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Novel potential inhibitors of the postsqualene portion of cholesterol synthesis were screened in HepG2 cells. 2-(4-Phenethylpiperazin-1-yl)-1-(pyridine-3-yl)ethanol (LK-980) was identified as a prospective compound and was characterized further in cultures of human primary hepatocytes from seven donors. In vitro kinetic measurements show that the half-life of LK-980 is at least 4.3 h. LK-980 does not induce CYP3A4 mRNA nor enzyme activity. Target prediction was performed by gas chromatography-mass spectrometry, allowing simultaneous separation and quantification of nine late cholesterol intermediates. Experiments indicated that human sterol Δ(7)-reductase (DHCR7) is the major target of LK-980 (34-fold increase of 7-dehydrocholesterol), whereas human sterol Δ(14)-reductase (DHCR14), human sterol Δ(24)-reductase (DHCR24), and human sterol C5-desaturase (SC5DL) represent minor targets. In the absence of purified enzymes, we used the mathematical model of cholesterol synthesis to evaluate whether indeed more than a single enzyme is inhibited. In silico inhibition of only DHCR7 modifies the flux of cholesterol intermediates, resulting in a sterol profile that does not support experimental data. Partial inhibition of the DHCR14, DHCR24, and SC5DL steps, in addition to DHCR7, supports the experimental sterol profile. In conclusion, we provide experimental and computational evidence that LK-980, a novel inhibitor from the late portion of cholesterol synthesis, inhibits primarily DHCR7 and to a lesser extent three other enzymes from this pathway.

The widely prescribed lipid-lowering statins are considered to be relatively safe drugs. However, the risk of severe myopathy and drug interactions as a consequence of statin therapy provides a challenge for development of novel cholesterol-lowering agents, targeting enzymes other than HMG-CoA reductase. The novel pyridylethanol-(phenylethyl)amine derivative, (2-((3,4-dichlorophenethyl)(propyl)-amino)-1-(pyridin-3-yl)ethanol (LK-935), blocking lanosterol 14alpha-demethylase, was demonstrated to efficiently reduce cholesterol biosynthesis. The drug interaction potential of LK-935 was investigated and compared with that of atorvastatin and rosuvastatin in primary human hepatocytes. Clear evidence was provided for the induction of CYP3A4 by LK-935. LK-935 was proved to be a potent human pregnane X receptor (hPXR) activator as a prerequisite for the transcriptional activation of CYP3A4 gene; however, the rapid metabolism of LK-935 in primary hepatocytes prevented maximal CYP3A4 induction. Therefore, the induction of CYP3A4 by LK-935 may be prone to mild or negligible drug interactions. However, because CYP3A4 and also CYP2C9 play a significant role in LK-935 metabolism, the inhibition of these cytochromes P450 by coadministered drugs may lead to some increase in the LK-935 concentration required for the potent induction of CYP3A4. Rosuvastatin was found to increase human constitutive androstane receptor (hCAR)-mediated transcription of CYP3A4, CYP2C9, and CYP2B6 genes, predicting the consequent potential for drug interactions with several coadministered drugs. Activation of hCAR and hPXR by atorvastatin and the subsequent induction of not only CYP2B6 and CYP3A4 but also of CYP2C9 present an additional target by which atorvastatin, a widely used cholesterol-lowering drug, can modify the kinetics of numerous drugs.

Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine- and tobacco-specific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a -95 to +12 region containing an hepatic nuclear factor 4 (HNF4) alpha response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4alpha- and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4alpha increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4alpha mRNA levels by 4-fold; however, the amount of HNF4alpha nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4alpha to the HNF4-RE and that an interaction of GR and HNF4alpha occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4alpha to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4alpha with an HNF4-RE rather than a glucocorticoid response element.

The xenobiotic-mediated induction of three major human liver cytochrome P450 genes, CYP2B6, CYP2C9, and CYP3A4, is known to be regulated by the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). CAR and PXR are regulated, at least in part, by the glucocorticoid receptor (GR) and the hypothesis of a signal transduction cascade GR-[CAR/PXR]-P450 has been proposed. This study was aimed at testing this hypothesis in primary human hepatocytes by using the tubulin network disrupting agent colchicine. Colchicine (COL) decreased both basal and rifampicin- and phenobarbital-inducible expression of CYP2B6, CYP2C8/9, and CYP3A4. A parallel down-regulation of mRNA expression of CAR, PXR, and tyrosine aminotransferase, a prototypic gene directly regulated by GR, was observed. COL affected neither the level of GR mRNA nor ligand binding to GR. To evaluate the effect of colchicine on GR-mediated gene transactivation, HeLa cells stably or transiently transfected with a GR-responsive element-dependent luciferase reporter gene were used. COL decreased the dexamethasone-induced luciferase expression in stably transfected cell line by 50%, whereas GR transactivation in transiently transfected cells was not affected by COL. In contrast, ligand-dependent GR translocation in the human embryonic kidney 293 cell line transiently transfected with GFP-GR was inhibited by COL. We conclude that alteration of the signal transduction mediated through the GR-[CAR/PXR]-P450 cascade by colchicine is responsible for the down-regulation of CYP2C9 and CYP3A4, implicating cytoskeleton as necessary for correct functioning of this cascade under physiological conditions.