率。每组实验重复三次(mean±s.d.；n＝3)，***表示p＜0.001。
图12表示SPA和LM-2I抑制瘤体体积的增长。SPA(10mg/kg/d)和LM-2I(5m/kg/d)分别给药18天，隔天给药，并记录瘤体最长径和最短径，通过公式计算得到瘤体体积，其中Vehicle为溶剂对照，实验结果采用mean±s.d.统计，n＝7，*表示p＜0.05，**表示p＜0.01。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1
靶点鉴定和确证实验
具体试验步骤如下：
1)合成基于生物素的多杀菌素探针多杀菌素-生物素共合物Xn-03-17A。
(1)N-(3-溴己烷基)多杀菌素的合成：向100mL单口圆底烧瓶中加入多杀菌素B1 g(1.39mmoL)、乙腈20ml和385mg(1.39mmol)碳酸钾，最后加入1,6-二溴己烷2.14 mL(13.9mmoL)，于45℃下搅拌48h。抽滤除去产生的固体，除去溶剂，用50mL水和 EA(3×60mL)萃取，合并有机相，无水硫酸钠干燥。除去溶剂后经硅胶柱层V(乙酸乙酯)：V(石油醚)＝4:1得白色固体840mg,产率73％。m.p.95～98℃；HRMS calcd.forC46H74BrNO10 880.4574，found880.4602。
(2)多杀菌素-己烷基-生物素(Xn-03-17A)的合成：向50mL单口圆底烧瓶中加入DMF1.5 mL、化合物5100mg(0.11mmoL)，和生物素31mg(0.13mmoL)，最后加入K2CO3 16mg(0.11mmoL)，室温搅拌12h。向反应液中加入15ml水，有大量白色沉淀析出，用EA(3× 25mL)萃取，合并有机相，无水硫酸钠干燥。除去溶剂后经硅胶柱层析V(乙酸乙酯)：V (甲醇)＝20:1纯化，再用V(正己烷)：V(异丙醇)＝6:1重结晶得白色粉末68mg，产率 57％，m.p.124～129℃；1HNMR(500MHz,CDCl3)δ:6.78(s,1H),5.90(d,J＝9.7Hz,1H),5.82 (m,1H),5.22(s,1H),4.97(s,1H),4.87(d,J＝1.3Hz,1H),4.73-4.64(m,1H),4.57-4.50(m,1H),4.44(d,J＝6.6Hz,1H),4.38-4.28(m,2H),4.08(t,J＝6.7Hz,2H),3.68-3.61(m,1H),3.59-3.56(m,4H),3.54-3.46(m,10H),3.33-3.27(m,1H),3.20-3.11(m,3H),3.06-2.99(m,1H),2.96-2.92(m,1H),2.91-2.86(m,1H),2.77(d,J＝12.8Hz,1H),2.43-2.33(m,5H),2.30-2.25(m,2H),2.19(s, 3H),1.99-1.92(m,2H),1.74-1.61(m,10H),1.59-1.53(m,4H),1.52-1.45(m,6H),1.39-1.32(m,6H),1.30(d,J＝6.2Hz,4H),1.26-1.25(m,3H),1.20(d,J＝6.8Hz,4H),0.95-0.88(m,1H),0.84(t, J＝7.5Hz,3H)；HRMS calcd.forC56H89N3O13S 1044.6194，found1044.6234。
2)探针Xn-03-17A探寻互作蛋白(pull-down实验)。
用Xn-03-17A与MCF-7细胞裂解液孵育4℃过夜，然后加入链霉亲和素磁珠室温孵育 4h，设置不加、10倍和20倍药物竞争组，磁场吸附磁珠，使用IP裂解液反复清洗磁珠 4次后，加入SDS-loading buffer煮沸10min，SDS-PAGE后硝酸银染色，实验独立重复三次结果一致(n＝3)。如图4结果显示Xn-03-17A泳道有特异性结合带，而10倍和20 倍药物组可以竞争，没有条带。特异性条带进行MS鉴定，结果显示多杀菌素及其衍生物的作用靶点为蛋白ASS1。其中图4A是使用Xn-03-17A探针从MCF-7细胞裂解液中 Pull-down产物银染结果图。图4B为使用ASS1抗体Western blot检测图。本发明确证 ASS1是多杀菌素衍生物的作用靶点。
3)随后，我们将特异性条带切割下来，胶内消化后进行MS/MS质谱分析，通过比对蛋白数据库发现，可信度最高的是ASS1，我们共检测到特异性肽段和特异性图谱，涵盖了117个氨基酸，ASS1共包含412个氨基酸，检测到的肽段占28.4％，而且分子量大小为47kDa(图3)，与银染结果匹配。为了确认质谱的结果，我们使用了ASS1特异性单克隆抗体对Pull-down产物进行Western blot检测，将图3所得到的特异性条带切胶、酶解后进行质谱分析，通过比对数据库，发现ASS1鉴定出来的肽段最多，评分最高，图中红色标记出来的表示鉴定到的肽段(图5)，结果表明拖下来的蛋白确实是ASS1。
实施例2
药物对蛋白ASS1酶活性影响
ASS1活性检测原理是：ASS1催化瓜氨酸与天冬氨酸反应，生成精氨酸代琥珀酸，该反应会消耗ATP生成焦磷酸(PPi)，生成的PPi在焦磷酸酶的催化作用下生产磷酸，磷酸与钼酸铵反应生成磷钼杂多酸，在维生素C的还原下生成蓝色的磷钼蓝，在OD660有特征吸收峰。
酶活性计算公式：
酶活性为：[(药物+ASS1)OD660-NCOD660]/(ASS1OD660-NCOD660)×100％。
(药物+ASS1)OD660：加药物和ASS1时溶液吸光度(波长660nm)；ASS1OD660:
加ASS1时溶液吸光度溶液吸光度(波长660nm)；NCOD660：空白参照溶液吸光度 (波长660nm)。
1)ASS1的原核表达
(1)原核表达载体的构建
提取人乳腺癌细胞系MCF-7细胞系总RNA，RT-PCR扩增ASS1基因，使用引物为 (5′-3′)：
ASS1-F：ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC(SEQ ID NO 3)；
ASS1-R：AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT
(SEQ ID NO 4)。
提取pET-28a质粒，酶切、胶回收后，按同源重组的方法将ASS1基因整合到pET-28a质粒中。
转化：将构建好的pET-28a质粒转化到大肠杆菌DH5a中，筛选出阳性克隆送测序，对于测序正确的克隆扩大培养后提取质粒，转化到BL21(DE3)菌株中，PCR鉴定阳性克隆，确定条带大小为1.0-1.5kb之间，送测序。选取测序正确的菌株-80℃保菌，备用。
(2)原核表达：取出-80℃保存的构建好的ASS1 BL21(DE3)菌株恢复培养至OD660到 0.4-0.6之间，加入终浓度为1mM IPTG进行诱导4h，离心收集菌液，-80℃保存备用。蛋白的提取：将-80℃保存的菌液冰上解冻，按每克2-5mL加入lysis buffer冲液重悬细菌，加溶菌酶至终浓度为1mg/mL，冰上放置30min，超声破壁，离心，取上清，冰上待用。
(3)蛋白的纯化：按每4mL裂解液加入1mL 50％Ni-NTA填料的比例加入填料，4℃旋转孵育60min，将混合物装柱，wash buffer洗两次，每次1mL，elution buffer洗4次，每次1mL，将四次洗脱的液体收集。
(4)蛋白的除盐和保存：将洗脱液放入超滤管中，离心除盐，根据蛋白的性质，调整储存缓冲液，根据所需的浓度，调整缓冲液体积。
(5)分装与保存：将测定好蛋白浓度的蛋白分装，每只10-20μL分装，液氮速冻后-80℃保存。
2)0.5μM ASS1与20μM药物4℃孵育过夜，加入缓冲液37℃孵育1min，之后加入等体积的Molybdate buffer显色3min，37℃，OD660检测吸光度。不予药物孵育的ASS1组作为阳性对照，没有ASS1的试剂组为NC。缓冲液组分为：20mM Tris.HCl(pH 7.8)、2mM ATP、10mM瓜氨酸、10mM天冬氨酸、6mM MgCl2、20mM KCl、0.2U焦磷酸酶。 Molybdate buffer组分为：10mM维生素C、2.5mM的钼酸铵、2％(V/V)硫酸(酶激活结果图6和表1)。
表1多杀菌素衍生物激活精氨酸代琥珀酸合成酶的相对活性
结果显示，多杀菌素衍生物能够不同程度增加ASS1酶活性，与ASS1阳性对照相比，酶活性增幅为5.8-203.9％。SPA和LM-2I的半数激活浓度AC50分别为18.6和2.0μΜ。
实施例3
药物对蛋白ASS1和ASS1G362V酶活性影响
为了检测SPA及衍生物LM-2I对蛋白ASS1和ASS1G362V酶活性影响，我们通过同源重组技术构建了ASS1 pET28a质粒，并在此基础上，采用基因定点突变技术，构建了 ASS1G362VpET28a质粒，构建好的质粒转入BL21(DE3)中进行原核表达并纯化蛋白ASS1 和ASS1G362V。
具体步骤为：
3.1ASS1和ASS1G362V氨基酸序列
ASS1氨基酸序列为：
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMSSKGSVVLAYSGGLDTSCIL VWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALYEDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAP WRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPPGLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGR IDIVENRFIGMKSRGIYETPAGTILYHAHLDIEAFTMDREVRKIKQGLGLKFAELVYTGFWHS PECEFVRHCIAKSQERVEGKVQVSVLKGQVYILGRESPLSLYNEELVSMNVQGDYEPTDATGFININSLRLKEYHRLQSKVTAK(SEQ ID NO 1)。
ASS1G362V氨基酸序列：
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMSSKGSVVLAYSGGLDTSCIL VWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALYEDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAP WRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPPGLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGR IDIVENRFIGMKSRGIYETPAGTILYHAHLDIEAFTMDREVRKIKQGLGLKFAELVYTGFWHS PECEFVRHCIAKSQERVEGKVQVSVLKGQVYILVRESPLSLYNEELVSMNVQGDYEPTDATGFININSLRLKEYHRLQSKVTAK(SEQ ID NO 2)。
ASS1在362位点为G，ASS1G362V在362位点为括号内的V。
3.2ASS1-pET-28a质粒构建
(1)提取人乳腺癌细胞系HCC1806细胞系总RNA，RT-PCR扩增ASS1基因，使用引物为(5′-3′)：
ASS1-F：ACCCTCGAGGGATCCGAATTCATGTCCAGCAAAGGCTCC(SEQ ID NO 3)；
ASS1-R：AGACTGCAGGTCGACAAGCTTTTATTTGGCAGTGACCTT(SEQ ID NO 4)。
(2)提取pET-28a质粒，酶切胶回收后按同源重组的方法将ASS1基因整合到pET-28a 质粒中。
(3)转化：将构建好的pET-28a质粒转化到大肠杆菌DH5a中，筛选出阳性克隆送测序，对于测序正确的克隆扩大培养后提取质粒，转化到BL21(DE3)菌株中，PCR鉴定阳性克隆，确定条带大小为1.0-1.5kb之间，送测序。
其中ASS1的核苷酸序列NCBI数据库中的ASS1 variant 1(ACCESSION： NM_000050)的CDS序列完全一致，当翻译为氨基酸时，其序列与Uniprot数据库中的 ASS1序列完全一致，因此，我们构建的ASS1原核表达载体可以用于后续的研究。选取测序正确的菌株-80℃保菌，备用。
3.3ASS1G362V-pET-28a表达质粒的构建
使用南京诺唯赞生物技术有限公司的Mut Express II Fast Mutagenesis KitV2试剂盒，具体操作如下：
设计点突变引物，根据同源重组的原理设计突变引物：
ASS1G362V-F：TACATCCTCGTCCGGGAGTCCCCACTGTCTCTCTACAAT (SEQ ID NO 5)
ASS1G362V-R：GGACTCCCGGACGAGGATGTACACCTGGCCCTTGAGGAC (SEQ ID NO 6)
PCR扩增：使用突变引物对pet28a-ASS1质粒进行扩增；扩增产物的Dpnl消化，去除甲基化模板质粒；进行同源重组反应，转化，测序鉴定。
3.4蛋白的表达与纯化
(1)原核表达：取出-80℃保存的构建好的ASS1 BL21(DE3)菌株恢复培养至OD660到0.4-0.6之间，加入终浓度为1mM IPTG进行诱导4h，离心收集菌液，-80℃保存备用。
(2)蛋白的提取：将-80℃保存的菌液冰上解冻，按每克2-5mL加入Lysis buffer冲液重悬细菌，加溶菌酶至终浓度为1mg/mL，冰上放置30min，超声破壁，离心，取上清，冰上待用。
(3)蛋白的纯化：按每4mL裂解液加入1mL 50％Ni-NTA填料的比例加入填料，4℃旋转孵育60min，将混合物装柱，wash buffer洗两次，每次1mL，elution buffer洗4次，每次1mL，将四次洗脱的液体收集。
(4)蛋白的除盐和保存：将洗脱液放入超滤管中，离心除盐，根据蛋白的性质，调整储存缓冲液，根据所需的浓度，调整缓冲液体积。
(5)分装与保存：将测定好蛋白浓度的蛋白分装，每只10-20μL分装，液氮速冻后-80℃保存。
其中：Lysis buffer(1L)(使用前加蛋白酶抑制剂，使用前加5mM巯基乙醇)：50 mMTris.HCl，500mM NaCl，10mM imidazole，Adjust pH to 8.0using NaOH；
Wash buffer(1L)(不加蛋白酶抑制剂，使用前加5mM巯基乙醇)：50mM Tris.HCl，500mM NaCl，20mM imidazole，Adjust pH to 8.0using NaOH；
Elution buffer(1L)(不加蛋白酶抑制剂，不加5mM巯基乙醇)：50mM Tris.HCl，500mM NaCl，250mM imidazole，Adjust pH to 8.0using NaOH
洗脱后脱盐换成(20mM的Tris.HCl+100-300mM NaCl)，加入终浓度为1mM TCEP 超滤浓缩至100μM(越高越好)，10μL每只分装，液氮速冻后-80℃保存。
纯化后用考马斯亮蓝染色检测ASS1和ASS1G362V表达纯化情况(图7)。结果显示ASS1和ASS1G362V两种蛋白均在上清液中表达，溶于水，且纯化后的纯度在90％以上。
3.5体外酶活实验
ASS1和ASS1 G362V浓度为0.5μM，与多杀菌素及其衍生物浓度LM-2I为10μM，在 4℃孵育过夜，加入缓冲液，37℃孵育1min，之后加入等体积的Molybdate buffer显色 3min，37℃，OD660检测吸光度。不予药物孵育的ASS1组作为阳性对照，没有ASS1的试剂组为NC。
相对酶活性为：[ASS1G362V OD660-NCOD660]/(ASS1OD660-NCOD660)×100％。
其中缓冲液组分为：20mM Tris.Hcl(pH 7.8)、2mM ATP、10mM瓜氨酸、10mM天冬氨酸、6mM MgCl2、20mM KCl、0.2U焦磷酸酶。Molybdate buffer组分为：10mM维生素C、2.5mM的钼酸铵、2％(V/V)硫酸。
通过ASS1体外酶活实验发现SPA和LM-2I可以恢复突变型ASS1活性。ASS1和ASS1G362V反应浓度为0.5μM，多杀菌素A(SPA)及其衍生物浓度LM-2I为10μM，反应时间为1min，单独的ASS1为阳性对照，结果显示，多杀菌素及其衍生物LM-2I能够不同程度增加ASS1G362V酶活性，其中ASS1为阳性对照，其酶活性标准化为100±6.49％，ASS1G362V酶活性为60.43±3.64％，10μM的SPA处理ASS1G362V后酶活性为70.8±4.22％，与 ASS1G362V本身酶活性相比，活性增加10.37％，不显著；与ASS1酶活性相比，显著降低。 10μM的LM-2I处理ASS1G362V后酶活性94.12±1.83％，与ASS1G362V本身酶活性相比，活性增加33.69％，显著增加；与ASS1酶活性相比，基本一致(图8)。
实施例4
化合物对不同ASS1表达水平肿瘤细胞的活性影响
1慢病毒过表达载体和shRNA干扰载体的构建
ASS1的过表达载体和shRNA干扰载体从吉玛基因购买，其shRNA有两条，序列分别为(5′-3′)：
ASS1 shRNA1：
TGGATGTCAGCAGGGAGTTTGTTTCAAGAGAACAAACTCCCTGCTGACATCCTTTTTTC( SEQ IDNO 7)。
ASS1 shRNA2：
TGGAGGATGCCTGAATTCTACATTCAAGAGATGTAGAATTCAGGCATCCTCCTTTTTTC(S EQ IDNO 8)。
The negative control shRNA sequence：
TGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAACTTTTTTC(SE Q ID NO9)。
ASS1过表达序列采用全合成的方式获得，其序列为人类ASS1 variant 1(ACCESSION：NM_000050)的CDS序列。
2慢病毒的包装
1)复苏293T工具细胞培养至状态良好，胰酶消化，接种到10cm培养皿中，培养过夜，待细胞生长到90％融合时，进行慢病毒包装。
2)取两只1.5mL无菌离心管，一只加入600μL无血清的DMEM培基，按比例加入目的质粒和包装质粒(pGag/Pol、pRev、pVSV-G)，混匀，另一只管加入600μL无血清的 DMEM培基，再加入120μL RNAi-Mate，混匀，室温放置5min后两管混匀，室温放置20 min。
3)将培养皿中的完全培养基换成无血清的DMEM培养基，在将配置好的转染体系逐滴加入培养皿中，轻轻的前后左右晃动混匀，继续在培养箱中培养。
4)继续培养6h后，去除无血清的DMEM培养基，换成完全培养基，继续培养72 h。
5)收取培养皿中上清液到50mL的离心管中，即为病毒原液，将病毒原液低温低速离心，4℃，4000rpm，15min，随后将离心管的上清液用0.45μm滤器过滤。
6)将过滤后的病毒液进行超速离心，4℃，20000rpm，2h，去除上清液，加入适量的培养基溶解病毒，分装到到1.5mL离心管中，贴上标记，-80℃保存。
3慢病毒滴度检测(GFP荧光计数法)
1)293T工具细胞培养至90％融合，胰酶消化，离心后重悬，计数。
2)以每孔3×104细胞/孔接种到96孔板中，混匀后继续培养24h。
3)将慢病毒原液10μL，用完全培养基按10倍稀释5个梯度，加入终浓度为5μg/mL的Polybrene增加感染效率。
4)去除96孔板中原有培基，加入含不同浓度梯度的病毒培养基，继续培养24h，设置好空白对照。
5)去除含病毒培养基，换上完全培养基继续培养72h。
6)荧光显微镜观察GFP荧光，统计荧光细胞，根据稀释倍数计算病毒滴度。
4慢病毒感染及单克隆筛选
1)复苏MDA-MB-231和MCF-7细胞，10cm培养皿培养至状态良好，待细胞长大 90％融合时，消化、离心和计数，MDA-MB-231(1×106/皿)和MCF-7(1.5×106/皿)传代，待细胞密度到40-50％时，进行慢病毒感染实验。
2)根据细胞数量，计算所需病毒量(两株细胞的MOI值均为50)和所需病毒的体积，加入终浓度为5μg/mL的Polybrene，用完全培养基补充体积到8mL，混匀。
3)去除细胞原有培养基，加入含病毒的培养基继续培养24h，24h后去除含病毒培养基，换完全培养基继续培养72h。
4)将感染后的细胞消化传代，加入终浓度为2μg/mL的嘌呤霉素(Puromycin)筛选阳性细胞，并在后续的培养过程中，始终保持培基中含有2μg/mL的嘌呤霉素。
5)存活下来的细胞进行消化，计数，按每孔0.5个细胞的数量接种到96孔板中，进行单克隆筛选。长出来的单克隆胰酶消化后接种到6孔板中，待其长到80-90％左右后，一半用于传代，一半用于提取蛋白Western blot检测目的蛋白的表达情况。对于符合要求的单克隆进行放大培养，液氮保存，并进行MTT实验。
6)MTT法细胞实验:用ASS1敲低或过表达的细胞系检测多杀菌素A(SPA)或LM-2I的药物敏感性，相同浓度的SPA或LM-2I同时处理MCF-7ASS1 sh和MCF-7NC或MDA- MB-231ASS1 OE和MDA-MB-231NC细胞48h。
结果显示：如图9a所示，ASS1在不同细胞系中表达量存在较大差异，在MDA-MB-231中表达最低，在MCF-7中表达最高，为了便于统计，我们使用Image J对条带进行灰度扫描，以ASS1表达量最低MDA-MB-231为参照，定义为1，进行相对定量，得到ASS1相对表达谱(图9b)，数值范围在1-11.16±0.57之间。
采用MTT的方法，以0h为参照，检测乳腺癌细胞系48h的相对增殖率，如图10所示，我们惊奇的发现：ASS1的相对表达量和细胞增殖率呈现显著的负相关(Spearman’sρ＝-0.783，p＝0.003)(图10)。以上结果提示我们，ASS1在乳腺癌中可能扮演肿瘤抑制因子的角色。
结果显示，在MDA-MB-231细胞系中(图11)，相同浓度的SPA或LM-2I处理，与 NC相比，ASS1 OE细胞存活率显著增加，药物敏感性减弱；与之相反，在MCF-7细胞系中(图11-b)，与NC相比，ASS1 sh细胞存活率显著减低，药物敏感性增强。显然，多杀菌素衍生物对低表达ASS1肿瘤具有更好的敏感性，可作为ASS1低表达型肿瘤的个体化治疗药物。ASS1低表达型肿瘤不限于乳腺癌，如黑色素瘤，肝细胞癌，前列腺癌，膀胱癌，间皮瘤，卵巢癌，肾癌，胰腺恶性肿瘤，鼻咽癌，骨肉瘤和粘液纤维肉瘤等。
实施例5
体内肿瘤抑制效果
选择了ASS1表达量低、恶性程度高，易转移，预后差且没有靶向药物的三阴性乳腺癌 MDA-MB-231细胞系做代表进行动物实验。
购买出生4周的免疫缺陷裸鼠BALB/c(nu/nu)，在SPF级动物房适应性饲养一周后，每只裸鼠注射MDA-MB-231细胞5×106到小鼠腋下，4-7天后长成米粒大小瘤体，表示建模成功。溶剂组为阴性对照，临床乳腺癌一线治疗药物5-FU作为阳性对照组，处理浓度为10 mg/kg/d，SPA处理组浓度为10mg/kg/d，LM-2I处理组分两组，浓度分别为5mg/kg.d和10 mg/kg.d，隔天给药，给药10次，每天观察小鼠的生活状态，每次给药前称量体重，测量肿瘤长径a和短径b，肿瘤体积使用公式：V＝1/2ab2 mm3。
给药18天后，Vehicle组肿瘤体积841.52±420.81mm3；SPA(10mg/kg/d)体积386.27 ±77.06mm3，与Vehicle相比降低54.10％，有统计学差异；LM-2I(5mg/kg/d)组体积为 306.41±79.08mm3，与Vehicle相比降低63.60％，有统计学差异。从抑制效果来看，LM-2I (5mg/kg/d)优于SPA(10mg/kg/d)，其中LM-2I药物浓度只有SPA的一半(图12)。
实验结果证实SPA和衍生物LM-2I能有效抑制ASS1低表达型肿瘤生长。多杀菌素及其衍生物可用于ASS1低表达等缺陷性肿瘤的个体化治疗。
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Claims (1)
1.多杀菌素衍生物及其在医学上可接受的盐在制备抑制三阴性乳腺癌的药物中的应用，其特征在于，所述多杀菌素衍生物为LM-2I；
所述LM-2I的结构式为：
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