基于超高效液相色谱串联质谱联用法的HMG-CoA还原酶抑制剂的筛选
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摘要
甲羟戊酸通路（mevalonate pathway，MVA）是一条极其复杂的生化通路，该通路在一系列酶的调控作用下最终能够生成对人体至关重要的终产物：胆固醇、多萜类、类异戊二烯、辅酶Q以及异戊烯腺嘌呤等。而对通路中的调控酶3-羟基-3-甲基戊二酰辅酶A还原酶（3‐Hydroxy‐3‐methylglutaryl coenzyme A，HMGCR）的研究是胆固醇代谢研究中的核心内容。HMGCR是肝脏中胆固醇合成的限速酶，是整个胆固醇代谢过程中第一个也是最重要的酶。研究发现抑制该酶的活力能够降低血液中胆固醇的含量，降低患心脑血管疾病的风险，尤其是能够有效控制高血压、冠心病以及动脉粥样硬化等疾病。
HMGCR基因广泛存在于生物界中，在原核、真核和古生物界中均有表达。通过序列比对分析HMGCR分为两个亚型：真核HMGCR（class I）和原核HMGCR（class II）。其中以人类为代表的真核生物的HMGCR归于还原酶class I。HMGCR基因位于人类第5号染色体，包括20个外显子，19个内含子，编码26kb的基因。所有哺乳动物的HMGCR都位于内质网膜上，人类的HMGCR蛋白由888个氨基酸残基组成。该蛋白被分为两个相邻的区域，N端是跨膜功能区，通过自身以短环连接的八个跨膜片段牢牢固定在内质网膜上。中间有109个氨基酸残基组成的连接区。C端是它的催化结构域，HMGCR通过这一区域发挥其催化活性。
目前，临床治疗高胆固醇症的药物主要为他汀类药物。该药能够竞争性的抑制肝脏中合HMGCR的活性，从而降低胆固醇的合成速率，达到降低血脂的目的。虽然他汀类药物在抑制HMGCR方面有显著效果，但其副作用近年来也屡见报道，主要体现在肌肉疼痛以及肌肉无力、抽搐、血清肌酸激酶（CK）水平偏高等，其中危害较大的是肌肉横纹肌溶解。此外，对肝脏以及肾脏的损伤主要是转氨酶升高，但其原因尚不明确。也有人认为长期肝脏酶类指标异常易导致非酒精性脂肪肝。基于上述他汀类药物可能引起的并发症，开发更安全更有效的HMGCR的抑制剂成为当下的研究热点。
天然产物诸如中药、茶叶等具有廉价易得，对身体危害较小等特点而被广泛关注。因此本研究克隆并纯化出人HMGCR的催化结构域，并从天然产物中筛选其有效的抑制剂。
我们以人肝癌细胞HepG2cDNA为模板，通过PCR扩增HMGCR催化结构域基因，然后将其克隆到pGex‐HisC3载体中，并将重组质粒转入大肠杆菌Rosstta DE3plysS中，加入IPTG诱导表达，最后通过亲和层析获得了融合蛋白GST‐△HMGCR。而后建立了用超高效液相色谱电喷雾串联质谱仪（UPLC‐ESI‐MS/MS）检测酶促反应体系中还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）浓度变化，来检测HMGCR酶活并快速筛选其抑制剂的方法。
以往针对该酶的主要检测方法有放射化学分析法、分光光度计法以及高效液相法（HPLC）。但前者由于对身体危害较大，较少被使用。分光光度计法是目前普遍使用的方法，该方法方便快速，但对底物溶液的损耗量较大且结果不够精确。NADPH极性太大，在C18色谱柱里几乎没有保留，且很多天然产物抑制剂都有很强的紫外吸收，因而HPLC法无法将NADPH同抑制剂等酶反应体系中的其他物质分开，导致在筛选HMGCR抑制剂的时候容易造成结果不准确的问题。故而我们利用UPLC‐ESI‐MS/MS法检测HMGCR酶反应体系中NADPH含量。该方法线性相关系数好，定量范围低，灵敏度高，节省了底物，降低了HMGCR抑制剂研发的成本。
我们考察了HMGCR酶的活性，结果表明，我们纯化的GST-△HMGCR比活力约为2400U/mg，GST‐△HMGCR在6分钟内显示了良好的线性关系。
茶叶是东亚，尤其是中国人的日常饮料，具有很好的保健功效，能够抗衰老、防癌、杀菌、消炎、瘦身等。并具有明显的抑制血脂增高的功效，且价格低廉，副作用小，是潜在的治疗与血脂偏高相关疾病的研究目标。虽然茶叶的功效日益受到关注，但是其降脂机理尚不清楚。为此，我们制备了不同种类的茶溶液，并从中筛选HMGCR的抑制剂，从实验结果看，同红茶以及普洱茶相比，绿茶能够明显的抑制HMGCR的活性，当其水提物浓度为1mg/mL时，抑制率可以达到42.9%。该结果提示绿茶中可能存在某些可以抑制HMGCR的物质。鉴于此，我们对绿茶的组份进行了进一步的筛选。
根据以往报导，绿茶的重要成份EGCG（表没食子儿茶素没食子酸酯）对癌症、糖尿病、肥胖以及心脑血管疾病均有一定的预防治疗作用。EGCG能够抑制诸如11β‐甾类脱氢酶、5α‐还原酶、芳香酶等与固醇相关的酶。经过实验发现，EGCG还对抑制HMGCR的活性有比较明显的作用。当EGCG浓度达到1mM时，抑制率可以达到46.3%。
但是，进一步研究表明，EGCG只有在存在丙三醇的条件下才能够抑制HMGCR的活性，而缺少丙三醇的反应体系中，EGCG则对HMGCR没有明显的抑制效果。并且随着丙三醇浓度的增高，EGCG对HMGCR的抑制作用逐渐加强。当丙三醇浓度达到600mM（5.4%）时，其抑制效果最为明显。但之后，抑制效果又随着丙三醇浓度的增大而逐步下降。应用分子docking软件进行分子动力模拟分析，结果显示，HMGCR‐EGCG‐丙三醇三者结合的自由能确实低于EGCG单独与HMGCR结合的自由能。因此，我们推测很可能是丙三醇促进了HMGCR同EGCG形成复合物，致使底物不能进入酶的活性中心。在丙三醇的协同作用下，EGCG同HMGCR的相互作用力明显增强。
最后我们以HepG2细胞为模型，在细胞水平上研究了EGCG的降胆固醇作用，以及对HMGCR mRNA和蛋白表达水平的影响。结果表明EGCG能够降低细胞胆固醇水平，但是不能降低HMGCR mRNA和蛋白表达水平，说明EGCG不是通过下调HMGCR转录以及翻译水平从而降低胆固醇内源性合成。提示其可能同他汀类药物一样，通过抑制HMGCR的活性以下调胆固醇的合成。
The MVA pathway is a complex biochemical pathway required for thegeneration of several fundamental end-products including cholesterol, isoprenoids,dolichol, ubiquinone, and isopentenyladenine. At the heart of this pathway is therate-limiting enzyme,3-hydroxy-3-methylglutaryl CoA reductase. It is widelyacknowledged that HMGCR has been recognized as the rate-limiting enzyme insynthesis of cholesterol. The degradation of the reductase can reduce serumcholesterol levels and dramatically decrease the risk of cardiovascular andcerebrovascular diseases, essential hypertension, coronary heart diseases, andatherosclerosis.
Comparison of the sequence of HMGR has revealed the existence of twoclasses of this enzyme. The class I enzymes was found in eukaryotes and somearchaea, and the class II found in certain eubacteria and the archaea．In allmammalian species that have been studied to datereductase localizes to membranesof the endoplasmic reticulum (ER) and consists of888amino acids that can beseparated into two contiguous domains. The N-terminal domain of reductase isintegrated into membranes by virtue of eight membrane-spanning segments that areseparated by short loops. The C-terminal domain of reductase projects into thecytosol and exerts all of the enzymatic activity.
Statins lower cholesterol levels by specifically inhibiting HMGCR, a keyenzyme in the cholesterol biosynthesis pathway. These drugs significantly reducecardiovascular end points and are among the most commonly prescribedmedications. but their side effects are occasionally serious. A well-knowncomplication of statins use is musculoskeletal symptoms. Few patients develop anautoimmune myopathy even after the statins are discontinued. In addition, the use ofstatins may elevate serum transaminases, induce a statin-induced inhibition ofproximal tubular reabsorption of protein and have deleterious effects on the peripheral nervous system. In addition, The damage on the liver and kidney ismainly elevated transaminase which occurs after the compensatory increasedaccompany with the resulting dose-dependent drug. but the reason is not yet clear. Itis suggested that long-term liver enzymes indicators abnormalities lead tonon-alcoholic fatty liver. On the basis of data available from above, statins therapymay involve some risks of potential complications. Therefore, the development ofsafer inhibitors of HMGCR with less serious side effects has become the main studyconcern in recent years.
Natural products such as traditional Chinese medicine, tea are widespreadconcern because their cheap and easy to get, smaller or even no toxic effects to thehealth. So in the present study, we cloned and purified the catalytic domain ofhuman HMGCR(△HMGCR) and screened its inhibitors from natural products.
Total RNAs were extracted from Human Liver Cells (HepG2), the cDNAs weresynthesized from the polyadenylated mRNA, the Catalytic Domain of HMGCR wasamplified by PCR and then the truncated enzyme was purified by GST Resincolumn. A rapid and accurate method has been developed for screening HMGCRinhibitors by measuring the concentration of NADPH in the enzymatic reactionsystem using ultra performance liquid chromatography-electrospray ionizationtandem mass spectrometry.
Traditional detection methods for the enzyme are radiochemical method,spectrophotometer and HPLC. But the former is less used for its great harm. thespectrophotometer method is commonly used method for its convenient and fast, butthe method is waste in the substrate solution and the results are not accurate enough.NADPH polarity is too high, so it was almost no reservations in the C18column,many of the natural product inhibitors have very strong UV absorption, and thusNADPH can not be separated from the other substances in the enzyme reactionsystem by HPLC. The UPLC-ESI-MS/MS we used has a good linear correlationcoefficient, it can save the substrate and reduce the cost of the HMGCR inhibitor developed for the lower quantitative range.
We analyzed activity of HMGCR and screened inhibitors in the traditionalChinese medicine, tea and other natural products. The results showed that△HMGCR shows a good linear relationship in the first6minutes.
Teas are the East Asia, especially the Chinese people's day-to-day drinks. theyhave many benefits, such as anti-aging, anti-cancer, sterilization, anti-inflammatory,slimming, significantly lower the blood lipids. So they become potential researchobjectives treatment to high blood lipids related diseases.
We prepared the different kinds of tea solution, and screened HMGCR inhibitor.It shows that green tea can inhibit HMGCR compared with Pu'er and black tea.when its concentration is100μg/mL, the inhibition rate reach42.95%. The resultsshow that green tea can inhibit cholesterol synthesis by inhibiting HMGCR enzymeactivity. we further screen inhibitors from its subdivided components.
Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol compoundin green tea. As a natural product with less serious side effects, it has been used toprevent cancer, obesity, diabetes and cardiovascular disease. EGCG can inhibit somesteroid-related enzymes, such as11β-hydroxysteroid dehydrogenase,5α-reductaseand aromatase, but it is ineffective in the inhibition of HMGCR. However, ourresults showed that EGCG exhibits exciting inhibitory ability against HMGCR.Subsequent study indicated that EGCG is able to inhibit HMGCR with the additionof some glycerol to the reaction system. The inhibitory effects of HMGCR enhancedwith the increase in concentration of glycerol. When the concentration reached up to600mM (5.4%), the best inhibition was observed. Afterwards, the inhibitory effectgradually reduced as the concentration surpassed600mM.
In order to explore to role that glycerol played in this process, the analysis ofmolecular dynamics simulation was conducted on HMGCR-EGCG-glycerolinteractions. The3D structure of HMGCR was adopted. Through the model ofdynamics simulation, we speculate that EGCG and HMGCR form certain complex so that substrates are prevented from penetrating into the active center. The resultsrevealed that the affinity between EGCG and HMGCR is significantly stronger inthe synergy of glycerol.
Moreover, EGCG can decrease cellular total cholesterol in HepG2cells withoutchanging HMGCR mRNA and protein levels after24hrs incubation compared tocontrol.
HMGCR基因广泛存在于生物界中，在原核、真核和古生物界中均有表达。通过序列比对分析HMGCR分为两个亚型：真核HMGCR（class I）和原核HMGCR（class II）。其中以人类为代表的真核生物的HMGCR归于还原酶class I。HMGCR基因位于人类第5号染色体，包括20个外显子，19个内含子，编码26kb的基因。所有哺乳动物的HMGCR都位于内质网膜上，人类的HMGCR蛋白由888个氨基酸残基组成。该蛋白被分为两个相邻的区域，N端是跨膜功能区，通过自身以短环连接的八个跨膜片段牢牢固定在内质网膜上。中间有109个氨基酸残基组成的连接区。C端是它的催化结构域，HMGCR通过这一区域发挥其催化活性。
目前，临床治疗高胆固醇症的药物主要为他汀类药物。该药能够竞争性的抑制肝脏中合HMGCR的活性，从而降低胆固醇的合成速率，达到降低血脂的目的。虽然他汀类药物在抑制HMGCR方面有显著效果，但其副作用近年来也屡见报道，主要体现在肌肉疼痛以及肌肉无力、抽搐、血清肌酸激酶（CK）水平偏高等，其中危害较大的是肌肉横纹肌溶解。此外，对肝脏以及肾脏的损伤主要是转氨酶升高，但其原因尚不明确。也有人认为长期肝脏酶类指标异常易导致非酒精性脂肪肝。基于上述他汀类药物可能引起的并发症，开发更安全更有效的HMGCR的抑制剂成为当下的研究热点。
天然产物诸如中药、茶叶等具有廉价易得，对身体危害较小等特点而被广泛关注。因此本研究克隆并纯化出人HMGCR的催化结构域，并从天然产物中筛选其有效的抑制剂。
我们以人肝癌细胞HepG2cDNA为模板，通过PCR扩增HMGCR催化结构域基因，然后将其克隆到pGex‐HisC3载体中，并将重组质粒转入大肠杆菌Rosstta DE3plysS中，加入IPTG诱导表达，最后通过亲和层析获得了融合蛋白GST‐△HMGCR。而后建立了用超高效液相色谱电喷雾串联质谱仪（UPLC‐ESI‐MS/MS）检测酶促反应体系中还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）浓度变化，来检测HMGCR酶活并快速筛选其抑制剂的方法。
以往针对该酶的主要检测方法有放射化学分析法、分光光度计法以及高效液相法（HPLC）。但前者由于对身体危害较大，较少被使用。分光光度计法是目前普遍使用的方法，该方法方便快速，但对底物溶液的损耗量较大且结果不够精确。NADPH极性太大，在C18色谱柱里几乎没有保留，且很多天然产物抑制剂都有很强的紫外吸收，因而HPLC法无法将NADPH同抑制剂等酶反应体系中的其他物质分开，导致在筛选HMGCR抑制剂的时候容易造成结果不准确的问题。故而我们利用UPLC‐ESI‐MS/MS法检测HMGCR酶反应体系中NADPH含量。该方法线性相关系数好，定量范围低，灵敏度高，节省了底物，降低了HMGCR抑制剂研发的成本。
我们考察了HMGCR酶的活性，结果表明，我们纯化的GST-△HMGCR比活力约为2400U/mg，GST‐△HMGCR在6分钟内显示了良好的线性关系。
茶叶是东亚，尤其是中国人的日常饮料，具有很好的保健功效，能够抗衰老、防癌、杀菌、消炎、瘦身等。并具有明显的抑制血脂增高的功效，且价格低廉，副作用小，是潜在的治疗与血脂偏高相关疾病的研究目标。虽然茶叶的功效日益受到关注，但是其降脂机理尚不清楚。为此，我们制备了不同种类的茶溶液，并从中筛选HMGCR的抑制剂，从实验结果看，同红茶以及普洱茶相比，绿茶能够明显的抑制HMGCR的活性，当其水提物浓度为1mg/mL时，抑制率可以达到42.9%。该结果提示绿茶中可能存在某些可以抑制HMGCR的物质。鉴于此，我们对绿茶的组份进行了进一步的筛选。
根据以往报导，绿茶的重要成份EGCG（表没食子儿茶素没食子酸酯）对癌症、糖尿病、肥胖以及心脑血管疾病均有一定的预防治疗作用。EGCG能够抑制诸如11β‐甾类脱氢酶、5α‐还原酶、芳香酶等与固醇相关的酶。经过实验发现，EGCG还对抑制HMGCR的活性有比较明显的作用。当EGCG浓度达到1mM时，抑制率可以达到46.3%。
但是，进一步研究表明，EGCG只有在存在丙三醇的条件下才能够抑制HMGCR的活性，而缺少丙三醇的反应体系中，EGCG则对HMGCR没有明显的抑制效果。并且随着丙三醇浓度的增高，EGCG对HMGCR的抑制作用逐渐加强。当丙三醇浓度达到600mM（5.4%）时，其抑制效果最为明显。但之后，抑制效果又随着丙三醇浓度的增大而逐步下降。应用分子docking软件进行分子动力模拟分析，结果显示，HMGCR‐EGCG‐丙三醇三者结合的自由能确实低于EGCG单独与HMGCR结合的自由能。因此，我们推测很可能是丙三醇促进了HMGCR同EGCG形成复合物，致使底物不能进入酶的活性中心。在丙三醇的协同作用下，EGCG同HMGCR的相互作用力明显增强。
最后我们以HepG2细胞为模型，在细胞水平上研究了EGCG的降胆固醇作用，以及对HMGCR mRNA和蛋白表达水平的影响。结果表明EGCG能够降低细胞胆固醇水平，但是不能降低HMGCR mRNA和蛋白表达水平，说明EGCG不是通过下调HMGCR转录以及翻译水平从而降低胆固醇内源性合成。提示其可能同他汀类药物一样，通过抑制HMGCR的活性以下调胆固醇的合成。
The MVA pathway is a complex biochemical pathway required for thegeneration of several fundamental end-products including cholesterol, isoprenoids,dolichol, ubiquinone, and isopentenyladenine. At the heart of this pathway is therate-limiting enzyme,3-hydroxy-3-methylglutaryl CoA reductase. It is widelyacknowledged that HMGCR has been recognized as the rate-limiting enzyme insynthesis of cholesterol. The degradation of the reductase can reduce serumcholesterol levels and dramatically decrease the risk of cardiovascular andcerebrovascular diseases, essential hypertension, coronary heart diseases, andatherosclerosis.
Comparison of the sequence of HMGR has revealed the existence of twoclasses of this enzyme. The class I enzymes was found in eukaryotes and somearchaea, and the class II found in certain eubacteria and the archaea．In allmammalian species that have been studied to datereductase localizes to membranesof the endoplasmic reticulum (ER) and consists of888amino acids that can beseparated into two contiguous domains. The N-terminal domain of reductase isintegrated into membranes by virtue of eight membrane-spanning segments that areseparated by short loops. The C-terminal domain of reductase projects into thecytosol and exerts all of the enzymatic activity.
Statins lower cholesterol levels by specifically inhibiting HMGCR, a keyenzyme in the cholesterol biosynthesis pathway. These drugs significantly reducecardiovascular end points and are among the most commonly prescribedmedications. but their side effects are occasionally serious. A well-knowncomplication of statins use is musculoskeletal symptoms. Few patients develop anautoimmune myopathy even after the statins are discontinued. In addition, the use ofstatins may elevate serum transaminases, induce a statin-induced inhibition ofproximal tubular reabsorption of protein and have deleterious effects on the peripheral nervous system. In addition, The damage on the liver and kidney ismainly elevated transaminase which occurs after the compensatory increasedaccompany with the resulting dose-dependent drug. but the reason is not yet clear. Itis suggested that long-term liver enzymes indicators abnormalities lead tonon-alcoholic fatty liver. On the basis of data available from above, statins therapymay involve some risks of potential complications. Therefore, the development ofsafer inhibitors of HMGCR with less serious side effects has become the main studyconcern in recent years.
Natural products such as traditional Chinese medicine, tea are widespreadconcern because their cheap and easy to get, smaller or even no toxic effects to thehealth. So in the present study, we cloned and purified the catalytic domain ofhuman HMGCR(△HMGCR) and screened its inhibitors from natural products.
Total RNAs were extracted from Human Liver Cells (HepG2), the cDNAs weresynthesized from the polyadenylated mRNA, the Catalytic Domain of HMGCR wasamplified by PCR and then the truncated enzyme was purified by GST Resincolumn. A rapid and accurate method has been developed for screening HMGCRinhibitors by measuring the concentration of NADPH in the enzymatic reactionsystem using ultra performance liquid chromatography-electrospray ionizationtandem mass spectrometry.
Traditional detection methods for the enzyme are radiochemical method,spectrophotometer and HPLC. But the former is less used for its great harm. thespectrophotometer method is commonly used method for its convenient and fast, butthe method is waste in the substrate solution and the results are not accurate enough.NADPH polarity is too high, so it was almost no reservations in the C18column,many of the natural product inhibitors have very strong UV absorption, and thusNADPH can not be separated from the other substances in the enzyme reactionsystem by HPLC. The UPLC-ESI-MS/MS we used has a good linear correlationcoefficient, it can save the substrate and reduce the cost of the HMGCR inhibitor developed for the lower quantitative range.
We analyzed activity of HMGCR and screened inhibitors in the traditionalChinese medicine, tea and other natural products. The results showed that△HMGCR shows a good linear relationship in the first6minutes.
Teas are the East Asia, especially the Chinese people's day-to-day drinks. theyhave many benefits, such as anti-aging, anti-cancer, sterilization, anti-inflammatory,slimming, significantly lower the blood lipids. So they become potential researchobjectives treatment to high blood lipids related diseases.
We prepared the different kinds of tea solution, and screened HMGCR inhibitor.It shows that green tea can inhibit HMGCR compared with Pu'er and black tea.when its concentration is100μg/mL, the inhibition rate reach42.95%. The resultsshow that green tea can inhibit cholesterol synthesis by inhibiting HMGCR enzymeactivity. we further screen inhibitors from its subdivided components.
Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol compoundin green tea. As a natural product with less serious side effects, it has been used toprevent cancer, obesity, diabetes and cardiovascular disease. EGCG can inhibit somesteroid-related enzymes, such as11β-hydroxysteroid dehydrogenase,5α-reductaseand aromatase, but it is ineffective in the inhibition of HMGCR. However, ourresults showed that EGCG exhibits exciting inhibitory ability against HMGCR.Subsequent study indicated that EGCG is able to inhibit HMGCR with the additionof some glycerol to the reaction system. The inhibitory effects of HMGCR enhancedwith the increase in concentration of glycerol. When the concentration reached up to600mM (5.4%), the best inhibition was observed. Afterwards, the inhibitory effectgradually reduced as the concentration surpassed600mM.
In order to explore to role that glycerol played in this process, the analysis ofmolecular dynamics simulation was conducted on HMGCR-EGCG-glycerolinteractions. The3D structure of HMGCR was adopted. Through the model ofdynamics simulation, we speculate that EGCG and HMGCR form certain complex so that substrates are prevented from penetrating into the active center. The resultsrevealed that the affinity between EGCG and HMGCR is significantly stronger inthe synergy of glycerol.
Moreover, EGCG can decrease cellular total cholesterol in HepG2cells withoutchanging HMGCR mRNA and protein levels after24hrs incubation compared tocontrol.
引文
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