2,2-Dialkyl-1,2-dihydroquinolines: cytochrome P 450 catalyzed N-alkylporphyrin formation, ferrochelatase inhibition, and induction of 5-aminolevulinic acid synthase activity

Abstract

Incubation of 2,4-diethyl-1,2-dihydro-2-methylquinoline (DMDQ) with hepatic microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, or dexamethasone results in minor loss of the cytochrome P-450 chromophore and accumulation of a hepatic pigment. The hepatic pigment consists of the four regioisomers of N-ethylprotoporphyrin IX and minor amounts of the corresponding N-methyl regioisomers. Exposure of chick embryo liver cells to DMDQ results in inhibition of their ferrochelatase activity, induction of their 5-aminolevulinic acid synthase activity, and accumulation of protoporphyrin IX. 1,2-Dihydro-2,2,4-trimethylquinoline (TMDQ) causes negligible loss of cytochrome P-450 in rat liver microsomes but in vivo still produces the four N-methylprotoporphyrin IX regioisomers in low yield. Furthermore, it inhibits ferrochelatase activity, elevates 5-aminolevulinic acid synthase activity, and causes protoporphyrin IX accumulation in cultured chick embryo hepatocytes. One-electron oxidation of the 2,2-dialkyl-1,2-dihydroquinolines to radical cations is postulated to result in N-alkylation of the prosthetic heme group of cytochrome P-450. The N-alkylprotoporphyrins IX thus formed are potent inhibitors of ferrochelatase. Inhibition of ferrochelatase causes the induction of 5-aminolevulinic acid synthase and the accumulation of protoporphyrin IX. Heme alkylation and ferrochelatase inhibition may be generally associated with substrates that are subject to cytochrome P-450 mediated oxidative extrusion of alkyl radicals.

Inhibition of Cytochrome P450 Enzymes

“…A variety of compounds containing an olefinic bond, such as ethylene, allylisopropylacetamide (AIA), and secobarbital, can form covalent adducts on the nitrogen of the porphyrin group of the prosthetic heme leading to inactivation [382][383][384][385] Secobarbital has been shown to completely inactivate CYP2B1 with only partial loss of the heme chromophore [384,386,387] Isolation of the modified CYP2B1 protein and the N-alkylated porphyrins indicates that the reactive compound partitions between protein modification, N-alkylation of the heme, and formation of an epoxide metabolite in the ratio of 02:08:59, respectively [387] (Fig 517) The formation of a heme adduct in the active site of CYP2B1 was confirmed spectrally based on its typical absorption maximum at ~ 445 nm, a characteristic fea- ture of iron complexed N-modified porphyrins [388] The modified CYP2B1 peptide has been isolated and shown to span residues 277-323 By sequence analogy, these residues correspond to the distal I helix in P450 cam [386,[389][390][391][392] Further digestion of the modified peptide has resulted in identification of the site for modification by secobarbital to a residue in the peptide G299-S304 [387] Although the identity of the adducted residue has not yet been determined, these results are consistent with modification of the CYP2B1 in the active site Specific mutations of CYP2B1 in the putative substrate-recognition site (SRS) 2,4,5, and 6, but not in SRS-1, cause a decrease in the inactivation by secobarbital Mutation of residue 367 from V to A in SRS-5 had a marked inhibitory effect on protein modification [387] Isolation of the N-modified porphyrins as the parent adducts as well as the corresponding dimethylesters and analysis by LC-MS demonstrated the formation of adducts of hydroxysecobarbital with protoporphyrin IX (Fig 517) [386] Like terminal olefins, terminal acetylenes can alkylate the P450 prosthetic heme However, compounds such as 10-undecynoic acid, 1-ethynylpyrene, 2-ethynylnaphthalene, 9-ethynylnaphthalene, 17β-ethynylprogesterone, and 17α-ethynylestradiol (EE) inactivate P450s primarily by covalently binding to the apoprotein with little or no effect on the heme group (Fig 518) [305, 310, 317, 318] Almost stoichiometric binding of 10-undecynoic acid to rat liver CYP4A1 (the ω-hydroxylase) as well as of 2-ethynylnaphthalene and 1-ethynylpyrene to CYP1A1 and -1A2, and of EE to CYP3A4 has been observed [305]…”

Autocatalytic Radical Reactions in Physiological Prosthetic Heme Modification

“…For example, horseradish peroxidase reacts with alkyl and arylhydrazines, 4-alkyldihydropyridines, cyclopropanone hydrate, nitromethane, and azide, to give adducts in which the substrate or a fragment of it is covalently attached to a meso -carbon of the heme. In the case of cytochrome P450, reaction with an even more diverse range of compounds, including alkyl and arylhydrazines, , 4-alkyldihydropyridines, , 2,2-dialkyldihydroquinolines, terminal olefins and acetylenes, − 1-aminoaryltriazoles, sydnones, and alkyl aldehydes results in covalent attachment of the xenobiotic or a fragment thereof to the heme iron atom, a pyrrole nitrogen atom of the porphyrin, or a porphyrin meso -carbon. Many of these “pathological” autocatalytic reactions of heme groups are covered in other reviews and are not discussed here. ,, …”

Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants

“…This spectral absorption was dependent on the olefinic moiety of the drug, because amobarbital, the saturated analog of SB, failed to destroy the enzyme as well as yield a corresponding spectrum, when it replaced SB in the incubation mixture. It is conceivable that this spectrum reflects the presence of SB-N-heme (Fe 3ϩ ) adduct in this in vitro system, because it is similar to that of the purified iron-complexed N-ethylporphyrins, that exhibit absorbance maxima at 442 nm (25). Furthermore, consistent with this possibility, the absolute spectra of phenylacetylene-or 3-alkylsydnone-inactivated P450 2B1 preparations that yield N-modified heme adducts also exhibit a time-dependent increase in a 445 nm shoulder (26,27).…”