Variability of drug metabolism, especially that of the most important phase I enzymes or cytochrome P450 (CYP) enzymes, is an important complicating factor in many areas of pharmacology and toxicology, in drug development, preclinical toxicity studies, clinical trials, drug therapy, environmental exposures and risk assessment. These frequently enormous consequences in mind, predictive and pre-emptying measures have been a top priority in both pharmacology and toxicology. This means the development of predictive in vitro approaches. The sound prediction is always based on the firm background of basic research on the phenomena of inhibition and induction and their underlying mechanisms; consequently the description of these aspects is the purpose of this review. We cover both inhibition and induction of CYP enzymes, always keeping in mind the basic mechanisms on which to build predictive and preventive in vitro approaches. Just because validation is an essential part of any in vitro–in vivo extrapolation scenario, we cover also necessary in vivo research and findings in order to provide a proper view to justify in vitro approaches and observations.

Inhibition and induction of human cytochrome P450 enzymes: current status.

Induction of drug metabolism was described more than 40 years ago. Progress in understanding the molecular mechanism of induction of drug-metabolizing enzymes was made recently when the important roles of the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of transcription factors, were discovered to act as sensors for lipophilic xenobiotics, including drugs. CAR and PXR bind as heterodimeric complexes with the retinoid X receptor to response elements in the regulatory regions of the induced genes. PXR is directly activated by xenobiotic ligands, whereas CAR is involved in a more complex and less well understood mechanism of signal transduction triggered by drugs. Most recently, analysis of these xenobiotic-sensing nuclear receptors and their nonmammalian precursors such as the chicken xenobiotic receptor suggests an important role of PXR and CAR also in endogenous pathways, such as cholesterol and bile acid biosynthesis and metabolism. In this review, recent findings regarding xenosensors and their target genes are summarized and are put into an evolutionary perspective in regard to how a living organism has derived a system that is able to deal with potentially toxic compounds it has not encountered before.

Induction of Drug Metabolism: The Role of Nuclear Receptors

CAR and PXR: the xenobiotic-sensing receptors.

Nuclear receptors CAR and PXR: Molecular, functional, and biomedical aspects.

Xenobiotics regulate the transcription of many genes in the liver, intestine, kidney and other organs to alter their own metabolism and excretion. The barbiturate phenobarbital (PB)isoneofthebest studied examples of a drug that potently induces cytochrome P450s (CYPs) and other genes in the liver. The mechanisms that control the induction of detoxifying genes and the receptors recognizing those have remained elusive for many years. This changed in 1998 when the pregnane X receptor [PXR, alternatively called pregnane activated receptor (PAR) or steroid and xenobiotic receptor (SXR); NR1I2] and the constitutive androstane receptor (CAR, alternatively called constitutive active receptor; NR1I3) were discovered to play a role in xenobiotic mediated induction of CYPs. It turned out that PXR and CAR could accommodate all the conundrums associated with induction of CYPs by xenobiotics. First, due to an
unusually flexible ligand-binding domain (LBD), PXR and CAR
are promiscuous towards many structurally diverse
compounds. Second, as heterodimers with the retinoid X receptor (RXR; NR2B1, 2 and 3), PXR and CAR are able to bind to different DNA-binding elements and thus directly activate many genes involved in drug detoxification and excretion. Third, the LBDs of PXR and CAR orthologs are unusually divergent between species, and therefore are activated by different chemicals in a very species-specific manner. A number of review articles provide an up-to-date description of the basic functions of PXR and CAR. This chapter is focused on the recent advances in the
characterization of PXR and CAR. In particular, it highlights the novel findings about endogenous functions
and their clinical implications.

Induction of drug metabolism : role for nuclear receptors

The constitutive androstane receptor (CAR) regulates mouse and humanCYP2B genes through binding to the direct repeat-4 (DR4) motifs present in the phenobarbital-responsive enhancer module (PBREM). The preference of PBREM elements for nuclear receptors and the extent of cross-talk between CAR and other nuclear receptors are currently unknown. Our transient transfection and DNA binding experiments indicate that binding to DR4 motifs does not correlate with the activation response and that mouse and human PBREM are efficiently ‘insulated’ from the effects of other nuclear receptors despite their substantial affinity for DR4 motifs. Certain nuclear receptors that do not bind to DR4 motifs, such as peroxisome proliferator-activated receptor-α and farnesoid X receptor, can suppress PBREM function via a coactivator-dependent process that may have relevance in vivo. In competition experiments, mouse PBREM is clearly more selective for CAR than human PBREM. Pregnane X, vitamin D, and thyroid hormone receptors can potentially compete with human CAR on human PBREM. In contrast to the selective nature of PBREM, CYP3A enhancers are highly and comparably responsive to CAR, pregnane X receptor, and vitamin D receptor. In addition, the ligand specificities of human and mouse CAR were defined by mammalian cotransfection and yeast two-hybrid techniques. Our results provide new mechanistic explanations to several previously unresolved aspects of CYP2B andCYP3A gene regulation.

Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors.

Amino acids important for ligand specificity of the human constitutive androstane receptor.

mCAR (mouse constitutive androstane receptor; NR1I3) controls the expression of cytochrome P450 as well as other enzymes involved in drug and steroid metabolism. The high basal activity of mCAR can be modulated by inhibitory steroids related to androstenol and by activating xenobiotic chemicals such as 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene and chlorpromazine. The ability of oestrogens and some other xenobiotics to activate mCAR is not clear. In the present study, co-transfection assays in HEK-293 cells indicated that oestrogens varied in their efficacy to activate mCAR, depending on variation at the steroid D-ring and position of hydroxy groups. In general, oestrogens were weaker activators of mCAR than 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene and chlorpromazine. Also, the induction of CYP2B10 mRNA by oestrogens was less pronounced in mouse primary hepatocytes. Yeast two-hybrid assays indicated that, unlike androstenol and the established activators, oestrogens attracted both nuclear receptor co-repressors and co-activators to the mCAR ligand-binding domain, thus limiting the extent of mCAR activation. This novel dual action is not limited to oestrogens, but is shared by some xenobiotic CYP2B inducers such as clotrimazole and methoxychlor. These findings offer an alternative explanation for the recently suggested nuclear activation step of mCAR.

Dual action of oestrogens on the mouse constitutive androstane receptor.

The constitutive androstane receptor (CAR) regulates drug and steroid metabolism through binding to cytochrome P450 2B, 2C, and 3A gene enhancers. Uniquely among nuclear receptors, mouse CAR (mCAR) can be suppressed by androstenol and activated by structurally diverse drugs, pesticides, and environmental pollutants. To gain insight into presently ill-defined structural requirements of mCAR ligands, we employed a mCAR inhibition assay in mammalian HEK293 cells to create a QSAR model that could well predict the inhibition by three unknown steroids. Two novel mCAR inhibitors were thus identified. Yeast two-hybrid assays indicated that steroids inhibit mCAR primarily by promoting association of mCAR with the corepressor NCoR, with only minor contribution from other mechanisms. Analysis of chimeric and mutant mCAR constructs suggested that androstenol sensitivity is controlled by residues between amino acids 201-263 (helices 5-7) and it does not depend on the residue 350 within helix 12, as previously suggested.

Molecular determinants of steroid inhibition for the mouse constitutive androstane receptor.

Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones. 

Janne Mäkinen

Papers

Modulation of mouse and human phenobarbital-responsive enhancer module by nuclear receptors.

Amino acids important for ligand specificity of the human constitutive androstane receptor.

Dual action of oestrogens on the mouse constitutive androstane receptor.

Molecular determinants of steroid inhibition for the mouse constitutive androstane receptor.

Fluorogenic RNA aptamers to probe transcription initiation and co-transcriptional RNA folding by multi-subunit RNA polymerases.