CN102512407A - 一种β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用 - Google Patents
一种β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明属于生物制药领域,具体涉及β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用。
背景技术
糖尿病是由于遗传和环境因素相互作用引起胰岛素绝对或者相对不足以及靶组织对胰岛素敏感度降低,从而导致蛋白质、脂肪、水和电解质等代谢紊乱的综合症。目前全球糖尿病人数3.47亿,中国高达9240万,占四分之一。糖尿病患者长期高血糖状态可导致多种并发症,如糖尿病性白内障、周围神经病变、视网膜病、肾脏病、动脉样硬化等。糖尿病并发症所致的高致死率和高致残率已成为严重的社会问题。从2001年起,连续5年,世界糖尿病日都将关注的重点放在糖尿病并发症的预防和控制上。研究普遍认为,糖尿病并发症与激活醛糖还原酶介导的多元醇代谢通路有关。
醛糖还原酶(EC.1.1.1.21,aldose reductase,ALR2)是多元醇代谢通路中的关键限速酶,催化葡萄糖转化为相应的还原产物山梨醇。在高血糖环境下,ALR2过表达,造成山梨醇在细胞内不断蓄积,导致一系列糖尿病并发,如白内障、神经组织传导阻滞、视网膜病变、肾脏病变、动脉样硬化等。因此,ALR2被认为是治疗糖尿病并发症的重要靶标。目前认为,ALR2抑制剂是防止糖尿病并发症的有效药物。先后有10余种ALR2抑制剂被批准上市,但由于临床上引起各种不良反应,而被撤出市场。目前,仅有依帕司他(Epalrestat,Microlabs Ltd)在中国和日本销售。当前,在研发中的ALR2抑制剂包括Ranirestat(Dainippon andSumitomo Pharm,Phase III)、QR-333(Quigley Pharm,Phase II)GRT-3983Y(Gruenenthal,Phase II)及Fidarestat(Sanwa Kagaku Kenkyusho,Phase III)。
目前,临床使用的一些药物存在副作用及比较差的选择性。因此,针对这一药物靶标,研究开发高效低度的药物是必要的,我们基于虚拟筛选的方法发现了全新的、高选择性、低细胞毒的醛糖还原酶抑制剂。
发明内容
本发明的一个目的是提供一种β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用;本发明的另一个目的在于提供一种含有上述β-苯丙氨酸类化合物的药物组合物及其在治疗和预防由醛糖还原酶引起的疾病方面的应用。
本发明通过下述方案达到上述目的:
一种β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用,其中所述β-苯丙氨酸类化合物的结构式为:
其中,R1、R4、R5为氢;
R2、R3为氢、烷基、烷氧基、巯基或卤素取代;
R6为萘基、嘧啶基、吡嗪基、苯基、四氢化萘基、吲哚基。
R6的基团也可以任选地被C1~4的烷基、烷氧基、羟基、氨基、硝基、氰基、或卤素取代。本发明R6基团中的“任选地被…取代”是指R6的基团可以被这些取代基取代,也可以不被这些取代基取代,即并不仅限制于被所列举的这些基团取代的情况,也包括不被所列举的这些取代基的情况。这种表达方式是扩大到所有所述的基团,即包括取代或者非取代的萘基、取代或者非取代的嘧啶基、取代或者非取代的吡嗪基、取代或者非取代的苯基、取代或者非取代的四氢化萘基、取代或者非取代的吲哚基等。
本发明上述结构式化合物存在手性中心,本发明包括其各种光学异构体。手性优选为S构型,也可以为R构型。
优选地,上述β-苯丙氨酸类化合物的结构式为:
本发明通过如下筛选方法获得上述β-苯丙氨酸类化合物:选取醛糖还原酶的三个晶体结构作为虚拟筛选的起始结构,采用分子动力学做出采样和结构优化验证,并用于基于分子对接的虚拟筛选;以本实验室自备的小分子实体库作为物质基础,采用自建的三个化合物选择标准,通过逐级排除,选择化合物;对分子对接得到的化合物,做基于CUDA的分子动力学,排除由对接产生的假阳性分子,最终得到有潜在活性的化合物;进一步进行醛糖还原酶活性测试,筛选出具有活性的化合物。
上述筛选方法的具体步骤如下:
(1)选取醛糖还原酶的三个晶体结构作为虚拟筛选的起始结构,采用分子动力学做出采样和结构优化验证,并用于基于分子对接的虚拟筛选。
(2)选择本实验室自备的化合物实体库作为筛选化合物库,对化合物数据库做去除离子、加氢、产生合理的三维构象,之后用于分子对接。
(3)化合物的选取,采用自建的三个化合物选择标准,通过逐级排除,最后得到136个化合物。
(4)对分子对接得到的136个化合物,通过分子动力学模拟,排除由对接产生的假阳性分子,最终得到71个潜在的化合物。
(5)对71个化合物进行醛糖还原酶活性测试,发现26个有活性的化合物,其中15个高活性的化合物测定出其IC50(半数抑制浓度)。
(6)在此15个高活性化合物中,β-苯丙氨酸类化合物表现出最好的活性,其活性与上市药物依帕司他相类似。因此,枚举一系列此类化合物,再进一步进行生物学测试,证明β-苯丙氨酸类化合物显示抑制醛糖还原酶活性。
(7)选择性研究:对β-苯丙氨酸类化合物进行人类醛糖还原酶(ALR1)活性测试,发现此类抑制剂和上市药物依帕司他相比,表现出更好的选择性。
(8)为了进一步确定该类化合物的可药性,进行细胞毒活性测试。采用人类胚胎肾细胞用于检测化合物的毒性,证明此类化合物毒性很小,接近上市药物依帕司他。
发明也提供一种药物组合物及其作为醛糖还原酶抑制剂的应用,该药物组合物含有上述的β-苯丙氨酸类化合物,可以在人类和哺乳动物中抑制醛糖还原酶,降低山梨(糖)醇水平从而降低果糖水平,可以作为治疗和预防由醛糖还原酶引起的疾病的药物。所述由醛糖还原酶引起的疾病为糖尿病性白内障、周围神经病变、视网膜病、肾脏病或动脉样硬化。
与现有的抑制剂相比,本发明具有如下优点:(1)对醛糖还原酶具有高活性抑制;(2)对人类醛糖还原酶(ALR1)的选择性高,毒性小;(3)对细胞无毒性;(4)化合物结构简单、易合成。
附图说明
图1为β-苯丙氨酸类化合物的筛选流程;
图2为SYSU-22363S、SYSU-22433S、SYSU-22439S、SYSU-22449S四个化合物对醛糖还原酶的抑制曲线;
图3为细胞毒性试验数据。
具体实施方式
实施例1:虚拟筛选
虚拟筛选具体步骤如图1所示。
从蛋白质晶体结构数据库获取三个结合口袋差异较大、能代表所有抑制剂结合口袋的三个醛糖还原酶晶体结构,其蛋白的代码为2PDK、1US0、2FZD。三个晶体结构作如下处理:去除水分子、修复丢失的氨基酸残基。以这三个结构作为初始结构进行动力学研究。动力学软件采用分子模拟软件包AMBER10来进行。蛋白给予FF03的amber分子力场,小分子采用GAFF力场,小分子的电荷采用RESP方法来拟合。动力学完成后做采样分析,采样利用簇分析方法找到能代表在水溶液里合理构象,随后再做构象优化。得到的三个结构作为平行虚拟筛选的优势构象。采用平行虚拟筛选主要因为该靶标存在诱导效应。
虚拟筛选化合物库为自备化合物数据库。
化合物库作如下处理:去除离子及络合水分子、加电荷、质子化、产生三维构象。这些流程均在药物设计软件包Discovery Studio 2.5中完成。其中质子化在PH6.5-8.5条件下进行。准备好的小分子库用于虚拟筛选。
平行虚拟筛选采用FlexX来进行,苗头化合物的选择遵循自建的三个化合物选择原则:
第一,苗头化合物必需满足对接打分在训练集之下,训练集采用992个活性小于50μM的醛糖还原酶抑制剂。对接到处理好的三个蛋白晶体结构中,得出三个平均打分值,此值作为化合物选择的阈值;
第二,满足阈值的化合物必须要满足一定的结合模式,即自建的蛋白-抑制剂作用的分子指纹图谱(protein ligand interaction fingerprints,PLIF)。此PLIF利用71个蛋白质晶体结构,采用药物设计软件包MOE(Molecular OperatingEnvironment)计算得到;
第三,进行蛋白质-配体动力学模拟研究,排除来自对接的假阳性分子。最终71个化合物作为苗头化合物进行下一步的生物学测试。
实施例2:醛糖还原酶的抑制测试
利用甘油醛作为底物,在醛糖还原酶和辅酶(NADPH)的作用下发生氧化还原反应,NADPH转化成NADP+,表现在紫外区340nM可检测的吸收峰下降的原理,通过改变待测化合物浓度,测得不同浓度药物作用后的醛糖还原酶催化底物得到的不同NADP+的吸收峰值判断药物对酶的抑制程度,从而计算得到药物对醛糖还原酶抑制的IC50值。
实验采用重组的人类醛糖还原酶,经过克隆、表达、纯化、测试得到。实验所用甘油醛、NADPH均购自Sigma公司。实验所测化合物由本实验室自备提供。实验体系为1mL,其中磷酸钠的缓冲液(0.1M,PH=6.8)、NADPH(0.15mM)、硫酸锂(0.4M)、醛糖还原酶(0.486μM)、甘油醛(10mM)。以上浓度均为终浓度。化合物为50mM,后面做具体的稀释。
1、药物配置:
准确称量化合物(结构与代码见表1),加入DMSO溶液溶解至50mM,用磷酸钠的缓冲液稀释至50μM,用于单点的筛选测试。
2、底物制备:
将1g甘油醛用缓冲液配置到0.1mol/L,测试时加入100μl到反应体系即可,终浓度为10mM。
3、反应体系制备:
将人类重组的醛糖还原酶(25mg/mL)以缓冲液溶稀释至4.86μM,采用1mL的比色皿,单点测试样品71个,使用已上市药物依帕司他为阳性对照物,同时空白组及本底对照组加入0.1μL的DMSO,30℃反应90秒。根据NADP+在340nM的吸收峰下降值来检测化合物对醛糖还原酶的抑制。此实验使用紫外分光光度计(UV)。
4、数据处理
计算扣除本底后化合物在50μM下所测到的吸收峰下降值,运用GraphPad Prism 5,计算化合物对醛糖还原酶的抑制常数(见表1)。
5、IC50的测定:
单点测试抑制常数大于50%的化合物,配成不同梯度的浓度,来进行IC50的测试,测试结果同样用GraphPad Prism 5来计算IC50。在测试的化合物中,其中四个β-苯丙氨酸类化合物(SYSU-22363S,SYSU-22433S,SYSU-22439S,SYSU-22449S)表现出很好的活性。表2列出了这四个β-苯丙氨酸类化合物对ALR2的抑制活性。图2给出了四个β-苯丙氨酸类化合物对醛糖还原酶的抑制曲线。
表1 16个β-苯丙氨酸类化合物的结构与对醛糖还原酶的抑制常数
实施例3:醛还原酶的选择性抑制测试
利用甘油醛作为底物,在醛还原酶(ALR1)和辅酶(NADPH)的作用下发生氧化还原反应,NADPH转化成NADP+,表现在紫外区340nM可检测的吸收峰下降的原理,通过改变SYSU-22363S,SYSU-22433S,SYSU-22439S,SYSU-22449S四个化合物浓度,测得不同浓度药物作用后的醛糖还原酶催化底物得到不同NADP+的吸收峰值判断药物对酶的抑制程度,从而计算得到药物对醛还原酶抑制的IC50值。反应体系与醛糖还原酶体系类似,不同之处如下:体系不加入硫酸锂、体系测试温度为37℃、体系所用醛还原酶的浓度为醛糖还原酶的一半(0.243μM)。数据处理同上所述。表2列出SYSU-22363S,SYSU-22433S,SYSU-22439S,SYSU-22449S四个化合物对醛还原酶的抑制活性。
表2四个化合物对醛糖还原酶的抑制活性数据及对醛还原酶的选择性数据
实施例4:细胞毒性测试
利用人类肾胚胎细胞(HEK293)模型,应用MTT法评估β-苯丙氨酸类化合物的细胞毒性。
1、试验方法:
MTT溶液:取125mg MTT粉末用50ml无菌PBS(0.01mM,pH=7.4)溶解,配成2.5mg/mL,在超声清洗仪振荡数十秒,滤过除菌,然后于4℃避光保存。两周内使用有效,最好现配现用。
2、HEK293细胞培养:
HEK293细胞使用含10%胎牛血清的DMEM培养基,在37℃、5%CO2培养箱进行培养。
3、接种细胞:
将生长良好的HEK293细胞,小心弃去培养皿内的大部分细胞培养液,用0.25%胰酶消化4min后,1000rpm/min离心5min;弃去离心后上清液,重新加入DMEM,用血细胞计数板计数,转移单细胞悬液至培养皿并调整细胞密度为8×104个/ml。用8道加样器吸取单细胞悬液加入96孔板中,100μl/孔,即8000个细胞/孔。
4、药物干预:
在37℃,5%CO2培养箱中培养24h,待细胞贴壁后吸去上清液,分别用培养基稀释的不同浓度药物处理细胞,设立不加任何处理因素的对照组,空白孔,每个浓度设3个平行复孔,待测药物(SYSU-22363S,SYSU-22433S,SYSU-22439S,SYSU-22449S)配置初始浓度100μM,采用四个浓度梯度等倍稀释,分别为100μM,50μM,25μM,12.5μM。加入10μM的待测药物溶液至100μl微孔中,与细胞于37℃,5%CO2培养箱中作用48h。每孔加入2.5mg/mL的MTT 20μL,于37℃,5%CO2培养箱中培养4h。弃去上清,每孔加入100μL的DMSO,振荡15秒,测量570nm处的吸收值(A)。
细胞毒性评价:各组细胞存活率(%)=(试验组A值/对照组A值)×100%,从而得出各组细胞存活率。
5、实验结果:
图3为细胞毒性试验数。可以看出SYSU-22363S,SYSU-22433S,SYSU-22439S,SYSU-22449S四个化合物对HEK293细胞无明显毒性,并且低浓度可以促进细胞增长。
Claims (8)
2.根据权利要求1所述的β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用,其特征在于R6进一步被取代,其取代基为C1~4的烷基、烷氧基、羟基、氨基、硝基、氰基或卤素。
3.根据权利要求1所述的β-苯丙氨酸类化合物作为醛糖还原酶抑制剂的应用,其特征在于化合物存在手性中心且为S构型。
5.一种药物组合物,其特征在于含有权利要求1所述的β-苯丙氨酸类化合物。
6.权利要求5所述的药物组合物作为醛糖还原酶抑制剂的应用。
7.根据权利要求5所述的药物组合物,其特征在于作为治疗和预防由醛糖还原酶引起的疾病的药物。
8.根据权利要求7所述的药物组合物,其特征在于所述由醛糖还原酶引起的疾病为糖尿病性白内障、周围神经病变、视网膜病、肾脏病或动脉样硬化。
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