CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

Selective photocytotoxicity of anthrols on cancer stem-like cells: The effect of quinone methides or reactive oxygen species.

Cancer stem cells (CSCs) are a subpopulation of cancer cells that share properties of embryonic stem cells like pluripotency and self-renewal and show increased resistance to chemo- and radiotherapy. Targeting CSC, rather than cancer cells in general, is a novel and promising strategy for cancer treatment. Novel therapeutic approaches, such as photodynamic therapy (PDT) have been investigated. A promising group of phototherapeutic agents are reactive intermediates - quinone methides (QMs). This study describes preparation of QM precursor, 2-hydroxy-3-hydroxymethylanthracene (2) and a detailed photochemical and photobiological investigation on similar anthracene derivatives 3 and 4. Upon photoexcitation with near visible light at λ > 400 nm 1 and 2 give QMs, that were detected by laser flash photolysis and their reactivity with nucleophiles has been demonstrated in the preparative irradiation experiments where the corresponding adducts were isolated and characterized. 3 and 4 cannot undergo photodehydration and deliver QM, but lead to the formation of phenoxyl radical and singlet oxygen, respectively. The activity of 1-4 was tested on a panel of human tumor cell lines, while special attention was devoted to demonstrate their potential selectivity towards the cells with CSC-like properties (HMLEshEcad). Upon the irradiation of cell lines treated with 1-4, an enhancement of antiproliferative activity was demonstrated, but the DNA was not the target molecule. Confocal microscopy revealed that these compounds entered the cell and, upon irradiation, reacted with cellular membranes. Our experiments demonstrated moderate selectivity of 2 and 4 towards CSC-like cells, while necrosis was shown to be a dominant cell death mechanism. Especially interesting was the selectivity of 4 that produced higher levels of ROS in CSC-like cells, which forms the basis for further research on cancer phototherapy, as well as for the elucidation of the underlying mechanism of the observed CSC selectivity based on oxidative stress activation.

In vitro and in vivo metabolic activation of berbamine to quinone methide intermediate.

Berbamine (BBM) is a bisbenzylisoquinoline alkaloid isolated from herbal medicine Berberis amurensis. BBM has been widely used for the treatment of leukemia. Recent studies demonstrated that exposure to BBM can give rise to cytotoxicity. The major objective of this study was to explore the metabolic activation of BBM in vitro and in vivo. Two oxidative metabolites (M1 and M2) and an N-acetylcysteine (NAC) conjugate (M3) were detected in human liver microsomal incubations of BBM supplemented with NAC, and the formation of all metabolites was NADPH dependent. Microsomal inhibition and recombinant P450 enzyme incubation studies demonstrated that P450 3A4 was the major enzyme responsible for the metabolic activation of BBM. In addition, a BBM-cysteine conjugate (M4) was detected in the urine of rats given BBM. The metabolism study will facilitate the understanding of the biochemical mechanisms of BBM-induced cytotoxicity.

Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products.

In vitro metabolism of oxymetazoline: evidence for bioactivation to a reactive metabolite.

Oxymetazoline (6-tert-butyl-3-(2-imidazolin-2-ylmethyl)-2,4-dimethylphenol) has been widely used as a nonprescription nasal vasoconstrictor for >40 years; however, its metabolic pathway has not been investigated. This study describes the in vitro metabolism of oxymetazoline in human, rat, and rabbit liver postmitochondrial supernatant fraction from homogenized tissue (S9) fractions and their microsomes supplemented with NADPH. The metabolites of oxymetazoline identified by liquid chromatography (LC)/UV/tandem mass spectrometry (MS/MS), included M1 (monohydroxylation of the t-butyl group), M2 (oxidative dehydrogenation of the imidazoline to an imidazole moiety), M3 (monohydroxylation of M2), M4 (dihydroxylation of oxymetazoline), and M5 (dihydroxylation of M2). Screening with nine human expressed cytochromes P450 (P450s) identified CYP2C19 as the single P450 isoform catalyzing the formation of M1, M2, and M3. Glutathione conjugates of oxymetazoline (M6) and M2 (M7) were identified in the liver S9 fractions, indicating the capability of oxymetazoline to undergo bioactivation to reactive intermediate species. M6 and M7 were not detected in those liver S9 incubations without NADPH. Cysteine conjugates (M8 and M9) derived from glutathione conjugates and hydroxylated glutathione conjugates (M10 and M11) were also identified. The reactive intermediate of oxymetazoline was trapped with glutathione and N-acetyl cysteine and identified by LC/MS/MS. M6 was isolated and identified by one-dimensional or two-dimensional NMR as the glutathione conjugate of a p-quinone methide. We have shown the tendency of oxymetazoline to form p-quinone methide species via a bioactivation mechanism involving a CYP2C19-catalyzed two-electron oxidation. Nevertheless, we conclude that the formation of this reactive species might not be a safety concern for oxymetazoline nasal products because of the typical low-dose and brief dosage regimen limited to nasal delivery.

Suppression of inflammatory responses by celastrol, a quinone methide triterpenoid isolated from Celastrus regelii.

BACKGROUND: Celastrol, a quinone methide triterpenoid isolated from the Celastraceae family, exhibits various biological properties, including chemopreventive, antioxidant and neuroprotective effects. In this study, we showed that celastrol inhibits inflammatory reactions in macrophages and protects mice from skin inflammation. MATERIALS AND METHODS: Anti-inflammatory effects of celastrol (0-1 microM) were examined in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. To investigate the effects of celastrol (0-50 microg per mice) in vivo, activation of myeloperoxidase (MPO) and histological assessment were examined in the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear oedema model. RESULTS: Our in vitro experiments showed that celastrol suppressed not only LPS-stimulated generation of nitric oxide and prostaglandin E(2), but also expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW264.7 cells. Similarly, celastrol inhibited LPS-induced production of inflammatory cytokines, including tumour necrosis factor-alpha and interleukin-6. In an animal model, celastrol protected mice from TPA-induced ear oedema, possibly by inhibiting MPO activity and production of inflammatory cytokines. CONCLUSIONS: Our data suggest that celastrol inhibits the production of inflammatory mediators and is a potential target for the treatment of various inflammatory diseases.

NRH:quinone oxidoreductase 2 (NQO2) catalyzes metabolic activation of quinones and anti-tumor drugs.

NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that utilizes NRH as electron donor. The present studies investigate the role of NQO2 in metabolic detoxification/activation of quinones and quinone based anti-tumor drugs. Chinese hamster ovary (CHO) cells stably overexpressing cDNA derived mouse NQO2 and mouse keratinocytes from DMBA-induced skin tumors in wild-type and NQO2-null mice were generated. The CHO cells overexpressing NQO2 and mouse keratinocytes expressing or deficient in NQO2 were treated with varying concentrations of mitomycin C (MMC), CB1954, MMC analog BMY25067, EO9, menadione and BP-3,6-quinone, in the absence and presence of NRH. The cytotoxicity of the drugs was evaluated by colony formation. The CHO cells overexpressing higher levels of mouse NQO2 showed significantly increased cytotoxicity to menadione, BP-3,6-quinone and to the anti-tumor drugs MMC and CB1954 when compared to CHO cells expressing endogenous NQO2. The cytotoxicity increased in presence of NRH. Similar results were also observed with BMY25067 and EO9 treatments, but to a lesser extent. The results on keratinocytes deficient in NQO2 supported the data from CHO cells. The inclusion of NRH had no effect on cytotoxicity of quinones and drugs in keratinocytes deficient in NQO2. Mouse NQO2 protein was expressed in bacteria, purified and used to study the role of NQO2 in MMC-induced DNA cross-linking. Bacterially expressed and purified NQO2 efficiently catalyzed MMC activation that led to DNA cross-linking. These results concluded that NQO2 plays a significant role in the metabolic activation of both quinones and anti-tumor drugs leading to cytotoxicity and cell death.

Dopamine-dependent iron toxicity in cells derived from rat hypothalamus.

We report a new and specific mechanism for iron-mediated neurotoxicity using RCHT cells, which were derived from rat hypothalamus. RCHT cells exhibit immunofluorescent-positive markers for dopamine beta-hydroxylase and the norepinephrine transporter, NET. In the present study, we observed that iron-induced neurotoxicity in RCHT cells was dependent on (i) formation of an Fe-dopamine complex (100 microM FeCl3:100 microM dopamine); (ii) specific uptake of the Fe-dopamine complex into RCHT cells via NET (79+/-2 pmol 59Fe/mg/min; P<0.05), since the uptake of the 59Fe-dopamine complex by the cells was inhibited by 30 microM reboxetine, a specific NET inhibitor (78% inhibition, P<0.001); and (iii) intracellular oxidation of dopamine present in the Fe-dopamine complex to aminochrome; (iv) inhibition of DT-diaphorase, since incubation of RCHT cells with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase, induced significant cell death (51+/-5%; P<0.001). However, this cell death was reduced by 75% when the cells were incubated in the presence of 30 microM reboxetine (P<0.01). No significant cell death was observed when the cells were incubated with 100 microM dopamine, 100 microM Fe-Dopamine complex, 100 microM dicoumarol, or 100 microM FeCl3 (8.3+/-2, 9+/-4, 8.5+/-3, or 9.7+/-2% of control, respectively). ESR studies using the spin trapping agent DMPO showed no formation of hydroxyl radicals when the cells were incubated with 100 microM FeCl3 alone. However, using the same ESR technique, the formation of hydroxyl radicals and a carbon-centered radical was detected when the cells were incubated with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol. These studies suggest that iron can induce cell toxicity by a mechanism that requires the formation and NET-mediated uptake of an Fe-dopamine complex, ultimately resulting in the intracellular formation of reactive species.