Screening Of3-Hydroxy-3-Methylglutaryl CoA Reductase Inhibitors By Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry

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.