Metabolism of 32-oxo-24,25-dihydrolanosterols by partially purified cytochrome P-450(14DM) from rat liver microsomes

Metabolism of 32-oxo-24,25-dihydrolanosterols (3 beta-hydroxylanost-8-en-32- al (4,delta 8-CHO) and 3 beta-hydroxylanost-7-en-32-al (5,delta 7-CHO)) was studied in a reconstituted system consisting of rat liver partially purified cytochrome P-450, which catalyzes lanosterol 14-demethylation (P-450(14DM)), and reduced nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase. The reconstituted system converted delta 8-CHO to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol (2, 8, 14-Diene), which corresponds to the 14-deformylated product. delta 7-CHO, the isomer of delta 8-CHO, was not converted to the corresponding 14-deformylated product. The apparent Km value of cytochrome P-450(14DM) for delta 8-CHO was about 1/20 of that for 24,25-dihydrolanosterol (1, DHL). The metabolism of delta 8-CHO was inhibited by 7-oxo-24,25-dihydrolanosterol (6, 7-oxo-DHL), which is a potent inhibitor of cholesterol biosynthesis from lanosterol or DHL. However, the metabolism of delta 8-CHO was less inhibited by 7-oxo-DHL than that of DHL.     

Metabolism of 24,25-dihydrolanosterol analogs by partially purified cytochrome P-450(14DM) from rat liver microsomes

27-Nor-24,25-dihydrolanosterol (27-nor-DHL), 26,27-dinor-24,25- dihydrolanosterol (26,27-dinor-DHL), and 25,26,27-trinor-24,25-dihydrolanosterol (25,26,27-trinor-DHL), analogs of 24,25-dihydrolanosterol (DHL) which have no C-27 carbon, C-26,27 carbons and C-25,26,27 carbons, were converted to the corresponding 14-demethylated products using a reconstituted monooxygenase system from rat liver microsomes which contained cytochrome P-450(14DM) catalyzing lanosterol 14-demethylation and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase in the presence of NADPH and molecular oxygen. Each metabolite showed a relative retention time (RtR) of 0.72 with respect to each substrate in high-performance liquid chromatography (HPLC) on a reversed-phase column. Comparison of each gas chromatography-mass spectrum and RtR value with those of the metabolite of DHL, 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, indicated that the metabolites could be inferred to be 27-nor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, 26,27-dinor-4,4- dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, and 25,26,27-trinor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol. However, 24,25,26,27-tetranor- and 23,24,25,26,27-pentanor analogs of DHL and 20-iso-24,25-dihydrolanosterol were not metabolized by the reconstituted enzyme system.     

Inhibition of acyl coenzyme A: cholesterol acyltransferase by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors

Relatively high concentrations of MK-733 (simvastatin) and MK-803 (lovastatin, mevinolin), which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, were found to inhibit acyl coenzyme A: cholesterol acyltransferase (ACAT) of rabbit intestinal microsomes with IC50's of 2.0 x 10(-5) and 3.6 x 10(-5) M, respectively. Dihydroxy acid forms of both MK-733 and MK-803 did not inhibit ACAT activity. A kinetic analysis using a Lineweaver-Burk plot indicated that MK-733 is a competitive inhibitor of ACAT, with a Ki value of 1.2 x 10(-5) M.     

Vinylogous amide analogues of diaminopimelic acid (DAP) as inhibitors of enzymes involved in bacterial lysine biosynthesis

[reaction: see text] Vinylogous amides 5 and 6 have been synthesized from L-propargyl glycine and tested against diaminopimelate (DAP) enzymes involved in bacterial lysine biosynthesis. Both are reversible inhibitors of DAP D-dehydrogenase and DAP epimerase with IC(50) values in the 500 microM range. Compound 5 shows competitive inhibition against the L-dihydrodipicolinate (DHDP) reductase with a K(i) value of 32 microM, which is comparable to the planar dipicolinate 16 (K(i) = 26 microM), the best known inhibitor of the enzyme.     

Inhibitor design for 3-hydroxy-3-methyl-glutaryl-CoA reductase enzyme; molecular docking and determination of molecular and electronic properties of ligands by density functional theory method

Designing new inhibitors having less side effects is a need which also could reduce cholesterol levels. To fulfill this aim, we have carried out a molecular docking study toward 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase. A set of designed structural derivatives of statin drugs, eight ligands which are used as HIV-1 integrase inhibitor candidates, a set of terpenoids, and ligands downloaded from Zinc15 database were docked to HMG-CoA reductase enzyme which contains atorvastatin in crystal structure. The analysis of docking studies revealed that statin derivative ligands are more appropriate for inhibition of HMG-CoA reductase. To define the contribution of the molecular properties to the binding of ligands to enzyme structure; the highest occupied molecular orbitals-lowest unoccupied molecular orbitals, hardness, electronegativity, and chemical potential properties of ligands have best score in their sets calculated by quantum mechanical tools.     

Synthesis and antiplasmodial activity of 3-furyl and 3-thienylquinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives

The aim of this study was to identify new compounds active against Plasmodium falciparum based on our previous research carried out on 3-phenyl-quinoxaline-2-carbonitrile 1,4-di-N-oxide derivatives. Twelve compounds were synthesized and evaluated for antimalarial activity. Eight of them showed an IC(50) less than 1 microM against the 3D7 strain. Derivative 1 demonstrated high potency (IC(50)= 0.63 microM) and good selectivity (SI=10.35), thereby becoming a new lead-compound.     

Statins: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) Reductase Inhibitors Demonstrate Anti-Atherosclerotic Character due to Their Antioxidant Capacity

Atherosclerosis is a chronic inflammatory disease of multiple etiologies. It is associated with the accumulation of oxidized lipids in arterial lesions leading to coronary heart disease. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (commonly known as statins) are widely used in cardiovascular disease prevention to lower the cholesterol. The antioxidant activity of HMG-CoA reductase inhibitors was studied by lipid peroxidation inhibition assay, DPPH, and hydroxyl radical scavenging-activity methods. The lovastatin (93%) and simvastatin (96%) showed significant action of lipid peroxidation inhibition compared to other HMG-CoA reductase inhibitors. The DPPH radical and hydroxyl radical scavenging activity of simvastatin was 38% and 33%, respectively. The oxidative modification of serum lipid due to reactive oxygen species causes atherosclerosis. This study revealed the importance of lovastatin and simvastatin to prevent oxidative stress-related cardiovascular diseases.     

Reactions of 26-iodopseudodiosgenin and 26-iodopseudodiosgenone with various nucleophiles and pharmacological activities of the products

26-Iodopseudodiosgenin (8) and 26-iodopseudodiosgenone (9) were reacted with various nucleophiles (KSCN, KOCN, NaCN, NaN(3) and various amines) to give pseudodiosgenin derivatives (4, 12, 16-20, 26) and pseudodiosgenone derivatives (5, 13, 21-25, 27), respectively. The reactions of 8 and 9 with KOCN gave the elimination products (10) and (11), respectively. The reaction of 9 with NaCN gave 5alpha,26- (14) and 5beta,26-dicyanocholestan-3-one (15). The reaction of 8 with NaN3 gave triazepine derivative (30), while that of 9 gave 26-azidopseudodiosgenone (31). Compound 31 was converted into triazepine derivative (32) by heating at 120 degrees C. The cytotoxicity of the pseudodiosgenins and pseudodiosgenones on P-gp-underexpressing HCT 116 cells and P-gp-overexpressing Hep G2 cells was examined by MTT assay. Pseudodiosgenins 2, 4, 12 and 30 showed strong cytotoxic activity (IC50 values: 2.6+/-0.3-6.7+/-1.4 microM), as did pseudodiosgenones 3, 5, 11, 13, 21-25 and 27 (IC50 values: 1.3+/-0.3-6.4+/-0.3 microM) toward HCT 116 cells. Pseudodiosgenins 12, 16 and 30 (IC50 values: 1.2+/-0.7-2.2+/-0.6 microM) and pseudodiosgenones 22, 23, 25 and 27 (IC50 values: 0.6+/-0.1-2.5+/-0.3 microM) were highly cytotoxic to Hep G2 cells. Compounds 3 and 27 showed efficient antibacterial activity (MIC: 15.6, 10.4 microg/ml) and (MIC: 7.8, 15.6 microg/ml) against Bacillus subtilis and Staphylococcus aureus, respectively.     

Synthesis and activity of a new series of (Z)-3-phenyl-2-benzoylpropenoic acid derivatives as aldose reductase inhibitors

During the course of studies directed towards the discovery of novel aldose reductase inhibitors for the treatment of diabetic complications, we synthesized a series of new (Z)-3-phenyl-2-benzoylpropenoic acid derivatives and tested their in vitro inhibitory activities on rat lens aldose reductase. Of these compounds, (Z)-3-(3,4-dihydroxyphenyl)-2-(4-methylbenzoyl)propenoicacid (3k) was identified as the most potent inhibitor, with an IC50 of 0.49 microM. The theoretical binding mode of 3k was obtained by simulation of its docking into the active site of the human aldose reductase crystal structure.     

Simultaneous assay of the activities of two key enzymes in cholesterol metabolism by gas chromatography-mass spectrometry

A very sensitive and specific method for the simultaneous assay of the activities of two key regulatory enzymes in cholesterol metabolism, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (EC 1.1.1.34), and cholesterol 7 alpha-hydroxylase (EC 1.14.13.7), is described. The assay is based on the measurement of [2H3]mevalonolactone and 7 alpha-hydroxycholesterol produced by the incubation of [2H3]HMG-CoA and endogenous cholesterol with hamster liver microsomes using isotope dilution mass spectrometry. The incubation mixture was purified by means of solid extraction cartridges, and the extract was treated with benzylamine followed by dimethylethylsilyl imidazole. The resulting ether derivatives of the mevalonylbenzylamide and 7 alpha-hydroxycholesterol were quantified by gas chromatography-mass spectrometry with selected-ion monitoring in a high resolution mode. The method made it possible to assay simultaneously the activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in hamster liver microsomes with high sensitivity and accuracy.     

Glutathione S-transferase inhibiting chemical constituents of Caesalpinia bonduc

Glutathione S-transferase inhibition assay-guided fractionations on the ethanolic extract of the bark of Caesalpinia bonduc resulted in the isolation of a new sterol, 17-hydroxy-campesta-4,6-dien-3-one (1) along with four known compounds, 13,14-seco-stigmasta-5,14-dien-3alpha-ol (2), 13,14-seco-stigmasta-9(11),14-dien-3alpha-ol (3), caesaldekarin J (4) and pipataline (5) as active constituents. Structures of compounds 1-5 were established on the basis of extensive NMR spectroscopic studies. The compounds (1-5) were isolated on the basis of their inhibitory activity against glutathione S-transferase, an enzyme that has been implicated in resistances during treatment of cancer and parasitic infections. Efforts to study structure-activity relationships of compounds 2 and 3 were also made by modifying their structures. The IC50 values of these compounds and their derivatives ranged from 57-380 microM and were compared to the inhibitory effects due to sodium taurocholate, an isoprene-derived GST inhibitor (IC50=398 microM). A plausible biosynthesis of 13,14-seco-steroids has also been proposed.     

Total synthesis of 12-methoxydihydrochelerythrine and anti-tumour activity of its quaternary base: toward an efficient synthetic route for 12-alkoxybenzo[c]phenanthridine bases via naphthoquinone monooxime from 2-benzofuranyl-1-tetralone derivative

A concise total synthesis of 12-methoxydihydrochelerythrine (6), isolated from Bocconia integrifolia, is described. The synthesis features an efficient route to a 12-alkoxybenzo[c]phenanthridine skeleton via naphthoquinone monooxime 11 as a key compound. Starting from 7-methoxy-2-methylbenzo[b]furan (9), 3-aryl-1-tetralone 10 was synthesised, followed by aromatisation to 3-aryl-1-naphthol 17. After oxidative cleavage of the furan ring, basic nitrosation of naphthol 22 gave the naphthoquinone 11. The benzo[c]phenanthridine skeleton was formed by reductive cyclisation of 11. Deoxygenation of the lactam moiety in 23 afforded nor-base 32 and methylation of 32 under reductive conditions gave the target dihydro base 6 (23 steps from benzofuran 9 in 10% overall yield). The corresponding quaternary base 7 showed moderate anti-tumour activity against cancer cell lines; on NCI-H460: IC50 4.5 microM and on MDA-MB-231: IC50 1.2 microM. Introduction of a methoxy group into the 12-position of the benzo[c]phenanthridine skeleton could cause enhanced activity against MDA-MB-231 by comparison of 7 with chelerythrine (35) (IC50 5.3 microM).     

Synthesis and biological evaluation of 5-arylfuro[2,3-d]pyrimidines as novel dihydrofolate reductase inhibitors

A series of about fifty novel 5-arylfuro[2,3-d]pyrimidine derivatives were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) arising from different species. Weak enzyme inhibition was observed for most of the compounds, with only a few reaching IC50 values less than 30 microM. With regards to antibacterial and anti-malarial potency, only seven compounds showed a modest in vitro activity against some bacteria strains and only three products proved significantly active against P. falciparum.     

Hydantoin derivatives of L- and D-amino acids: synthesis and evaluation of their antiviral and antitumoral activity

3,5-Disubstituted hydantoin (1,3-imidazolidinedione) derivatives 5a-h were prepared by base induced cyclization of the corresponding N-(1-benzotriazolecarbonyl)-L- and D-amino acid amides 4a-h. Compounds 5a-h were evaluated for their cytostatic and antiviral activities. Among all the compounds evaluated, 3-benzhydryl-5-isopropyl hydantoin (5a) showed a weak but selective inhibitory effect against vaccinia virus (EC(50) = 16 microg/mL; selectivity index: 25). 3-Cyclohexyl-5-phenyl hydantoin (5g) showed inhibitory activity against cervical carcinoma (HeLa, IC(50) = 5.4 microM) and breast carcinoma (MCF-7, IC(50) = 2 microM), but also cytotoxic effects towards human normal fibroblasts (WI 38). On the contrary, the 3-benzhydryl-5-phenyl substituted hydantoin derivative 5h showed moderate inhibitory activity towards HeLa, MCF-7, pancreatic carcinoma (MiaPaCa-2), lung carcinoma (H 460) and colon carcinoma (SW 620) (IC(50) = 20-23 microM), but no effect on WI 38.     

Proteomics to display lovastatin-induced protein and pathway regulation in rat liver

Lovastatin is a lipid lowering agent that acts by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key regulatory enzyme in cholesterol biosynthesis. In this study the pattern of gene network regulation induced in hepatic proteins as a response to lovastatin treatment was analyzed by proteomics. In livers of male F344 rats treated with 1.6 mg/kg/day lovastatin or 150 mg/kg/day lovastatin for seven days, 36 proteins were found to be significantly altered (p<0.001) in relation to treatment. The changed proteins were classified according to their cellular function and participation in biochemical pathways. The following observations were made: (i) inhibition of HMG-CoA reductase provoked a regulatory response in the cholesterol synthesis pathway including the induction of cytosolic HMG-CoA synthase and of isopentenyl-diphosphate delta-isomerase, (ii) manipulation of the lipid metabolism triggered alterations in key enzymes of the carbohydrate metabolism, and (iii) lovastatin treatment was associated with signs of toxicity as reflected by changes in a heterogeneous set of cellular stress proteins involved in functions such as cytoskeletal structure, calcium homeostasis, protease inhibition, cell signaling or apoptosis. These results present new insights into liver gene network regulations induced by lovastatin and illustrate a yet unexplored application of proteomics to discover new targets by analysis of existing drugs and the pathways that they regulate.     

Pharmacophore identification by molecular modeling and chemometrics: The case of HMG-CoA reductase inhibitors

Summary A methodology based on molecular modeling and chemometrics is applied to identify the geometrical pharmacophore and the stereoelectronic requirements for the activity in a series of inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme involved in cholesterol biosynthesis. These inhibitors present two common structural features—a 3,5-dihydroxy heptanoic acid which mimics the active portion of the natural substrate HMG-CoA and a lipophilic region which carries both polar and bulky groups. A total of 432 minimum energy conformations of 11 homologous compounds showing different levels of biological activity are calculated by the molecular mechanics MM2 method. Five atoms are selected as representatives of the relevant fragments of these compounds and three interatomic distances, selected among 10 by means of a Principal Component Analysis (PCA), are used to describe the three-dimensional disposition of these atoms. A cluster analysis procedure, performed on the whole set of conformations described by these three distances, allows the selection of one cluster whose centroid represents a geometrical model for the HMG-CoA reductase pharmacophore and the conformations included are candidates as binding conformations. To obtain a refinement of the geometrical model and to have a better insight into the requirements for the activity of these inhibitors, the Molecular Electrostatic Potential (MEP) distributions are determined by the MNDO semiempirical method.     

An improved assay of 3-hydroxy-3-methylglutaryl-CoA reductase activity in Reuber H35 hepatoma cells without microsomes isolation

The optimal conditions for measuring 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity in Reuber H35 hepatoma cells are described in this paper. Cells in the exponential phase of growth were lysed by incubation with Brij 97 detergent for 30 min. We used imidazole buffer supplemented with EDTA and leupeptine, two inhibitors of proteases. Disrupted cells were then centrifuged at 12,000 g. Although microsomes are usually reported as enzyme preparations for measuring HMG-CoA reductase, our data showed that hepatoma cells may be used without previous isolation of microsomes. The 12,000 g supernatant showed similar levels of total and specific activities to those found in the microsomal fraction obtained after 105,000 g centrifugation. The soluble fraction showed less than 10% of reductase activity. Reductase activity from Reuber H35 hepatoma cells increased proportionally to the reaction time from 30 to 90 min and to the amount of protein added in a range of 50-500 micrograms. Our modified method was very sensitive and reproducible, because very low specific activity (about 15-100 pmol min-1 per mg protein) could be quantified in different assay conditions obtaining similar values.     

HMG-CoA还原酶抑制剂三维药效团的构建

以作用于鼠肝脏细胞的21个3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)还原酶抑制剂(RI)为训练集, 训练集化合物具备结构多样性, 来源于相同药理模型, 活性值IC50范围在0.3-8000 nmol·L-1. 利用Catalyst 计算HMG-CoA还原酶抑制剂最优药效团由一个氢键受体, 一个氢键给体, 一个疏水基团和一个芳香环特征组成. 药效团模型Fixed cost值, Total cost值和Configuration cost值分别为88.75、111.5 和16.98. 训练集化合物活性计算值与实测值相关系数为0.8883, 偏差值为1.269, 交叉验证结果表明, 药效团模型具有较高的置信度, 对测试集化合物活性值的预测结果显示有较好的预测能力, 可用于数据库搜索发现新的具有该活性的化合物, 也可用于中药或天然产物药物的研究开发.     

Structural requirements of flavonoids and related compounds for aldose reductase inhibitory activity

The methanolic extracts of several natural medicines and medicinal foodstuffs were found to show an inhibitory effect on rat lens aldose reductase. In most cases, flavonoids were isolated as the active constituents by bioassay-guided separation, and among them, quercitrin (IC(50)=0.15 microM), guaijaverin (0.18 microM), and desmanthin-1 (0.082 microM) exhibited potent inhibitory activity. Desmanthin-1 showed the most potent activity, which was equivalent to that of a commercial synthetic aldose reductase inhibitor, epalrestat (0.072 microM). In order to clarify the structural requirements of flavonoids for aldose reductase inhibitory activity, various flavonoids and related compounds were examined. The results suggested the following structural requirements of flavonoid: 1) the flavones and flavonols having the 7-hydroxyl and/or catechol moiety at the B ring (the 3',4'-dihydroxyl moiety) exhibit the strong activity; 2) the 5-hydroxyl moiety does not affect the activity; 3) the 3-hydroxyl and 7-O-glucosyl moieties reduce the activity; 4) the 2-3 double bond enhances the activity; 5) the flavones and flavonols having the catechol moiety at the B ring exhibit stronger activity than those having the pyrogallol moiety (the 3',4',5'-trihydroxyl moiety).