Diclofenac and its derivatives as tools for studying human cytochromes P450 active sites: particular efficiency and regioselectivity of P450 2Cs.
A comparison of the oxidations of diclofenac with microsomes of yeasts expressing various human liver cytochromes P450 showed that P450 2C9 regioselectively led to 4'-hydroxy diclofenac (4'-OHD) whereas P450 3A4 only led to 5-hydroxy diclofenac (5-OHD). P450 2C19, 2C18, and 2C8 led to the simultaneous formation of 4'-OHD and 5-OHD (respective molar ratios of 1.3, 0.37, and 0.17), and P450 1A1, 1A2, 2D6, and 2E1 failed to give any detectable hydroxylated metabolite under identical conditions. P450 2C9 was found to be much more efficient for diclofenac hydroxylation than all the other P450s tested (k(cat)/K(M) of 1.6 min(-1) microM(-1) instead of 0.025 for the second more active P450), mainly because of markedly lower K(M) values (15 +/- 8 instead of values between 170 and 630 microM). Oxidation of diclofenac with chemical model systems of cytochrome P450 based on iron porphyrin catalysts exclusively led to the quinone imine derived from two-electron oxidation of 5-OHD, in an almost quantitative yield. Two derivatives of diclofenac lacking its COO(-) function were then synthesized; their oxidation by recombinant human P450 2Cs always led to a major product coming from their 5-hydroxylation. Substrate 2, which derives from reduction of the COO(-) function of diclofenac to the CH(2)OH function, was studied in more detail. All the P450s tested (1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, and 3A4) almost exclusively led to its 5-hydroxylation. P450s of the 2C subfamily were found to be the most efficient catalysts for this reaction, with k(cat)/K(M) values between 0.2 and 1.6 min(-1) microM(-1). Oxidation of 2 with an iron porphyrin-based chemical model of cytochrome P450 also led to a product derived from the oxidation of 2 at position 5. These results show that oxidation of diclofenac and its derivative 2, either with chemical model systems of cytochrome P450 or with recombinant human P450s, generally occurs at position 5. This position, para to the NH group on the more electron-rich aromatic ring of diclofenac derivatives, is thus, as expected, the privileged site of reaction of electrophilic, oxidant species. The most spectacular exception to this chemoselective 5-oxidation of diclofenac derivatives was found for oxidation of diclofenac itself with P450 2C9 (and P450 2C19 and 2C18 to a lesser extent), which only led to 4'-OHD. A likely explanation for this result is a strict positioning of diclofenac in the P450 2C9 active site, via its COO(-) function, to completely orientate its hydroxylation toward position 4', which is not chemically preferred. P450 2C19, 2C18, and 2C8 would not lead to such a strict positioning as they give mixtures of 4'-OHD and 5-OHD. The above results show that diclofenac derivatives are interesting tools to compare the active site topologies of human P450 2Cs.
Mancy
A
A
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris V, Paris, France.
Antignac
M
M
Minoletti
C
C
Dijols
S
S
Mouries
V
V
Duong
N T
NT
Battioni
P
P
Dansette
P M
PM
Mansuy
D
D
D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism*; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism; D018716:Molecular Mimicry; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; D020122:tert-Butylhydroperoxide; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C082185:CYP2C18 protein, human; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D039181:Steroid 16-alpha-Hydroxylase