CN116327768A - 阿立哌唑在制备LAMP2a蛋白抑制剂以及具有抗癌作用的药物中的应用 - Google Patents
阿立哌唑在制备LAMP2a蛋白抑制剂以及具有抗癌作用的药物中的应用 Download PDFInfo
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Abstract
本发明涉生物医药技术领域,具体公开了阿立哌唑在制备LAMP2a蛋白抑制剂以及具有抗癌作用的药物中的应用。本发明首次发现阿立哌唑可直接与LAMP2a蛋白结合,抑制LAMP2a蛋白激活;因此,将阿立哌唑可作为LAMP2a蛋白抑制剂,可用于解决LAMP2a蛋白激活所引起的促进肿瘤生长的问题;可以将其作为活性成分用于制备抗癌药物。此外,阿立哌唑直接作用于LAMP2a蛋白抑制该蛋白活性,且相互作用位点已经研究清楚;同时,阿立哌唑安全性高,价格低廉,具有较好的开发利用前景。
Description
技术领域
本发明涉生物医药技术领域,具体涉及阿立哌唑在制备LAMP2a蛋白抑制剂以及具有抗癌作用的药物中的应用。
背景技术
目前临床上普遍采用手术、化疗、放疗甚至免疫治疗来清除大部分的肿瘤细胞,但是许多病人术后仍无法得到根治,其原因是:(1)大部分患者确诊时已经属于中晚期,而手术切除效果有限,术后残余的癌细胞会形成新的病灶,从而引发肿瘤复发和转移;(2)常见的靶向药物(Regorafenib、Bevacizumab、Cetuximab、Panitumumab等)存在耐药或毒副作用等各种缺陷,导致部分患者无法手术、无药可用。急需开发新的靶向抗癌制剂。
CMA是一种独特的具有选择性的细胞自噬过程,主要通过 LAMP2a 蛋白发挥降解底物功能,其底物都具备 KFERQ 样基序,被HSC70 识别结合后与溶酶体膜上的 LAMP2a 结合,通过转运复合体转入溶酶体内进行降解。 LAMP2a 的相关研究受到科研工作者的重视,越来越多的研究也证明癌组织中 LAMP2a往往处于高表达水平,可调控糖代谢、内质网应激等和癌症密切相关的生物过程,是目前较有潜力的癌症治疗靶点。然而目前尚未有直接靶向LAMP2a的生物制剂。因此,开发在体内外均能发挥效应的LAMP2a蛋白的抑制剂,是很有必要的。
美国食品与药物管理局(FDA)批准的药物库,其具有药物代谢动力学已知、毒副作用小、价格相对低廉等明显优点。从FDA药物分子库中筛选抗癌药物已被证实是一种切实可行的思路,如已经用于癌症治疗的warfarin(华法林)和 leflunomide(来氟米特)等都已有研究可用于癌症的预防或治疗。
阿立哌唑(aripiprazole)是一种抗精神病药,主要用于治疗精神分裂症,也可用于情感障碍的躁狂发作或混合发作急性期或维持期的治疗,及抽动障碍患者的激惹等。早先有报告阿立哌唑在乳腺癌中有一定的抑制效果,然而到目前为止,尚未有阿立哌唑靶标LAMP2a蛋白的相关报道,LAMP2a为自噬相关蛋白,目前也尚没有阿立哌唑对癌细胞中自噬通路的影响研究。
发明内容
为了克服现有技术中存在的至少之一的技术问题,本发明首先提供了阿立哌唑在制备LAMP2a蛋白抑制剂中的应用。
本发明的技术方案如下:
本发明首先提供了阿立哌唑在制备LAMP2a蛋白抑制剂中的应用。
发明人在研究中首次发现,阿立哌唑可直接与LAMP2a蛋白结合,抑制LAMP2a蛋白激活;因此,将阿立哌唑可作为LAMP2a蛋白抑制剂,可用于解决LAMP2a蛋白激活所引起的促进肿瘤生长的问题。
优选地,所述的LAMP2a蛋白抑制剂以阿立哌唑作为有效成分,还包含药学上可接受的载体。
优选地,所述的LAMP2a蛋白抑制剂制成口服或注射制剂。
优选地,所述的口服或注射制剂包括胶囊剂、片剂、颗粒剂、散剂、丸剂、滴丸剂、缓控释制剂、口服液、合剂、糖浆剂、液体注射剂、注射用粉剂和注射用片剂。
本发明还提供了阿立哌唑在制备具有抗癌作用的药物中的应用。
优选地,所述的癌为肠癌或肺癌。
优选地,阿立哌唑与抗癌化合物联用在制备具有抗癌作用的药物中的应用。
进一步优选地,所述的癌为肺癌。
进一步优选地,所述的抗癌化合物选自莪术烯醇和乌药醚内脂的组合。
进一步优选地,阿立哌唑与莪术烯醇和乌药醚内脂的重量比为3~5:1~2:2~4。
最优选地,阿立哌唑、莪术烯醇以及乌药醚内脂的重量比为4:1:3。
本发明还提供了一种具有抗癌作用的组合物,其包含阿立哌唑、莪术烯醇和乌药醚内脂。
优选地,阿立哌唑与莪术烯醇和乌药醚内脂的重量比为3~5:1~2:2~4。
最优选地,阿立哌唑、莪术烯醇以及乌药醚内脂的重量比为4:1:3。
优选地,所述的癌为肺癌。
发明人在进一步研究中惊奇的发现,将阿立哌唑与莪术烯醇和乌药醚内脂联用后,其具有十分优异的抗肺癌作用;其抗肺癌作用要远远高于单独的阿立哌唑以及单独的莪术烯醇和乌药醚内脂;将阿立哌唑与莪术烯醇和乌药醚内脂联用后可以产生显著的协同抗肺癌作用。
有益效果:阿立哌唑可直接与LAMP2a蛋白结合,抑制LAMP2a蛋白激活;因此,将阿立哌唑可作为LAMP2a蛋白抑制剂,可用于解决LAMP2a蛋白激活所引起的促进肿瘤生长的问题;可以将其作为活性成分用于制备抗癌药物。
此外,阿立哌唑直接作用于LAMP2a蛋白抑制该蛋白活性,且相互作用位点已经研究清楚;同时,阿立哌唑安全性高,价格低廉,具有较好的开发利用前景。
附图说明
图1为等温滴定实验(ITC)证明阿立哌唑可与LAMP2a蛋白直接结合图。
图2为LAMP2a敲除细胞及对照细胞图。
图3为细胞增殖实验证明敲除LAMP2a后可减弱阿立哌唑对肠癌增殖的抑制效果图。
图4为克隆形成实验证明敲除LAMP2a后可减弱阿立哌唑对肠癌增殖的抑制效果图。
图5为阿立哌唑与LAMP2a蛋白的结合模式实验结果图;其中A为分子对接模拟阿立哌唑与LAMP2a蛋白的结合模式图; B为阿立哌唑与LAMP2a蛋白结合位点预测图。
具体实施方式
以下结合具体实施例来进一步解释本发明,但实施例对本发明不做任何形式的限定。
实施例1阿立哌唑抑制LAMP2a蛋白激活以及抑制肠癌肿瘤细胞生长实验
1、首先确定阿立哌唑与LAMP2a蛋白是否结合,我们采用ITC实验来分析(图1):
我们纯化出LAMP2a蛋白,具体过程为:以HT29细胞(购自购于美国标准生物品收藏中心细胞库)的RNA逆转录而成的cDNA为模板,进行PCR扩增,获得LAMP2a基因,然后将其构建至pET-28b表达载体(Addgene)中,并将构建好的载体(LAMP2a-WT载体)转化于BL-21表达菌中,用IPTG(异丙基-β-D-硫代半乳糖苷)以终浓度为0.5mM的条件诱导表达4~6h后收集菌体。采用碧云天公司的His亲和层析纯化柱对重组蛋白进行纯化。通过相应的免疫印迹分析及考染分析,获得LAMP2a蛋白。获得重组的LAMP2a蛋白后,采用等温滴定实验(ITC)实验手段[参考文献:L.Zhang et al.,Crucial residue Trp158 of lipoprotein PiaAstabilizes the ferrichrome-PiaA complex in Streptococcus pneumoniae.Journalof inorganic biochemistry 167,150(Feb,2017)]进行分析,结果如图1所示,表明阿立哌唑与LAMP2a蛋白存在直接相互作用,即阿立哌唑可与LAMP2a蛋白结合。
LAMP2a质粒的引物序列:
2、通过实验证实阿立哌唑确实通过LAMP2a发挥抗癌效果
我们购买了LAMP2a的敲除质粒(IGEbio ,Guangzhou, China),在HT29细胞中构建了LAMP2a稳定敲除细胞系(sgLAMP2a)及对照细胞(sgcon) (图2).构建稳转细胞过程如下:
(1)将293T细胞铺板,待密度达到50%左右时准备进行质粒转染;
(2)293T细胞同时转染LAMP2a的敲除质粒与pMDLg/pRRE(Addgene #12251)、pRSV-Rev (Addgene#12253)和Pmd2.G (Addgene#12259)的混合质粒,三个包装质粒与目的质粒(miR-331稳定过表达质粒)质量配比为1:1:1:1.5 (500ng:500ng:500ng;1.5ug))质粒;转染过夜后将上清移除,加入不含双抗的1640培养基;
(2)24 h,48 h后两次收集细胞上清,此时上清中已经包含病毒,将两次收集的上清混合;
(3)将收集的上清离心去除细胞沉淀,并用0.45 nm的滤膜进行过滤;
(4)将过滤好的上清中按照1:500的比例加入溴化己二甲铵后加入提前铺好板的肠癌细胞中进行感染;
(5)感染48 h后,移除含病毒培养基,更换正常的完全培养基继续培养;
(6)待细胞密度长到50%左右时,加入嘌呤霉素使其总浓度为1 µg/mL进行筛选;
(7)持续观察细胞死亡情况,等到细胞不再发生死亡的时候停止加药;
(8)收集已筛选好的稳转敲除LAMP2a的细胞系进行后续实验。
将构建成功的LAMP2a敲除细胞及对照细胞进行细胞增殖实验(见图3-4),发现敲除LAMP2a后阿立哌唑无法发挥抗癌效果,说明在肠癌中LAMP2a确实为阿立哌唑发挥抗癌效果的作用靶点。细胞增殖实验步骤如下:
分别将LAMP2a敲除细胞(HT29-sgLAMP2a)及对照细胞(HT29-sgcon)铺入96孔板内,每孔3000个细胞;将阿立哌唑溶于DMSO(二甲 基 亚 砜) 中 , 配 制成浓度为1 0 m M的储备液 , 再用1640细胞培养基 (购自 于 L i f e Technologies ,Gaithersburg ,MD,USA)稀释储备液配制成不同浓度的工作液;待步骤LAMP2a敲除细胞(HT29-sgLAMP2a)及对照细胞(HT29-sgcon)贴壁后,按每孔100μL体积加入不同浓度(10、20μM)的工作液到对应孔中,以加入等量DMSO作为空白对照,工作液处理48小时后,用WST-1细胞增殖及细胞毒性检测试剂盒(购自碧云天生物技术有限公司)检测细胞活性。以上3个实验步骤进行3次生物学重复。结果如图3所示:结果表明随着阿立哌唑的浓度升高,细胞的生存能力降低,阿立哌唑浓度越大,细胞生存能力越弱,即阿立哌唑可以抑制肠癌细胞生长。然而在敲除LAMP2a细胞中则无此作用。
另外,我们还采用了克隆形成实验评估不同浓度阿立哌唑对LAMP2a敲除细胞(HT29-sgLAMP2a)及对照细胞(HT29-sgcon)的增值能力的影响。向6孔板中加2ml DMEM培养基,取2000个细胞于6孔板中,加入20μM 的阿立哌唑工作液(配制方法同上),培养箱培养2周,期间换液两次。克隆形成检测加入一定量甲醇(覆盖细胞表面),固定10min,吸出甲醇,加入结晶紫染色5min,用自来水冲洗干净后,倒扣6孔板,晾干,扫描。结果如图4所示,表明随着阿立哌唑使对照细胞的增值能力减弱,而对LAMP2a敲除细胞无作用。
3、为了进一步研究阿立哌唑与LAMP2a蛋白的结合模式,使用Discovery Studio软件对阿立哌唑和LAMP2a的相互作用情况进行计算模拟,并得出潜在作用位点。结果如图5所示,预测阿立哌唑可以与LAMP2a蛋白结合,预测结合位点如图5所示。以上结果确定了阿立哌唑可作为LAMP2a蛋白的抑制剂。预示着阿立哌唑通过抑制LAMP2a酶活是一个有效的肠癌治疗手段,在肠癌临床治疗方面具有广阔的应用价值。
实施例2 阿立哌唑及其组合物抗肺癌实验
(1)取收集对数生长期的A549细胞,用完全培养基配制成浓度为1ⅹ105个/mL的细胞液,按100 μL/孔的接种量接种至96孔板中,并于细胞培养箱(37 ℃,5% CO2)中培养24小时;
(2)步骤(1)培养结束后,在加药孔中加入100 μL不同浓度的待测药物(用完全培养基配制),对照孔中加入不含药物的培养液,每个浓度做6个复孔;加药完毕后继续于细胞培养箱(37 ℃,5% CO2)中培养48小时;
(3)步骤(2)培养结束后,每孔加入20 μL的MTT(5 mg/mL)后,继续于细胞培养箱(37 ℃,5% CO2)中培养4小时;
(4)取出96孔板,吸去MTT溶液;每孔再加入150 μL的DMSO,放置摇床上振摇5min使得紫色结晶完全溶解;然后用酶标仪在570 nm下检测各孔的吸光度值,记录吸光度值,计算待测药物的IC50值;测试结果见表1。
待测的药物如下:
实验组1的待测药物为阿立哌唑;实验组2的待测药物为莪术烯醇;实验组3的待测药物为乌药醚内脂;实验组4的待测药物为由重量比为4:1:3的阿立哌唑、莪术烯醇以及乌药醚内脂组成的组合物;实验组5的待测药物为由重量比为4:1的阿立哌唑和莪术烯醇组成的组合物;实验组6的待测药物为由重量比为4:3的阿立哌唑和乌药醚内脂组成的组合物;实验组7的待测药物为由重量比为1:3的莪术烯醇和乌药醚内脂组成的组合物。
表1.阿立哌唑及其组合物抗肺癌实验结果
从表1实验数据可以看出,实验组4将阿立哌唑与莪术烯醇以及乌药醚内脂三者联用后;其对A549肺癌细胞的IC50值大幅小于单独的阿立哌唑以及单独的莪术烯醇和乌药醚内脂;这说明:将阿立哌唑与莪术烯醇以及乌药醚内脂联用后,其抗肺癌作用得到了大幅的提高,得到的组合物具有十分优异的抗肺癌作用;三者联用可以产生显著的协同抗肺癌作用。
从表1实验数据可以看出,实验组5~6的IC50值与单独的阿立哌唑以及单独的莪术烯醇和乌药醚内脂相比,并未得到降低或大幅的降低;其IC50值远远高于实验组4;这说明:只有将阿立哌唑与莪术烯醇以及乌药醚内脂三者联用后,才具有十分优异的抗肺癌作用;只有将三者联用后才可以产生协同抗肺癌作用。而阿立哌唑与莪术烯醇以及乌药醚内脂中的任意二者联用并不能产生协同抗肺癌作用;阿立哌唑与莪术烯醇以及乌药醚内脂中的任意二者组合后并不具有十分优异的抗肺癌作用。
Claims (4)
1.阿立哌唑在制备LAMP2a蛋白抑制剂中的应用。
2.根据权利要求1所述的应用,其特征在于,所述的LAMP2a蛋白抑制剂以阿立哌唑作为有效成分,还包含药学上可接受的载体。
3.根据权利要求1所述的应用,其特征在于,所述的LAMP2a蛋白抑制剂制成口服或注射制剂。
4.根据权利要求3所述的应用,其特征在于,所述的口服或注射制剂包括胶囊剂、片剂、颗粒剂、散剂、丸剂、滴丸剂、缓控释制剂、口服液、合剂、糖浆剂、液体注射剂和注射用粉剂。
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