酶法制备羟甲基戊二酰CoA还原酶抑制剂手性中间体的研究进展

羟甲基戊二酰CoA(HMG-CoA)还原酶抑制剂是降血脂药物。综述了近年来国内外酶法制备HMG-CoA还原酶抑制剂手性中间体的研究进展,从不对称合成和外消旋体拆分2个方面介绍了酶法制备HMG-CoA还原酶抑制剂手性中间体的工艺路线和研究水平,对其工业化前景进行了展望。     

Inhibitory effects of tangeretin and trans-ethyl caffeate on the HMG-CoA reductase activity: Potential agents for reducing cholesterol levels

One of the pathways to reduce cholesterol production in the liver is through the inhibition of HMG-Coa reductase (HMGCR) by current drugs, statins. However, these have side effects if consumed in prolonged periods. Tangeretin and trans-ethyl caffeate as alternative drugs in reducing hypercholesterolemia and preventing atherosclerosis have never been reported. Their effects on inhibiting HMGCR activity were investigated through enzymatic method (in vitro and in vivo). The toxicity property was analyzed on the Serum Glutamate Oxalate Transaminase (SGOT)/Serum Glutamate Piruvate Transaminase (SGPT) levels and rat liver histology. The results showed that both compounds inhibited HMGCR activity significantly compare to the control simvastatin (p < 0.05). Tangeretin which showed very good activity in inhibiting HMGCR (83.8 of % inhibition, equal to simvastatin) was selected and used for anti-hypercholesterolemia in vivo assessment. Furthermore, tangeretin was shown to effectively reduced Total Cholesterol (TC) and Low Density Lipoprotein (LDL), and increased High Density Lipoprotein (HDL) levels significantly compared to the simvastatin group (p < 0.05). Tangeretin group was also proven to inhibit HMGCR rat liver activity significantly compare to the control simvastatin (p < 0.05). The toxicity study on the SGOT/SGPT levels and liver histology revealed that there were no side effects after administration by tangeretin. Results found that both tangeretin and trans-ethyl caffeate are potent candidates as anti-hypercholesterolemia agent in vitro. In addition, tangeretin was also shown to be safe and suitable as an alternative treatment for controlling hypercholesterolemia in vivo as well as have potency for preventing atherosclerosis.     

茶树HMG-CoA还原酶基因全长cDNA克隆及序列分析

香气是茶叶的重要品质之一,萜类物质不仅香气好,而且沸点普遍较高,是构成茶叶香气的重要物质基础,决定着茶叶的香气品质,也可作为茶叶香型划分的依据。在植物中,倍半萜、多萜醇等通过胞质中的甲瓦龙酸(MVA)途径合成。HMG-Co A还原酶(HMGR)催化HMG-Co A(3-羟基-3-甲基戊二酸单酰辅酶A)生成甲瓦龙酸,是依赖MVA萜类合成途径的关键限速反应。为了有助于理解茶树萜类合成的分子遗传机制,通过RACE-PCR方法从茶树中克隆了一个编码HMG-Co A还原酶的c DNA全长序列(命名为Cs HMGR1),该序列由1 979 bp组成,包含一个1 722 bp的完整开放阅读框,编码573个氨基酸。其推定的编码蛋白与橡胶树、旱莲木、人参、荔枝、西洋参、丹参、罗汉果及龙眼的同源蛋白具有80%~82%的序列一致性。利用Cs HMGR1和其它物种HMGR同源蛋白的催化区域构建系统发育树,表明其属于真核生物I类HMGR家族。结构分析表明,Cs HMGR1含有两个跨膜区,推测其与其它真核生物同源蛋白类似地定位于内质网上;含有两个HMG-Co A结合位点、两个NADPH结合位点、四个保守的催化活性残基及一个磷酸化位点,说明磷酸化/去磷酸化很可能也是其活性调节的重要方式。表达分析表明,Cs HMGR1在"大叶龙"叶芽、母株叶芽及花芽都有较强的表达。其表达调控及生理活性对茶叶品质可能有重要影响,并在其功能解析的基础上,有可能作为茶叶品质鉴定及育种的一个依据。     

Inhibition of the class II HMG-CoA reductase of Pseudomonas mevalonii

There are two structural classes of HMG-CoA reductase, the third enzyme of the mevalonate pathway of isopentenyl diphosphate biosynthesis-the Class I enzymes of eukaryotes and the Class II enzymes of certain eubacteria. Structural requirements for ligand binding to the Class II HMG-CoA reductase of Pseudomonas mevalonii were investigated. For conversion of mevalonate to HMG-CoA the -CH(3), -OH, and -CH(2)COO(-) groups on carbon 3 of mevalonate were essential for ligand recognition. The statin drug Lovastatin inhibited both the conversion of HMG-CoA to mevalonate and the reverse of this reaction. Inhibition was competitive with respect to HMG-CoA or mevalonate and noncompetitive with respect to NADH or NAD(+). K(i) values were millimolar. The over 10(4)-fold difference in statin K(i) values that distinguishes the two classes of HMG-CoA reductase may result from differences in the specific contacts between the statin and residues present in the Class I enzymes but lacking in a Class II HMG-CoA reductase.     

Modulation of hypercholesterolemia-induced alterations in apolipoprotein B and HMG-CoA reductase expression by selenium supplementation

Various studies demonstrated a significant association between the trace element selenium (Se), hypercholesterolemia and the risk of cardiovascular disorders. Present study was aimed to reveal the role of Se supplementation in modulation of hypercholesterolemia-induced changes in apolipoprotein B (apoB) and 3-hydroxy 3-methylglutaryl co-enzyme A (HMG-CoA) reductase expression during experimental hypercholesterolemia in Sprague-Dawley male rats. Animals were fed 0.2 and 1 ppm Se-supplemented control diet as well as 2% cholesterol-supplemented diet for 3 months. Apolipoprotein B levels were measured by ELISA and Western blot. HMG-CoA reductase mRNA expression was studied by RT-PCR. ApoB levels increased significantly on 2% cholesterol-supplemented diet feeding. On 1 ppm Se supplementation apoB levels decreased significantly. HMG-CoA reductase mRNA expression decreased significantly on cholesterol-supplemented diet feeding and on 1 ppm Se supplementation the mRNA expression further decreased. So the present results demonstrate that 1 ppm Se supplementation is responsible for down regulation of apoB and HMG-CoA reductase expression during hypercholesterolemia. These findings highlight the therapeutic potential of selenium supplementation in lipid metabolism.     

响应面法优化海洋镰刀腐皮菌产出HMG-CoA还原酶抑制剂的培养条件

【目的】采用响应面法对海洋微生物镰刀腐皮菌FG319发酵产生MFS(Metabolite of Fusarium solani FG319)培养条件进行优化。【方法】在单因素试验结果的基础上,依据Box-Behnken中心组合原则设计诱导物添加量、培养时间、培养温度的3因素3水平响应面实验,以MFS产出量为响应值优化镰刀腐皮菌FG319的培养条件。【结果】镰刀腐皮菌FG319最优培养条件为诱导物添加量0.6%、培养时间7 d、培养温度22°C,在此培养条件下,MFS最高产量达到20.11 mg/L,是优化前的4倍,与理论预测的相对误差为0.64%,实测值与响应面预测值拟合良好。镰刀腐皮菌FG319代谢产物MFS在HMG-CoA还原酶和NADPH构筑的分子评价反应体系,当MFS添加到100 mg/L时,具有类似洛伐他汀抑制HMG-CoA还原酶的最大抑制率。【结论】响应面试验设计对镰刀腐皮菌FG319培养条件的优化是有效的,其次生代谢产物MFS体外抑制HMG-CoA还原酶的效果也是明显的。     

Highly sensitive assay of HMG-CoA reductase activity by LC-ESI-MS/MS

We have developed a new sensitive and specific nonradioisotope assay method to measure the activity of HMG-CoA reductase, the rate-controlling enzyme in the cholesterol biosynthetic pathway. This method was based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry using electrospray ionization in positive mode. Mevalonic acid, the product of HMG-CoA reductase, was converted to mevalonolactone (MVL) in an incubation mixture, extracted by a salting-out procedure, derivatized into the mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide, and then purified using a disposable silica cartridge. The resulting mevalonylamide was quantified by selected reaction monitoring using the positive electrospray ionization mode. The detection limit of this mevalonylamide was found to be 240 amol (signal-to-noise ratio=3), approximately 833 times more sensitive than that of MVL measured by a conventional radioisotope (RI) method (200 fmol). The variances between sample preparations and between measurements by this method were analyzed by one-way layout and calculated to be 3.2% and 1.8%, respectively. The recovery experiments were performed using incubation mixtures spiked with 0.77-2.31 nmol MVL/mg protein and were validated by a polynomial equation. These results showed that the estimated concentration within a 95% confidence limit was 0.47+/-0.07 nmol/mg protein, which coincided completely with the observed X0 nmol/mg protein with a mean recovery of 94.6%. This method made it possible to measure HMG-CoA reductase activity with a high degree of reproducibility and reliability, and especially with sensitivity superior to that of the conventional RI method.     

Homeostatic restoration of microsomal lipids and enzyme changes in HMG-CoA reductase and Acyl-CoA : cholesterol acyltransferase in chick liver

We have studied the correlation between changes in the lipid composition in chick liver microsomes and the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acyl-CoA : cholesterol acyltransferase (ACAT) by in vivo and in vitro experiments with 21-day-old chicks. A 5% cholesterol diet for 3 hr produced an increase in the microsomal and plasmatic cholesterol content, a decrease in HMG-CoA reductase activity and a concomitant increase in ACAT activity. The effect produced by the short-term treatment virtually disappeared 27 hr after ending the cholesterol diet. In vitro experiments were carried out by using vesicles constituted by phosphatidycholine/cholesterol and phosphatidylcholine.     

Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors

In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl phenols (6a–c and 7a–c) and their saturated side-chain alkyl phenols (4a–c and 5a–c), and on the evaluation of their hypolipidemic activity using a murine Tyloxapol-induced hyperlipidemic protocol. The whole series of compounds 4–7 greatly and significantly reduced elevated serum levels of total cholesterol, LDL-cholesterol, and triglycerides, with series 6 and 7 showing the greatest potency ever found in our laboratory. At the minimum dose (25 mg/kg/day), the latter compounds lowered cholesterol by 68–81%, LDL by 72–86%, and triglycerides by 59–80%. This represents a comparable performance than that shown by simvastatin. Experimental evidence and docking studies suggest that the activity of these derivatives is associated with the inhibition of HMG-CoA reductase.     

Anti-lipid deposition effect of HMG-CoA reductase inhibitor, pitavastatin, in a rat model of hypertension and hypercholesterolemia

Since the rat is an atherosclerosis-resistant species, the study of atherosclerosis using rats is limited. The present study was undertaken to develop an atherosclerotic model in rats, to investigate the effect of nitric oxide (NO) inactivation and hyperlipidemia, and to evaluate the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor, on NO inactivation and on hyperlipidemia-induced changes in the cardiovascular system. Four-month-old male spontaneously hypertensive hyperlipidemic rats (SHHR) and Sprague-Dawley (SD) rats were used to study 1) the effect of the period of treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/L) on high fat diet (HFD)-treated SHHR and SD rats, and 2) the effect of pitavastatin (Pit, 0.3 mg/kg/day) on the changes in the aorta of L-NAME- and HFD-treated SHHR and SD rats. L-NAME administration for 1 month then HFD feeding for 2 months markedly increased the deposition of lipids and the thickness of the endothelium in SHHR. Continuous L-NAME treatment with HFD produced severe injury and stripped of endothelium in both strains. The plasma total cholesterol of L-NAME + HFD-treated and L-NAME + HFD + Pit-treated SHHR was significantly higher than that of control SHHR. Lipid deposition, however, was comparatively less in the aorta of L-NAME + HFD + Pit-treated SHHR. The concentration of cholesterol in the aorta of control SHHR was significantly lower than that in the aorta of L-NAME + HFD-treated SHHR, whereas that of L-NAME + HFD + Pit-treated SHHR was the same as that in control SHHR. These data indicated that Pit blocked lipid deposition in the aorta of L-NAME + HFD treated SHHR without changing plasma lipid profiles. In conclusion, NO inactivation and HFD induce lipid deposition in the endothelium, and the HMG-CoA reductase inhibitor blocks the deposition in SHHR.     

Supernatant protein factor stimulates HMG-CoA reductase in cell culture and in vitro

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase in vitro and, unexpectedly, cholesterol synthesis in cell culture. Because squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, we investigated the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway. Substitution of [(14)C]mevalonate for [(14)C]acetate in McARH7777 hepatoma cells expressing SPF reduced the 1.8-fold increase in cholesterol synthesis by half, suggesting that SPF acted on or prior to mevalonate synthesis. This conclusion was supported by the finding that substitution with [(14)C]mevalonate completely blocked an SPF-induced increase in squalene synthesis. Evaluation of 2,3-oxidosqualene synthesis from [(14)C]mevalonate demonstrated that SPF also stimulated squalene monooxygenase (1.3-fold) in hepatoma cells. Immunoblot analysis showed that SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels, indicating a direct effect on enzyme activity. Addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold, and the SPF-concentration/activation curve paralleled that for the SPF-mediated stimulation of squalene monooxygenase. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.     

Discovery of novel hepatoselective HMG-CoA reductase inhibitors for treating hypercholesterolemia: a bench-to-bedside case study on tissue selective drug distribution

The design of drugs with selective tissue distribution can be an effective strategy for enhancing efficacy and safety, but understanding the translation of preclinical tissue distribution data to the clinic remains an important challenge. As part of a discovery program to identify next generation liver selective HMG-CoA reductase inhibitors we report the identification of (3R,5R)-7-(4-((3-fluorobenzyl)carbamoyl)-5-cyclopropyl-2-(4-fluorophenyl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoic acid (26) as a candidate for treating hypercholesterlemia. Clinical evaluation of 26 (PF-03491165), as well as the previously reported 2 (PF-03052334), provided an opportunity for a case study comparison of the preclinical and clinical pharmacokinetics as well as pharmacodynamics of tissue targeted HMG-CoA reductase inhibitors.     

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment

Aims:  Statins – inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs.
Methods and results:  We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins.
Conclusion:  The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes.
Significance and Impact of the Study:  These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.     

Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent

In contrast with the accelerated degradation observed in tumor cells in response to sterols, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase turnover in whole animals was not increased by dietary cholesterol. Furthermore, treating rats with lovastatin to lower hepatic cholesterol levels did not decrease the rate of degradation. The half-life remained in the 6 h range. Co-immunoprecipitation studies revealed that the amount of ubiquitin associated with the reductase was entirely dependent upon the amount of microsomal protein subjected to immunoprecipitation. The results indicate that in liver, neither the rate of reductase protein degradation nor the ubiquitin-proteasome system appear to play roles in mediating changes in HMG-CoA reductase protein levels in response to dietary cholesterol.     

Osmoadaptation-dependent activity of microsomal HMG-CoA reductase in the extremely halotolerant black yeast Hortaea werneckii is regulated by ubiquitination

We have investigated regulation of HMG-CoA reductase (HMGR) in one of the most salt-tolerant fungi, Hortaea werneckii, under different salinities and at the level of protein degradation. Two different HwHMGR isoenzymes were identified, specific to mitochondria and endoplasmic reticulum: HwHmg1 and HwHmg2, respectively. The activity of microsomal HwHmg2 is highest under hypo-saline and extremely hyper-saline conditions, and down-regulated under optimal growth conditions. We show that this is due to intense ubiquitination and proteasomal degradation of HwHmg2. The activity of the truncated mitochondrial HwHmg1 is constant under different growth conditions, suggesting an osmoadaptation-directed fate for mevalonate utilization in H. werneckii.     

Kinetic characterization of an oxidative,cooperative HMG-CoA reductase from Burkholderia cenocepacia

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a key enzyme in endogenous cholesterol biosynthesis in mammals and isoprenoid biosynthesis via the mevalonate pathway in other eukaryotes, archaea and some eubacteria. In most organisms that express this enzyme, it catalyzes the NAD(P)H-dependent reduction of HMG-CoA to mevalonate. We have cloned and characterized the 6x-His-tagged HMGR from the opportunistic lung pathogen Burkholderia cenocepacia. Kinetic characterization shows that the enzyme prefers NAD(H) over NADP(H) as a cofactor, suggesting an oxidative physiological role for the enzyme. This hypothesis is supported by the fact that the Burkholderia cenocepacia genome lacks the genes for the downstream enzymes of the mevalonate pathway. The enzyme exhibits positive cooperativity toward the substrates of the reductive reaction, but the oxidative reaction exhibits unusual double-saturation kinetics, distinctive among characterized HMG-CoA reductases. The unusual kinetics may arise from the presence of multiple active oligomeric states, each with different V_max values.     

The inhibition of degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by sterol regulatory element binding protein cleavage-activating protein requires four phenylalanine residues in span 6 of HMG-CoA reductase transmembrane domain

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway. This endoplasmic reticulum membrane protein contains a cytosolic catalytic domain and a transmembrane domain with eight membrane spans that are necessary for sterol-accelerated degradation. Competition experiments showed that wild-type transmembrane domains of HMGR and sterol regulatory element binding protein cleavage-activating protein (SCAP) blocked sterol-accelerated degradation of intact HMGR and HMGal, a model protein containing the membrane domain of HMGR linked to Escherichia coli beta-galactosidase. However, mutant transmembrane domains of HMGR and SCAP whose sterol-sensing functions were abolished did not inhibit sterol-accelerated degradation of HMGR and HMGal. In addition, our mutagenesis studies on HMGal indicated that four Phe residues conserved in span 6 of HMGR and the sterol-sensing domains of other sterol-related proteins are required for the regulated degradation of HMGR. These results suggest that HMGR and SCAP compete for binding to a sterol-regulated regulator protein, and this binding may need the four Phe residues.