Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes

Human liver microsomes catalyze an efficient 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. The hydroxylation is involved in a minor, alternative pathway for side-chain degradation in the biosynthesis of cholic acid. The enzyme responsible for the microsomal 25-hydroxylation has been unidentified. In the present study, recombinant expressed human P-450 enzymes have been used to screen for 25-hydroxylase activity towards 5β-cholestane-3α,7α,12α-triol. High activity was found with CYP3A4, but also with CYP3A5 and to a minor extent with CYP2C19 and CYP2B6. Small amounts of 23- and 24-hydroxylated products were also formed by CYP3A4. The V_max for 25-hydroxylation by CYP3A4 and CYP3A5 was 16 and 4.5 nmol/(nmol×min), respectively. The K_m was 6 μM for CYP3A4 and 32 μM for CYP3A5. Cytochrome b₅ increased the hydroxylase activities. Human liver microsomes from ten different donors, in which different P-450 marker activities had been determined, were incubated with 5β-cholestane-3α,7α,12α-triol. A strong correlation was observed between formation of 25-hydroxylated 5β-cholestane-3α,7α,12α-triol and CYP3A levels (r²=0.96). No correlation was observed with the levels of CYP2C19. Troleandomycin, a specific inhibitor of CYP3A4 and 3A5, inhibited the 25-hydroxylase activity of pooled human liver microsomes by more than 90% at 50 μM. Tranylcypromine, an inhibitor of CYP2C19, had very little effect on the conversion. From these results, it can be concluded that CYP3A4 is the predominant enzyme responsible for 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol in human liver microsomes.     

Five 3β, 6α-dihydroxysterols in organ-pipe cactus

The sterol diol fraction from the lipids of organ-pipe cactus, Stenocereus thurberi, was separated into five compounds: macdougallin, peniocerol, cyclostenol, stenocereol and thurberol. The last three compounds have not been described before. All compounds were characterized by physical and spectroscopic properties.     

Triterpenoids from Salvia phlomoides, three new lupane derivatives

Three new triterpenoids have been isolated from the aerial parts of Salvia phlomoides. Their structures have been established by chemical and spectroscopic means and by correlation with known products as lupane-3β,11α,20-triol, 3β-acetoxy-lupane-11α,20-diol and 3-keto-lupane-11α,20-diol.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

The involvement of cytochrome b5 in 5β-cholestane-3α,7α,12α-triol 25-hydroxylation and taurodeoxycholate 7α-hydroxylation of rat liver

Role of cytochrome b₅ in NADPH-supported 5β-cholestane-3α,7α,12α-triol 25-hydroxylation and taurodeoxycholate 7α-hydroxylation of rat liver microsomes was investigated using highly purified antibodies against cytochrome b₅. Anti-b₅ antibody strongly inhibited both hydroxylation reactions indicating that cytochrome b₅ is a functional component in these steroid hydroxylation systems. It was shown that the involvement of cytochrome b₅ in these systems could be altered by the conditions of the reaction systems.     

Inhibition of ergosterol biosynthesis in saccharomyces cerevisiae and Ustilago maydis by tridemorph,fenpropimorph and fenpropidin

The structurally related fungicides, tridemorph, fenpropimorph and fenpropidin have been shown to inhibit the sterol Δ¹⁴-reductase and Δ⁸→Δ⁷-isomerase during ergosterol biosynthesis in Saccharomyces cerevisiae and Ustilago maydis. However, although the three fungicides are able to inhibit both enzymes, tridemorph inhibits the Δ⁸-Δ⁷-isomerase better than the Δ¹⁴-reductase whilst the reverse is true for fenpropidin and to a lesser extent for fenpropimorph.     

3,5,7,3′,5′-Pentahydroxyflavan and 3α-methoxyfriedelan from Humboldtia laurifolia

The leaf, bark and timber extractives of Humboldtia laurifolia were investigated and the following compounds have been isolated: O-acetyloleanolic aldehyde, a sitosteryl ester, lupeol, sitosterol, a fatty acid, 5,7,4′-trihydroxyflavone (apigenin), (2R,3R)-3,5,7,3′,5′-pentahydroxyflavan and 3α-methoxyfriedelan. The latter two compounds are new natural products.     

Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates containing piperazine as inhibitors of PI3Kα

PI3K pathway has been heavily studied and is one of the most potential targets for various cancer treatment. Herein, we designed and synthesized a series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates contained piperazine based on our previous research. They were evaluated for their PI3Kα wild-type and H1047R mutant inhibitory activities and anticancer effects in vitro. Most of these compounds displayed the potential antiproliferative activities against four cancer cell lines (HCT-116, A549, Huh7 and HL60). Among them, Compound 4p revealed the remarkable antiproliferative activity and was selected for further biological evaluation. Compound 4p displayed the potent activity against both PI3Kα wild-type and H1047R mutant, and a certain degree of selectivity for PI3Kα over PI3Kβ, γ and δ, and meanwhile it can remarkable down-regulate the phosphorylation of Akt. In addition, compound 4p was found to induce cell apoptosis via upregulation of Bax and cleaved-caspase 3/9, and downregulation of Bcl-2. The above results suggested that compound 4p could be considered as a promising PI3Kα inhibitor.     

Two triterpenoid carboxylic acids from Acanthopanax trifoliatus

Two new triterpenoid carboxylic acids have been isolated from leaves of Acanthopanax trifoliatus and their structures elucidated as 3α, 11α-dihydroxylup-20(29)-en-28-oic acid and 30α, 11α,23-trihydroxylup-20(29)-en-28-oic acid by physical data and chemical transformations.     

Design,synthesis and biological evaluation of novel series of 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold derivatives as PI3Kα inhibitors

The abnormal activation of PI3K signaling pathway leads to the occurrence of various cancers. The PI3Kα is frequently mutated and overexpressed in many human cancers. Therefore, the PI3Kα was considered as a promising target in therapeutic treatment of cancer. In this study, two series of compounds containing 2H-benzo[b][1,4]oxazin-3(4H)-one and 2H-benzo[b][1,4]oxazine scaffold were synthesized and evaluated antiproliferative activities against three cancer cell lines, including HCT-116, MDA-MB-231 and SNU638. Compound 7f with the most potent antiproliferative activity was selected for further evaluation on normal cells and PI3K kinase. Studies indicated that compound 7f could decrease the phospho-Akt (T308) in a dose-dependent manner. Four key hydrogen bonding interactions were found in the docking of 7f with PI3K enzyme. All the results suggested that 7f was a potent PI3Kα inhibitor.     

3α,11α-Dihydroxylup-20(29)-ene-23,28-dioic acid from Schefflera octophylla

The structure of 3α,11α-dihydroxylup-20(29)-ene-23,28-dioic acid, a new triterpene isolated from Schefflera octophylla, has been determined by spectroscopic methods.     

A lupenediol from Euonymus revolutus

A new lupane derivative isolated from Euonymus revolutus (Celastraceae) has been established to be 2α,3α-dihydroxy-lup-20(29)-en-28-oic acid on the basis of chemical interconversions and spectroscopic data.     

Triterpenoids from Salvia pinnata

A new and four known triterpenoids have been isolated from the leaves of Salvia pinnata. The structures of these compounds have been established by spectral data and by some chemical reactions.     

Triterpenes of five euphorbiaceae species of Sri Lanka

Five endemic species of Euphorbiaceae have been investigated. Aleuritolic acid and its derivatives were isolated from P. thwaitesii. A. cardiosperma had friedelan derivatives. The Glochidion species contained the same triterpenes, viz. glochidonol, lup-20(29)-ene-3α,23-diol and glochidiol. The bark extractives of a new Glochidion species had, in addition, glochidone and probably lup-20(29)-ene-1α,3α,23-triol. The latter is a new natural product. Glochidone was isolated for the first time outside the genus Glochidion from Bridelia moonii.     

′6β-propanoyloxy-3α-tigloyloxytropane,a new alkaloid from Datura innoxia

A new alkaloid, shown by spectroscopic and degradative means to be 6β-propanoyloxy-3α-tigloyloxytropane has been isolated from Datura innoxia r     

Cloning and functional characterization of gpd and α-tubulin promoters from Annulohypoxylon stygium,a companion fungus of Tremella fuciformis

Annulohypoxylon stygium is an ascomycete that helps Tremella fuciformis produce the fruiting body in wild state or artificial cultivation. Although the interaction between these two fungi is well known, the underlying molecular mechanism is limited. In this study, the 981 bp and 886 bp promoter sequences of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and α-tubulin gene have been cloned, respectively. Sequence analysis showed that gpd promoter contained nine CAAT boxes, and single TGACG-motif, TATA box, ABRE motif, STRE motif, MYB motif, and W box. The α-tubulin promoter included eight CAAT boxes, three STRE, two TATA boxes and MYB, single Box 4, CAT-box, CCAAT-box, TGA-element, and ABRE. Subsequently, we evaluated the promoters' function by constructing four vectors pGEH, pGRH, pTEH, and pTRH to drive fused enhanced green fluorescent protein and hygromycin B phosphotransferase (egfp-hph) or red fluorescent protein and hygromycin B phosphotransferase (rfp-hph) expression under the control of gpd or α-tubulin promoters in A. stygium. The integration of the transformed DNA into A. stygium genome was verified by PCR, Southern blot, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). All the results indicated that the two promoters could drive egfp-hph and rfp-hph expression. This result could provide help in gene functional studies by using gpd and α-tubulin promoters to direct gene over-expression or build dual promoter silencing systems.     

Biosynthesis of the tigloyl esters of Datura: Cis-trans isomerism

Datura innoxia plants were wick fed with angelic acid-[1-¹⁴C] and l-isoleucine-[U-¹⁴C] to act as a positive control. After 7 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, and 3α,6β-ditigloyloxytropan-7β-ol were isolated and it was determined that angelic acid is not a precursor for the tigloyl moiety of these alkaloids. Tiglic acid-[1-¹⁴C] which was fed via the roots to hydroponic cultures of Datura innoxia, was incorporated to a considerable degree after 8 days.     

Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells

It has been shown that the cultured cells of Digitalis purpruea are capable of transforming progesterone (I) to 5α-pregnane-3,20-dione (II), 5α-pregnan-3β-ol-20-one (III), its glucoside (IV), 5α-pregnane-3β,20α-diol (V), its glucoside (VI), 5α-pregnane-3β,20β-diol (VII), its glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one (IX), its glucoside (X), Δ-pregnen-20β-ol-3-one (XI) and its glucoside (XII). 5α-Pregnan-3β-ol-20-one glucoside (IV), 5α-pregnane-3β,20α-diol glucoside (VI), 5α-pregnane-3β,20β-diol glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one glucoside (X) and Δ⁴-pregnen-20β-ol-3-one glucoside (XII) have been found for the first time as new metabolises by plant tissue cultures. A scheme for the biotransformation of progesterone (I) has been proposed, and the reduction and glucosidation activities distinctly have been observed in these cultured cells.     

Aromatic monoterpenes from Lavandula gibsonii

Three aromatic monoterpenes, not reported previously as natural products, together with ursolic acid, were isolated from Lavandula gibsonii. They were characterized as 3-hydroxy-α,α,4-trimethyl benzyl alcohol, 3-hydroxy- α,α,4-trimethyl benzyl methyl ether and 3-hydroxy-α,4-dimethyl styrene.     

Metabolism of 3β-hydroxy-5α- and 3β-hydroxy-5β-pregnan-20-one by leaf homogenates

Leaf homogenates of Cheiranthus cheiri, Nerium oleander, Strophanthus kombé, Digitalis purpurea, and Corchorus capsularis were ex