The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells

RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), which is currently in clinical trials, is a diaziridinyl benzoquinone bioreductive anticancer drug that was designed to be activated by the obligate two-electron reductive enzyme NAD(P)H quinone oxidoreductase 1 (NQO1). In this electron paramagnetic resonance (EPR) study we showed that RH1 was reductively activated by the one-electron reductive enzyme NADPH cytochrome P450 reductase and by a suspension of HCT116 human colon cancer cells to yield a semiquinone free radical. As shown by EPR spin trapping experiments RH1 was reductively activated by cytochrome P450 reductase and underwent redox cycling to produce damaging hydroxyl radicals in reactions that were both H₂O₂- and iron-dependent. Thus, reductive activation by cytochrome P450 reductase or other reductases to produce a semiquinone that can redox cycle to produce damaging hydroxyl radicals and/or DNA-reactive alkylating species may contribute to the potent cell growth inhibitory effects of RH1. These results also suggest that selection of patients for treatment with RH1 based on their expression levels of NQO1 may be problematic.     

Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed‐function oxidase system that participates in the metabolism of endo‐ and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD‐to‐FMN, but not FMN‐to‐P450, electron transfer. Here, we report the 2.5 Å resolution crystal structure of a functionally competent yeast–human chimeric CPR in an open conformation, compatible with FMN‐to‐P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 Å.     

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.     

Establishment of a yeast system that stably expresses human cytochrome P450 reductase: application for the study of drug metabolism of cytochrome P450s in vitro

Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity. A novel system of Saccharomyces cerevisiae useful for expression studies of mammalian microsomal CYPs was established. Human CPR (hCPR) was co-expressed with human CYP3A4 (hCYP3A4) in this system, and two expression plasmids pTpLC and pYeplac195-3A4 containing the cDNA of hCPR and hCYP3A4 were constructed, respectively. The two plasmids were applied first and controlled by phosphoglycerate kinase (PGK) promoter. S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig. The activity of hCPR in yeast BWG-CPR was 443.2 nmol reduced cytochrome c/min/mg, which was about three times the CPR activity of the microsome prepared from the parental yeast. The protein amount of hCYP3A4 in BWG-CPR/3A4 was 35.53 pmol/mg, and the 6beta-hydroxylation testosterone formation activity of hCYP3A4 expressed was 7.5 nmol/min/nmol CYP, 30 times higher than the activity of hCYP3A4 expressed in the parental yeast, and almost two times the activity of hCYP3A4 from homologous human liver microsome. Meanwhile, BWG-CPR/3A4 retained 100 generations under nonselective culture conditions, indicating this yeast was a mitotically stable transformant. BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome. This special expression host for CYPs was validated to be stable and efficient for the expression of CYPs, applying as an effective selection model for the drug metabolism in vitro.     

Cloning and characterization of the NADPH cytochrome P450 reductase gene (CPR) from Candida bombicola

Candida bombicola is a yeast with at least two appealing features. The species can grow on alkanes when provided as the sole carbon source, and it produces glycolipids, which have several industrial, cosmetic and pharmaceutical applications. Both metabolic processes require in their pathway the activity of cytochrome P450 monooxygenase. This enzyme needs and gets reducing equivalents from NADPH cytochrome P450 reductase (CPR). The CPR gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes an enzyme of 687 amino acids, which shows homology with known CPRs of other species. The functionality of the gene was proven by heterologous expression in Escherichia coli. The recombinant protein exhibited NADPH-dependent cytochrome c reducing activity. Cloning and characterization of this enzyme is an important step in the study of the cytochrome P450 monooxygenase system of Candida bombicola. The GenBank accession number of the sequence described in this article is EF050789.     

Heme oxygenase-1 and NADPH cytochrome P450 reductase expression in experimental allergic encephalomyelitis: an expanded view of the stress response

Oxidative stress is implicated in the pathogenesis of experimental allergic encephalomyelitis (EAE), a model for multiple sclerosis. Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress. HO-1 metabolizes the pro-oxidant heme to the antioxidant biliverdin and CO. HO-1 requires electrons, donated by NADPH cytochrome P450 reductase (henceforth, reductase), for catalytic activity. EAE was induced with a peptide of proteolipid protein in SJL mice, and the expression of HO-1 and reductase in the hindbrain was analyzed. HO-1 protein levels were significantly increased in EAE animals compared with control mice. HO-1 expression was present in ameboid macrophages, reactive microglia, and astrocytes in white matter tracks. Bergmann glia and ameboid macrophages also were occasionally stained in the molecular layer of the cerebellum. Unlike HO-1, reductase protein levels decreased with disease severity. HO-1 and reductase were associated with each other in endoplasmic reticulum micelles, suggesting that the decrease in reductase does not interfere with its association with HO-1. In cells that express HO-1, the association of reductase with HO-1 should competitively inhibit the interaction of reductase with cytochrome P450 isozymes and thereby limit free radical production as the latter two enzymes act cooperatively to produce superoxide. The increase in HO-1 together with the decrease in reductase may be part of a common defense mechanism attempting to minimize tissue damage in several neurological conditions.     

The effect of dimethyl sulfoxide on the function of cytochrome P450 2D6 in HepG2 cells upon the co-expression with NADPH-cytochrome P450 reductase

HepG2 cells, a human hepatoma cell line, stably expressing NADPH-cytochrome P450 reductase (OR) and/or cytochrome P450 2D6 wild-type (CYP2D6-WT) or its variants (Pro34Ser, Gly42Arg, Arg296Cys and Ser486Thr) were established in the present study. The cultivation of HepG2 cells expressing CYP2D6-WT in the culture medium containing dimethyl sulfoxide (DMSO, 0.1% of final concentration) markedly increased the bufuralol (BF) 1'-hydroxylase activity compared with that of control cells when cultivated without DMSO. A similar effect was also observed in HepG2 cells stably expressing CYP2D6 and OR. The addition of hemin in place of DMSO to the culture medium resulted in no increase in the enzyme activity. Western blot analysis revealed that the levels of CYP2D6 protein were similar between DMSO-treated and non-treated HepG2 cells regardless of OR expression. Spectrophotometric analysis of reduced carbon monoxide-difference spectra of HepG2 cells expressing CYP2D6-WT and/or OR demonstrated that the addition of DMSO increased the peak height of functional CYP2D6 at 450 nm. These results suggest that the increase in CYP2D6 activity is attributable to the radical-scavenging effect of DMSO. The HepG2 cell lines stably expressing OR and CYP2D6 or its variants in combination with DMSO treatment may be useful for screening the cytotoxicity of chemical compounds which undergo oxidation by CYP2D6.     

Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using ³²P-labeled human DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5′-GG-3′, 5-GGGG-3′, 5′-GGGGG-3′ sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H₂O₂ and Cu(I). The formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could beproduced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.     

Structure-activity relationships of organosulfur compounds as inhibitors of cytochrome P450 1-mediated activation of benzo[a]pyrene in a human cell model

Twelve naturally-occurring organosulfur compounds were investigated as inhibitors of cytochrome P450 1 (CYP450 1)-mediated activation of benzo[a]pyrene (B[a]P) in human hepatoma (HepG2) cells. Inhibition depended on the presence of a diallyl group and the number of S atoms. Diallyl trisulfide (DATS), with a diallyl group and three S atoms, had the highest activity with an IC₅₀ of 0.4 mM, and 1.5-fold higher potency than diallyl disulfide (DADS) containing a diallyl group and two S atoms. Organosulfur compounds containing an alkyl group were less effective, or even ineffective, inhibitors of both CYP450 1 and B[a]P-induced cytotoxicity than DADS and DATS. Alliin and S-allyl cysteine containing the S-cysteinyl group had no inhibition.     

Anionic phospholipid-induced regulation of reactive oxygen species production by human cytochrome P450 2E1

We suggest that the cytochrome P450 2E1 (CYP2E1)-induced formation of reactive oxygen species (ROS) can be regulated by anionic phospholipids and the presence of the N-terminal region of the enzyme. When the content of cardiolipin (CL) in membranes at the expense of phosphatidylcholine matrix was increased, the ROS produced by recombinant human CYP2E1 was decreased as a function of CL concentration. On the contrary, the N-terminally truncated CYP2E1 had a decreased effect on the lipid-induced reduction of ROS formation. These results suggest that specific phospholipids can regulate the function of CYP2E1 by interaction with the enzyme including the N-terminal region(s).     

Electron transfer from pyridinyl radicals, hydrated electrons, CO2.- and O2.- to bacterial cytochrome P450.

Several rate constants for one-electron reduction of cytochrome P450 are more rapid in the absence than in the presence of the specific substrate. The respective values for methyl viologen, nicotinamide adenine dinucleotide and the 1-methyl-4-(and -3-)carbamidopyridinium radicals are 2.6, 3.4, 6 and 35 × 10⁷ M^?1 s^?1 without camphor, and 0.15, 0.1, 1.8 and 110 × 10⁷ M^?1 s^?1 for the camphor complex. Hydrated electrons react with cytochrome P450 with a rate constant of 3.0 × 10¹⁰ M^?1 s^?1 whether camphor is bound or not, but little of the reduction takes place at the haem iron. No reduction of the haem iron by $\text{CO}{}_2{}^{\mathrm{?-}}$ or $\text{O}{}_2{}^{\mathrm{?-}}$ could be detected, whether camphor is bound or not.     

Intratumoral activation of cyclophosphamide by retroviral transfer of the cytochrome P450 2B1 in a pancreatic tumor model. Combination with the HSVtk/GCV system

Background

Pancreatic cancer is one of the most aggressive human tumors and the development of new therapeutic approaches is particularly urgent since current therapies are not effective. The use of pro‐drug‐activating genes is a possible approach for cancer gene therapy.

Methods

The present study evaluated the efficiency of the cytochrome P4502B1 (CYP2B1) suicide gene that encodes the enzyme responsible for activating the pro‐drug cyclophosphamide (CPA), in pancreatic tumor cells invitro and in vivo. The effects on tumor growth of the combination of two suicide systems, CYP2B1/CPA and herpes simplex virus thymidine kinase gene/ganciclovir (HSVtk/GCV), were also studied.

Results

Retroviral CYP2B1 transfer followed by CPA treatment highly sensitized pancreatic tumor cells NP‐9, NP‐18, and NP‐31, and led to stabilization of tumor growth in a pancreatic tumor model. Differences in tumor volume at the end of the treatment were statistically significant when compared with animals injected with CPA alone. The combination of both suicide systems CYP2B1/CPA and HSVtk/GCV in vitro resulted in a potentiation of the killing effect. However, no potentiation was achieved in vivo, although retardation in tumor growth was evident.

Conclusions

The results show that in situ transduction of pancreatic tumor cells with the CYP2B1 gene by retroviral vectors clearly increases the sensitivity to CPA. Moreover, they suggest that in order to achieve a potentiation on cell killing when the two suicide systems HSVtk/GCV and CYP2B1/CPA are combined, co‐expression of both genes in the same tumor cell would be necessary. Copyright © 2002 John Wiley & Sons, Ltd.

     

Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR

Spectrophotometric assays of Mycobacterium aurum MO1 cells extracts gave evidence of a soluble cytochrome P450, involved in the degradative pathway of morpholine, a waste product from the chemical industry. In order to get further information, the kinetics of the biodegradation of the sulfur analogue thiomorpholine was monitored by using in situ nuclear magnetic resonance (NMR). This technique allowed the identification of two intermediates: the sulfoxide of thiomorpholine resulting from S-oxidation and thiodiglycolic acid owing to ring cleavage. The S-oxidation (S SO) represents one of the well-known reactions catalyzed by cytochromes P450. The inhibitory effect of metyrapone, a cytochrome P450 inhibitor, on the thiomorpholine and morpholine degradative abilities of M. aurum MO1 confirmed the involvement of a cytochrome P450. These results and the decrease of the rate of formation of the first intermediate during the morpholine degradation, 2-(2-aminoethoxy) acetate, proved the key role of the cytochrome P450 in the early events of the biodegradation, i.e, in the C–-N bond cleavage.     

Comparative study of the binding mode between cytochrome P450 17A1 and prostate cancer drugs in the absence of haem iron

Abstract

According to the X-ray crystal structures of CYP17A1 (including its complexes with inhibitors), it is shown that a hydrogen bond exists between CYP17A1 and its inhibitors (such as abiraterone and TOK-001). Previous short MD simulations (50?ns) suggested that the binding of abiraterone to CYP17A1 is stronger than that of TOK-001. In this work, by carrying out long atomistic MD simulations (200?ns) of CYP17A1 and its complexes with abiraterone and TOK-001, we observed a binding mode between CYP17A1 and abiraterone, which is different from the binding mode between CYP17A1 and TOK-001. In the case of abiraterone binding, the unfilled volume in the active site cavity increases the freedom of movement of abiraterone within CYP17A1, leading to the collective motions of the helices G and B′ as well as the breaking of hydrogen bond existing between the 3β-OH group of abiraterone and N202 of CYP17A1. However, the unfilled volume in the active site cavity can be occupied by the benzimidazole ring of TOK-001, restraining the motion of TOK-001. By pulling the two inhibitors (abiraterone and TOK-001) out of the binding pocket in CYP17A1, we discovered that abiraterone and TOK-001 were moved from their binding sites to the surface of protein similarly through the channels formed by the helices G and B′. In addition, based on the free energy calculations, one can see that it is energetically favorable for the two inhibitors (abiraterone and TOK-001) to enter into the binding pocket in CYP17A1.     

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.     

Inhibition of human cytochrome p450 1b1 further clarifies its role in the activation of dibenzo[a,l]pyrene in cells in culture

Metabolic activation and DNA adduct formation of the carcinogenic aromatic hydrocarbon dibenzo[a,l]pyrene (DBP) was investigated in human mammary carcinoma MCF-7 cells and human cytochrome P450 (CYP) 1B1-expressing Chinese hamster V79 cells in culture. It has been shown that DBP is metabolically activated to DNA-binding diol epoxides both in vitro and in vivo. To further establish the role of human CYP1B1 in the activation of DBP, both cell lines were cotreated with DBP and a selective chemical inhibitor of CYP1B1, 2,4,3' ,5'-tetramethoxy-stilbene (TMS). Results from DBP-DNA adduct analyses revealed the complete inhibition of DNA binding when cells were cotreated with DBP and TMS in comparison to DBP alone. Inactivation of CYP1B1 by TMS was also demonstrated through a decrease in the 7-ethoxyresorufin O-deethylase (EROD) activity in microsomes isolated from these cells. Emodin, 3-methyl-1,6,8-trihydroxyanthraquinone, an active ingredient of an herb, has been recently shown of being able to induce CYP1 gene expression. Examination of human CYP1B1 induction and EROD activity confirmed an increase in protein levels upon cotreatment with emodin and DBP. Despite increases in protein levels and enzyme activity, there was no significant change in DBP-DNA binding levels at very low substrate concentrations (17 nM). The data obtained in this study emphasize the central role of CYP1B1 in the activation of DBP in human cells in culture.     

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides as new antimitotic prodrugs activated by cytochrome P450 1A1 in breast cancer cells

The role and the importance of the sulfonate moiety in phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) were assessed using its bioisosteric sulfonamide equivalent leading to new cytochrome P450 1A1 (CYP1A1)-activated prodrugs designated as 4-(3-alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs). PAIB-SAs are active in the submicromolar to low micromolar range showing selectivity toward CYP1A1-expressing MCF7 cells as compared to cells devoid of CYP1A1 activity such as MDA-MB-231 and HaCaT cells. The most potent, PAIB-SA 13, bearing a trimethoxyphenyl group on ring B blocks the cell cycle progression in G2/M phase, disrupts the microtubule dynamics and is biotransformed by CYP1A1 into CEU-638, its potent antimicrotuble counterpart. Structure-activity relationships related to PAIB-SOs and PAIB-SAs evidenced that PAIB-SOs and PAIB-SAs are true bioisosteric equivalents fully and selectively activatable by CYP1A-expressing cells into potent antimitotics.