Cytochromes P450 are a superfamily of proteins [1] which are involved in the oxidative metabolism of both foreign and endogenous compounds [2]. The cytochrome P450 4A family is known to be highly induced by peroxisome proliferators in mouse liver [3,4], although there is constitutive expression of one gene [5]. The CYP4A [6], CYP4B [7,8] and CYP4F [9,10] proteins are known to have fatty acid hydroxylase activity, and there is extensive speculation that the formation of hydroxylated fatty acids by cytochrome P450 leads to the production of physiologically active metabolites that regulate physiological function [11][12][13][14][15][16].Cytochrome P450 metabolism of fatty acids may also be of fundamental importance in brain [17][18][19], and it is known that neurotransmitters and fatty acids can be actively metabolized by cytochrome P450 in brain [18,20,21]. Although the specific content of cytochrome P450 in brain is relatively low compared with liver [17,22,23], this level of cytochrome P450 can be induced by various agents [24]. A peculiar feature of brain P450 is that it is difficult to account for the total P450 content with previously characterized P450 proteins [17].We describe the cloning of human and mouse cDNAs for the CYP4x1 P450, a molecular model of the protein, and tissue-specific localization of the RNA in mouse and human. The Cyp4x1 protein was localized by immunohistochemistry and shown to be a major P450 protein in mouse brain. A novel cytochrome P450, CYP4x1, was identified in EST databases on the basis of similarity to a conserved region in the C-helix of the CYP4A family. The human and mouse CYP4x1 cDNAs were cloned and found to encode putative cytochrome P450 proteins. Molecular modelling of CYP4x1 predicted an unusual substrate binding channel for the CYP4 family. Expression of human CYP4x1 was detected in brain by EST analysis, and in aorta by northern blotting. The mouse cDNA was used to demonstrate that the Cyp4x RNA was expressed principally in brain, and at much lower levels in liver; hepatic levels of the Cyp4x1 RNA were not affected by treatment with the inducing agents phenobarbital, dioxin, dexamethasone or ciprofibrate, nor were the levels affected in PPARa-⁄ -mice. A specific antibody for Cyp4x1 was developed, and shown to detect Cyp4x1 in brain; quantitation of the Cyp4x1 protein in brain demonstrated 10 ng of Cyp4x1 proteinAEmg )1 microsomal protein, showing that Cyp4x1 is a major brain P450. Immunohistochemical localization of the Cyp4x1 protein in brain showed specific staining of neurons, choroids epithelial cells and vascular endothelial cells. These data suggest an important role for Cyp4x1 in the brain.Abbreviations DAB, 3,3¢-diaminobenzidine; TCDD, 2,3,7,8 tetrachlorodibenzo-p-dioxin.
The guinea pig does not undergo peroxisome proliferation in response to peroxisome proliferators, in contrast with other rodents. To understand the molecular basis of this phenotype, the peroxisome proliferator activated receptor alpha (PPARalpha) from guinea-pig liver was cloned; it encodes a protein of 467 amino acid residues that is similar to rodent and human PPARalpha. The guinea-pig PPARalpha showed a high substitution rate: maximum likelihood analysis was consistent with rodent monophyly, but could not exclude rodent polyphyly (P approximately 0.06). The guinea-pig PPARalpha cDNA was expressed in 293 cells and mediated the induction of the luciferase reporter gene by the peroxisome proliferator, Wy-14,643, dependent on the presence of a peroxisome proliferator response element. Moreover the PPARalpha RNA and protein were expressed in guinea-pig liver, although at lower levels than in a species which is responsive to peroxisome proliferators, the mouse. To determine whether the guinea-pig PPARalpha mediated any physiological effects, guinea pigs were exposed to two selective PPARalpha agonists, Wy-14, 643 and methylclofenapate; both compounds induced hypolipidaemia. Thus the guinea pig is a useful model for human responses to peroxisome proliferators.
Highly selective CYP1B1 inhibitors have potential in the treatment of hormone-induced breast and prostate cancers. Mimicry of potent and selective CYP1B1 inhibitors, α-naphthoflavone and stilbenes, revealed that two sets of hydrophobic clusters suitably linked via a polar linker could be implanted into a new scaffold 'biphenyl ureas' to create potentially a new class of CYP1B1 inhibitors. A series of sixteen biphenyl ureas were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™, yeast-derived recombinant microsomal enzymes. The most active human CYP1B1 inhibitors were further studied for their selectivity against human CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. The meta-chloro-substituted biphenyl urea 5h was the most potent inhibitor of CYP1B1 with IC value of 5 nM. It displayed excellent selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 (IC >10 μM in the four CYP assays, indicating >2000-fold selectivity). Similarly, two methoxy-substituted biphenyl ureas 5d and 5e also displayed potent and selective inhibition of CYP1B1 with IC values of 69 and 58 nM, respectively, showing >62 and >98-fold selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. In order to probe if the relatively insoluble biphenyl ureas were cell permeable and if they could at all be used for future cellular studies, their CYP1B1 inhibition was investigated in live recombinant human and yeast cells. Compound 5d displayed the most potent inhibition with ICs of 20 nM and 235 nM, respectively, in the two cell-based assays. The most potent and selective CYP1B1 inhibitor (compound 5h) from Sacchrosomes, also displayed potent inhibition in live cell assays. Molecular modeling was performed to understand the trends in potency and selectivity observed in the panel of five CYP isoenzymes used for the in vitro studies.
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