CN109745311A - RNase L酶抑制剂的应用 - Google Patents
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Abstract
本发明公开了RNase L酶抑制剂的应用。本发明发现了六种化合物显著抑制RNase L酶的活性,可用于制备RNase L抑制剂、治疗或辅助治疗肿瘤的产品。
Description
技术领域
本发明属于分子生物学、医药生物技术领域,具体涉及抑制RNase L酶的活性的化合物,并涉及该化合物在治疗或辅助治疗肿瘤中的应用。
背景技术
目前有一种新的抗癌疗法,称为溶瘤病毒疗法(Oncolytic Virus Therapy)。溶瘤病毒选择性地靶向肿瘤细胞并在肿瘤细胞内复制增殖而在正常细胞内少量存在或不能增殖,最终导致肿瘤细胞溶解和死亡而对正常细胞没有影响,并且溶解和死亡的肿瘤细胞释放出增殖的病毒颗粒,产生级联效应,扩增溶瘤效果,直至肿瘤细胞被清除。由于溶瘤病毒具有很强的肿瘤识别特异性、复制能力、杀伤力,这种溶瘤病毒疗法在癌症治疗领域备受青睐。2015年,美国食品药品监督局(FDA)正式批准Amgen公司用于治疗手术后残余的黑色素瘤的溶瘤病毒疗法,该疗法采用的药物的名字叫Talimogene laherparepvec(又名或者T-VEC),是一种经过基因改造的单纯疱疹病毒1(Herpes Simplex Virus-1)。
但是,利用体外病毒对人体内的肿瘤细胞进行攻击时,人体自身免疫系统会对病毒的入侵进行抵抗,因此削弱病毒对肿瘤细胞的攻击。其中,RNase L蛋白分子起着重要作用。病毒入侵时,由免疫细胞合成并分泌的干扰素诱导2-5A合成酶(Oligo-adenylateSynthetase,OAS)的表达,2-5A合成酶与病毒双链RNA结合后被激活,激活后的2-5A合成酶利用ATP合成2′,5′-磷酸二酯键连接寡腺苷酸(2′-5′linked oligoadenylate,2-5A),2-5A的化学结构如图1所示,包含三个线性连接的腺苷酸(A):px5′A(2′p5′A)n,其中x=1–3;n≥2]。2-5A继而与RNase L酶结合并使之激活,激活后的RNase L酶降解病毒及宿主细胞的单链RNA,倾向于UN^N(N代表任意核苷酸)序列的酶切位点,在尿嘧啶核糖核苷酸U之后的两个N之间进行剪切,病毒及宿主细胞的很多单链RNA被降解,导致病毒无法复制,入侵人体的病毒得以被清除。被病毒的双链RNA间接激活的RNase L酶在人体自身免疫系统清除入侵病毒的过程中发挥着关键作用,成为了溶瘤病毒疗法的一大障碍。2013年Jha BK等人在《Molecular Therapy:the journal of the American Society of Gene Therapy》(21(9):1749-1757)上发表的文章报道了,在溶瘤病毒疗法的开发中,RNase L酶会对使用的水疱性口炎病毒(Vesicular Stomatitis Virus,VSV)进行攻击,导致VSV病毒对癌细胞的抑制效果不佳。因此,亟需开发出效应强和特异性好的抑制RNase L酶的活性或者抑制RNaseL酶的表达的药物分子,通过抑制RNase L酶的活性或表达,削弱自身免疫系统对溶瘤病毒的攻击,从而提高溶瘤病毒的抗癌效应。
发明内容
本发明的目的在于提供RNase L酶抑制剂及其在制备抗肿瘤产品或溶瘤病毒抗肿瘤增效剂中的应用。
本发明所采用的技术方案是:
以下至少一种化合物在制备RNase L酶抑制剂中的应用:
5-(2-呋喃基)噻吩-2-羧酸(CAS号:868755-62-8),其结构式为:
反-2-氨基-4-环己烯-1-羧酸乙酯盐酸盐(CAS号:142547-16-8),其结构式为:
噻吩-2-磺酰乙腈(CAS号:175137-62-9),其结构式为:
3-(1H-吡洛-1-基)噻吩-2-羧酸(CAS号:74772-17-1),其结构式为:
2-苯基-2-(1H-吡咯-1-基)乙酸(CAS号:105264-23-1),其结构式为:
3-氧代-1-环戊烷羧酸(CAS号:98-78-2),其结构式为:
进一步的,所述RNase L酶抑制剂抑制RNase L酶降解单链RNA序列的活性。
进一步的,所述单链RNA序列含有至少一个UNN序列。
RNase L酶的底物选择性较弱,可以降解很多单链RNA序列,但是倾向于UN^N(N代表任意核苷酸)序列的酶切位点,即在尿嘧啶核糖核苷酸U之后的两个N之间进行剪切;并且,针对不同的核苷酸其降解的速率有所不同:UU>UA>>UG>UC。总的来说,RNase L酶倾向于在UU与UA位点之后进行剪切。
进一步的,所述RNase L酶抑制剂在制备抗肿瘤产品中的应用,其特征在于,所述抗肿瘤产品还包括负载了激活剂的抗肿瘤药物,所述激活剂为RNase L酶或/和2-5A合成酶的激活剂。
进一步的,所述激活剂包括外源双链RNA。
在一些技术方案中,所述抗肿瘤药物负载了能够靶向或杀伤肿瘤细胞的所述激活剂,比如,外源的双链RNA,但是所述激活剂诱使RNase L酶的激活,导致RNase L直接(通过直接降解该激活剂)或者间接失效所述抗肿瘤药物。取所述RNase L酶抑制剂加入到所述抗肿瘤药物中,即可弥补所述抗肿瘤药物的缺陷。
进一步的,所述RNase L酶抑制剂在制备溶瘤病毒抗肿瘤增效剂中的应用,其特征在于,其可减弱人体细胞对溶瘤病毒的抵抗作用,加强溶瘤病毒对肿瘤细胞的裂解,增强人体的免疫系统激活,扩大抗肿瘤效应。
进一步的,所述溶瘤病毒来源于以下至少一种病毒:单纯疱疹病毒、溶瘤腺病毒、新城疫病毒、牛痘病毒、水泡性口炎病毒、脑心肌炎病毒、西尼罗河病毒、冠状病毒。
RNase L酶具有较广的抗病毒谱,包括炎病毒、单纯泡疹病毒I型、牛痘病毒、柯萨奇B4病毒、西尼罗病毒和冠状病毒等。
有益效果是:
本发明中所述的化合物有效抑制RNase L酶的活性,可应用于制备RNase L酶抑制剂。
进一步的有益效果是:
本发明优选的实施例中,所述RNase L酶抑制剂用于制备溶瘤病毒抗肿瘤增效剂。溶瘤病毒的双链RNA直接激活2-5A合成酶的活性,激活后的2-5A合成酶将ATP合成为2′,5′-磷酸二酯键连接寡腺苷酸(2-5A,分子结构如图1所示),2-5A与RNase L结合并将其激活,活化后的RNase L酶降解病毒和宿主的大部分单链RNA,使得溶瘤病毒无法复制和无法发挥清除肿瘤细胞的功效。本发明的RNase L酶抑制剂有效抑制RNase L酶的活性,使得溶瘤病毒得以在宿主肿瘤细胞中复制并杀伤肿瘤细胞,大大发挥溶瘤病毒的抗肿瘤功效。
附图说明
图1. 2-5A的结构式
图2.体外表达具有生物活性的野生型RNase L蛋白的验证
图3.RNase L酶抑制剂的半数有效抑制浓度(IC50)曲线
具体实施方式
下面结合具体实施例对发明作进一步的说明。
本发明采用赛默飞旗下Maybridge公司的Maybridg Ro3片段文库(含1000种类药分子)。该1000种类药分子含有强药效团和具有优越的ADME(吸收(absorption)、分布(distribution)、代谢(metabolism)及排泄(excretion))性质;且结构多样性广、含有容易结合的基团、符合“Rule of Three”性能(脂水分配系数系数logP小于3、分子量小于300Da、不超过三个氢键供体、不超过三个氢键受体、不多于三个可旋转的键等);因此,采用该Maybridge Ro3片段文库筛选出来的具有生物活性的片段分子具有优良的先导化合物性能和药物开发潜力。
实施例中,各原料试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
实施例1
1材料与方法
1.1Maybridge Ro3片段文库
1.2RNase L蛋白样品的制备
1.2.2RNase L表达载体的构建
将RNase L的核苷酸序列片段NM_001097512插入载体pGEX-4-T1的BamHI和XhoI多克隆位点,构建pGEX-FL-RNase L重组质粒(FL:Full length,全长)。
1.2.3RNase L的体外表达及活性鉴定
将酶切和测序鉴定正确的重组质粒pGEX-FL-RNase L转化至大肠杆菌BL-21感受态菌株,筛选阳性克隆,挑取单克隆接种至5mL LB液体培养基培养过夜,然后将所得菌液转接至LB液体培养基进行扩增培养,当菌体密度OD600达到0.6-1.0时加入0.4mM IPTG,18℃诱导培养,过夜。离心收集菌体,高压破碎,高速离心,获得蛋白上清液。将所得上清液与GST亲和填料低温下混合,孵育3小时,冲洗填料,再加入TEV蛋白酶,低温过夜。收集经TEV酶切后的蛋白,最后通过阴离子交换以及SEC分子层析纯化获得野生型RNase L蛋白。取样进行SDS-PAGE测定和酶活性检测。
1.3RNase L活性检测方法
采用基于荧光共振能量转移(FRET)技术的酶活性检测方案
1.3.1底物材料
RNA荧光探针底物:6-FAM-UUA UCA AAU UCU UAU UUG CCC CAU UUU UUU GGUUUA-BHQ-1;FAM(羧基荧光素)为荧光报告基团,BHQ1为荧光猝灭基团,该序列源自呼吸道合胞病毒基因组的RNA序列,包含多个切割位点(UU和UA),对RNase L酶的切割具有高度敏感性。
1.3.2酶活性检测
混合RNase L蛋白(25pg/μl)、2-5A(1nM)、RNA荧光探针底物(100nM),22℃孵育1h。利用多孔酶标仪(PerkinElmer Envision 2104)在480nm激发光和535nm发射光波长下检测荧光信号,测定相对荧光单位(RFU)值。
2结果与分析
2.1RNase L蛋白的体外表达及活性鉴定
经大肠杆菌的原核表达系统表达、层析、纯化后获得野生型RNase L蛋白。
SDS-PAGE分析结果如图2A所示,野生型RNase L蛋白的相对分子质量为83.9kDa,获得了高浓度的野生型RNase L蛋白,纯度可达到95%以上。
将所得野生型RNase L蛋白进行酶活性检测实验,对照组采用的是BSA蛋白,利用多孔酶标仪测定相对荧光单位(RFU)值,根据不同反应时间及其对应的RFU值绘制曲线,如图2B所示,野生型RNase L蛋白所在的实验组具有很好的酶活性曲线特征,说明采用本实施例中所述的体外表达方法成功获得了具有生物活性的野生型RNase L蛋白。
2.2RNase L酶抑制剂的筛选
(1)从制造商购买Maybridge Ro3文库的化合物(1000种化合物,200mM)。将化合物在多孔板中稀释成500μM,取野生型RNase L蛋白(25pg/μl)与孔内对应的化合物(500μM)混合,冰上孵育10min,加入2-5A(1nM)和RNA荧光探针底物(100nM),22℃孵育1h。设置DMSO组(25pg/μl)、不加入激活配体2-5A组作为阴性对照。利用多孔酶标仪(PerkinElmerEnvision 2104)在480nm激发光和535nm发射光波长下检测荧光信号。根据所在孔的荧光信号强度,初步筛选出低于DMSO组的信号强度值一半以上的实验组,确定了26种化合物为阳性候选物,针对该26种化合物重新进行上述酶活实验,重复三次,其中6种化合物的抑制效果具有较佳的可重复性,优选该6种化合物用作RNase L酶抑制剂。
(2)测定化合物的半数有效抑制浓度(IC50)
设置的对照组加入野生型RNase L蛋白(25pg/μl)、RNA荧光探针底物(100nM)和2-5A(1nM),测得其荧光强度为F0;设置的实验组加入一定浓度的上述化合物、野生型RNase L蛋白(25pg/μl)、RNA荧光探针底物(100nM)和2-5A(1nM),测得其荧光强度为F,在抑制率(1-F/F0)为50%时所对应的化合物的浓度就即为IC50。
设置实验组的化合物的浓度梯度为1000μM,500μM,250μM,125μM,61.25μM,30.6μM,15.3μM,7.6μM,3.8μM,每个梯度平行三组,分别与25pg/μl野生型RNase L蛋白混合,冰上孵育10min,加入2-5A(1nM)和RNA荧光探针底物(100nM),22℃孵育1h。设置DMSO组(25pg/μl)、不加入激活配体2-5A组作为阴性对照。利用多孔酶标仪(PerkinElmer Envision 2104)在480nm激发光和535nm发射光波长下检测荧光信号,测定荧光强度值。
以化合物浓度的以10为底的对数值为横坐标,抑制率为纵坐标,采用GraphPadPrism 6.0绘制IC50曲线(如图3所示)并计算出各化合物的半数有效抑制浓度(IC50),如下表所示:
在本发明中,采用国际先进的基于片段的药物开发方法(FBDD),筛选出了能有效抑制靶点蛋白RNase L酶的活性的类药分子,提供了可以应用于增强溶瘤病毒疗法抗癌效应的RNase L酶的特异性抑制剂。
以上是对本发明的较佳实施进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明精神的前提下还可做出种种的等同变形或替换,这些等同的变形或替换均包含在本申请权利要求所限定的范围内。
Claims (7)
1.以下至少一种化合物在制备RNase L酶抑制剂中的应用:
5-(2-呋喃基)噻吩-2-羧酸(CAS号:868755-62-8),
反-2-氨基-4-环己烯-1-羧酸乙酯盐酸盐(CAS号:142547-16-8),
噻吩-2-磺酰乙腈(CAS号:175137-62-9),
3-(1H-吡洛-1-基)噻吩-2-羧酸(CAS号:74772-17-1),
2-苯基-2-(1H-吡咯-1-基)乙酸(CAS号:105264-23-1),
3-氧代-1-环戊烷羧酸(CAS号:98-78-2)。
2.根据权利要求1中所述的化合物在制备RNase L酶抑制剂中的应用,其特征在于,所述RNase L酶抑制剂抑制RNase L酶降解单链RNA序列的活性。
3.根据权利要求2所述的化合物在制备RNase L酶抑制剂中的应用,其特征在于,所述单链RNA序列含有至少一个UNN序列。
4.权利要求1-3中任一项所述的RNase L酶抑制剂在制备抗肿瘤产品中的应用,其特征在于,所述抗肿瘤产品还包括负载了激活剂的抗肿瘤药物,所述激活剂为RNase L酶或/和2-5A合成酶的激活剂。
5.根据权利要求4所述的应用,其特征在于,所述激活剂包括外源双链RNA。
6.权利要求1-3中任一项所述的RNase L酶抑制剂在制备溶瘤病毒抗肿瘤增效剂中的应用,其特征在于,所述溶瘤病毒负载了激活剂,所述激活剂为RNase L酶或/和2-5A合成酶的激活剂。
7.根据权利要求6所述的应用,其特征在于,所述溶瘤病毒来源于以下至少一种病毒:单纯疱疹病毒、溶瘤腺病毒、新城疫病毒、牛痘病毒、水泡性口炎病毒、脑心肌炎病毒、心肌炎病毒、西尼罗河病毒、冠状病毒。
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CN112022855A (zh) * | 2020-06-08 | 2020-12-04 | 北京大学深圳研究生院 | PLpro蛋白抑制剂在治疗或预防新型冠状病毒感染的药物中的应用 |
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CN112022855A (zh) * | 2020-06-08 | 2020-12-04 | 北京大学深圳研究生院 | PLpro蛋白抑制剂在治疗或预防新型冠状病毒感染的药物中的应用 |
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