Forms Of Cytochrome P450 Enzymes Research Articles

The binding of nitrogen-donor ligands to the ferric and ferrous forms of cytochrome P450 enzymes

The cytochrome P450 superfamily of heme-thiolate monooxygenase enzymes can catalyse various oxidation reactions. The addition of a substrate or an inhibitor ligand induces changes in the absorption spectrum of these enzymes and UV–visible (UV–vis) absorbance spectroscopy is the most common and readily available technique used to interrogate their heme and active site environment. Nitrogen-containing ligands can inhibit the catalytic cycle of heme enzymes by interacting with the heme. Here we evaluate the binding of imidazole and pyridine-based ligands to the ferric and ferrous forms of a selection of bacterial cytochrome P450 enzymes using UV–visible absorbance spectroscopy. The majority of these ligands interact with the heme as one would expect for type II nitrogen directly coordinated to a ferric heme-thiolate species. However, the spectroscopic changes observed in the ligand-bound ferrous forms indicated differences in the heme environment across these P450 enzyme/ligand combinations. Multiple species were observed in the UV–vis spectra of the ferrous ligand-bound P450s. None of the enzymes gave rise to the isolation of a single species with a Soret band at ∼442–447 nm, indicative of a 6-coordinate ferrous thiolate species with a nitrogen-donor ligand. A ferrous species with Soret band at ∼427 nm coupled with an α-band of increased intensity was observed with the imidazole ligands. With some enzyme-ligand combinations reduction resulted in breaking of the iron‑nitrogen bond yielding a 5-coordinate high-spin ferrous species. In other instances, the ferrous form was readily oxidised back to the ferric form on addition of the ligand.

Cytochrome P450-Activated Prodrugs: Targeted Drug Delivery

Cytochrome P450 (CYP) enzymes are a superfamily of heme containing proteins that catalyze xenobiotic metabolism phase I reactions. Oxidation reactions are the most common CYP-catalyzed reactions for both endogenous substrates and exogenous compounds, including drugs, although CYP enzymes are capable also to catalyze reduction reactions. Whereas the majority of clinically used drugs are inactivated by CYPs, several prodrugs are bioconverted to their active species by these enzymes. Therefore, this mechanism could be exploited to a greater extend, e.g. by taking advantage of the different CYP enzymes to achieve targeted drug delivery, to improve efficacy or to decrease the unwanted adverse effects of existing and novel drug molecules. This review describes the potential of CYP enzymes in prodrug design and summarizes a wide variety of CYP-activated prodrug structures, which are on the market or under the development. The bioactivation mechanisms of each CYP-activated prodrug structure are described and the specificity for the different forms of CYP enzymes is discussed.

Cytochrome P450 enzymes: Novel options for cancer therapeutics

Abstract The concept of overexpression of individual forms of cytochrome P450 enzymes in tumor cells is now becoming well recognized. Indeed, a growing body of research highlights the overexpression of P450s, particularly CYP1B1, in tumor cells as representing novel targets for anticancer therapy. The purpose of this review is to outline the novel therapeutic options and opportunities arising from both enhanced endogenous expression of cytochrome P450 in tumors and cytochrome P450-mediated gene therapy.

Drug Metabolizing Enzymes in Primates: Focused on Cytochrome P450.

To date, it is very difficult to extrapolate the data obtained from experimental animals (for example rodents) to humans, since there are species differences in the characteristics of drug metabolising enzymes. The monkeys is often used for the preclinical study. However, the similarities for properties and capacities of drug metabolizing enzymes between monkeys and humans were not well recognized. Therefore, we started to characterize the drug metabolizing enzymes, especially cytochrome P450s, in non-human primates. Four forms of cytochrome P450 enzymes (termed P450 CMLa-d) were purified from hepatic microsomes of cynomolgus monkeys. P450 CMLa was classified into the CYP2B subfamily, in its N-terminal amino acid sequence, catalytic activities, and immunochemical properties and act as a testosterone 16, 8-hydroxylase in cynomolgus monkseys. P450 CMLa is expressed constitutively as a minor form of P450 and is one of the forms inducible by phenobarbital. The nucleotide sequence of a cDNA coding for the monkey CYP2B was also determined and is now called CYP2B17. P450 CMLb and P450 CMLc purified from hepatic microsomes of cynomolgus monkeys were classified into CYP2A and CYP3A subfamilies, respectively. The content of the protein immunoreactive with anti-P450 CMLc in cynomolgus monkeys was decreased by the administration of aztreonam. P450 CMLd was one of CYP2C enzymes and was the S-mephenytoin 4'-hydroxylase in cynomolgus monkeys. The cDNA coding for monkey CYP2C (reffered to as CYP2C37) was isolated and was expressed in yeast. The expressed protein act as S-mephenytoin 4'-hydroxylase.

Metabolism of benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.

Biotransformation of terodiline. III. Opposed stereoselectivity in the benzylic and aromatic hydroxylations in rat liver microsomes.

1. Terodiline (N-tert-butyl-4,4-diphenyl-2-butylamine) is a racemic drug with anticholinergic and/or calcium antagonistic activity, which is subject to renewed interest as a potential remedy for urinary incontinence. As part of the current investigations on terodiline, the metabolism of its enantiomers is being investigated. 2. The metabolism of the enantiomers of terodiline in rat liver microsomes is slow, as for the racemate, though the S-enantiomer is metabolized more rapidly than its optical antipode. Phenobarbitone pretreatment of the rats enhances the metabolism with a marked increase in the conversion of the S-enantiomer. 3. While aromatic p-hydroxylation greatly exceeds benzylic oxidation in the metabolism of R-terodiline, this situation is reversed in the metabolism of S-terodiline. Moreover, the rate of aromatic p-hydroxylation of racemic terodiline follows that of R-terodiline, while the rate of benzylic hydroxylation of racemic terodiline follows that of S-terodiline. Phenobarbital pretreatment of the rats had little or no effect on aromatic p-hydroxylation but markedly increased benzylic oxidation. 4. Separation of the mixture of p-hydroxylated metabolites into diastereomeric pairs showed that their composition is highly dependent on which form of terodiline is used as substrate. 5. The results from the study are compatible with the participation of multiple forms of cytochrome P-450 enzymes.

Effect of retinoids on carcinogen-induced mutagenesis in Salmonella tester strains

The ability of retinol (Rol) in altering mutation frequencies induced by 7 carcinogens was studied in Salmonella/microsome assay using 4 tester strains namely TA98, TA100, TA102 and TA1535. The 7 carcinogens used were aflatoxin B 1 (AFB), cyclophosphamide (CPP), 3-methylcholanthrene (MCA), benzo[ a]pyrene (BP), benz[ a]anthracene (BA), 9,10-dimethyl-1,2-benz[ a]anthracene (DMBA) and mitomycin C (MMC). As reported previously, Rol significantly reduced the number of His + revertants induced by AFB. It also reduced mutations induced by CPP or MCA but not that by BP, BA, DMBA or MMC. The abilities of Rol, retinoic acid, retinyl acetate and a known inhibitor for certain P-450 isozymes, 7,8-benzo-flavone (BF) in inhibiting mutations caused by AFB and BP were studied and compared. All the 3 retinoids caused significant reduction of AFB-induced His + revertants in a dose-dependent manner, but there was no effect on BP-induced mutation. BF strongly inhibited both AFB- and BP-induced revertants. The possibility of retinoids in exerting their effects on mutagenesis of precarcinogens by inhibiting only certain forms of cytochrome P-450 enzymes is discussed.