Analysis of CYP2D6 substrate interactions by computational methods.

Cytochrome P450 CYP2D6 is involved in the oxidation of well over 150 drugs and, in general, those which contain a basic nitrogen atom in the molecule. To clarify how the residues of CYP2D6 are utilized for orientating a wide range of its specific substrates and distinguishing them from a variety of other organic compounds, docking studies by AutoDock and molecular dynamics (MD) simulations were conducted. Specific ligands were docked to both the homology model and crystal structures optimally to estimate the site of reaction on the ligand molecule and the binding energy for the complex, which were generally in good agreement with the experimental data. MD simulation for the CYP2D6-propranolol complex was then carried out to reveal the amino acid residues interacting with the substrate at the active site. Phe-120, Glu-216, Asp-301, and Phe-483 are identified as the substrate-binding residues in agreement with previously reported site-directed mutagenesis data and the crystal structure reported recently (PDB code: 2F9Q). As well as these residues, our theoretical prediction suggests that Phe-219 and Glu-222 are also important residues for mediating oxidation of substrates, especially propranolol.

Investigating human P450s involved in drug metabolism via homology with high-resolution P450 crystal structures of the CYP2C subfamily.

The important role of high-resolution crystal structures of cytochrome P450 (CYP) enzymes for the generation of P450 models by homology is discussed. The main focus is on human P450 enzymes involved in drug metabolism, where the role of homology modelling has been emphasized in the recent literature. Report of the first human P450 crystal structure has provided an opportunity for comparison between those modelled from other crystallographic templates, and the recent substrate-bound rabbit CYP2C5 structure exemplifies the relevance of high-resolution template structures to generating 3-D models of P450s where the homology is relatively high. In particular, the homology models of human CYP1 and CYP2 family enzymes are presented, where good agreement with experiment findings are apparent.

Structural modelling of the human drug-metabolizing cytochromes P450.

The structural and functional aspects of cytochrome P450 (CYP) enzymes are reviewed in the light of current developments in X-ray crystallography and other physical evidence, together with recent findings on the regulation of, and polymorphisms in, the human drug-metabolizing CYPs. It is emphasized that the crystal structures of eukaryotic CYPs are particuarly useful for constructing homology models of the human enzymes associated with drug metabolism, and that these models can aid in the high-throughput screening of novel compounds destined for human exposure.

Are food and environmental toxicants 'overdetected' by bioassay?

The definition of clean bioprocessing of foods should relate to the discharge of clean effluents that do not disturb functional ecosystems in the environment. Clean effluents should not pollute aquatic or terrestrial environments by increasing the levels or determined bioavailibility of reactive oxygen species (ROS), traces of heavy metals (e.g. arsenic, mercury, lead, cadmium) or radionuclides or other ecotoxicants such as the endocrine disrupting chemicals (e.g. xenoestrogens), herbicides and pesticides. Some saleable foodstuffs can contain very small amounts of potentially toxic components. Strategies dealing with potential toxins should be aimed at targeting remedial bioprocessing to safe limits as stipulated by regulatory agencies, rather than trying to eliminate all toxic components of food so that they can no longer be detected by bioassay or other highly sensitive techniques. The ability to detect even the tiniest amounts of toxicants may not be necessary. This 'overdetection' could lead to inappropriate courses of action in some situations.

Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP.

Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

Investigation of enzyme selectivity in the human CYP2C subfamily: homology modelling of CYP2C8, CYP2C9 and CYP2C19 from the CYP2C5 crystallographic template.

Homology modelling of human CYP2C subfamily enzymes, CYP2C8, CYP2C9 and CYP2C19, based on the rabbit CYP2C5 crystal structure template is reported. The relatively high sequence homologies (75-80%) between the rabbit CYP2C5 and human CYP2C subfamily enzymes tend to indicate that the resulting structures should prove adequate models of these major catalysts of human drug metabolism. Selective substrates of all three human CYP2C enzymes are found to fit closely within the putative active sites in a manner which is consistent with site-directed mutagenesis experiments and known positions of substrate metabolism. The specific interactions between substrates and enzymes can be used to rationalize the variation in substrate binding affinity and generate QSAR models for both inhibition and metabolism via CYP2C family enzymes, yielding a generally good agreement with experimental binding data obtained from Km values, with correlation coefficients (R values) of between 0.97 and 0.99 depending on the QSAR equation produced.

A molecular model of CYP2D6 constructed by homology with the CYP2C5 crystallographic template: investigation of enzyme-substrate interactions.

The results of homology modelling of CYP2D6 based on the mammalian P450 crystal structure of rabbit CYP2C5 are reported. It is found that many CYP2D6-selective substrates are able to fit closely within the putative active site of the enzyme where there are favourable contacts with complementary amino acid residues, including aspartate-301 which has been probed via site-directed mutagenesis. The homology model of CYP2D6 is consistent with available experimental evidence from selective substrate metabolism and site-specific mutation data. Quantitative structure-activity relationships (QSARs) with substrate binding affinity based on KD values and inhibition data (Ki values) demonstrate the importance of hydrogen bonding, pi-pi stacking and relative molecular mass in describing variations in avidity towards the CYP2D6 enzyme, although the compound lipophilicity (log D(7.4)) appears to be the most important single descriptor for CYP2D6 inhibition. Calculation of substrate binding affinity based on contributions from active site interactions and lipophilic character gives satisfactory agreement with experimentally determined KD values.

Molecular modelling of CYP2B6 based on homology with the CYP2C5 crystal structure: analysis of enzyme-substrate interactions.

The results of homology modelling of CYP2B6 based on the CYP2C5 crystal structure is described in terms of substrates and inhibitors binding within the putative active site. In general these results are in agreement with currently available evidence from substrate metabolism, mode of inhibitor action and site-directed mutagenesis experiments within the CYP2B subfamily of enzymes. Consequently, the model based on the CYP2C5 template represents an advance on those models produced from bacterial P450s, such as CYP101 and CYP102. Quantitative Structure-Activity Relationships (QSARs) for substrates binding to CYP2B6 indicate a key role for hydrogen bonding, and lipophilic character, as determined by the log P parameter (where P is the octanol/water partition coefficient), is also of importance for explaining the variation in experimental binding affinity for CYP2B6 substrates. It is possible to estimate the binding energies for typical CYP2B6 substrates based on their properties and interactions with the enzyme, which show good concordance with experimental data in the form of apparent Km values.

An evaluation of ondansetron binding interactions with human cytochrome P450 enzymes CYP3A4 and CYP2D6.

The results of an evaluation study of ondansetron binding to human cytochromes P450 CYP3A4 and CYP2D6 is reported. The methodology includes NMR spectroscopic measurements of substrate to heme iron distances together with molecular modelling of the enzyme-substrate interactions. It is shown that there is a generally good agreement between the experimental and calculated binding affinities for ondansetron towards CYP2D6 and CYP3A4 enzymes, based on interactive docking studies. Moreover, the modelled binding orientations for ondansetron in CYP2D6 and CYP3A4 are largely consistent with the NMR data and with the known routes for P450-mediated metabolism of this compound.

Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitro.

The focus on the refinement, reduction and replacement of animal use in toxicity testing requires the development of cell-based systems that mimic the effects of xenobiotics in human tissues. The human adrenocortical carcinoma cell line, H295R, has been proposed as a model for studies on adrenal steroidogenesis and its disruption. In this study, expression profiles for nine adrenal steroidogenic genes were characterized in H295R cells using real-time RT-PCR. Treatment with forskolin increased cortisol secretion and stimulated transcription of all the steroidogenic genes except SULT2A1. The transcript profile from H295R cells in the presence and absence of forskolin was compared with the transcript profile from human adrenal glands. The gene expression pattern observed in the forskolin-treated H295R cells was more similar to that in the human adrenal gland, than the expression pattern in untreated cells. To examine H295R cells as a possible in vitro system for the assessment of adrenal disruption using molecular endpoints, the insecticide lindane (gamma-hexachlorocyclohexane) was used. In vivo, lindane has been shown to inhibit testicular, ovarian and adrenal steroidogenesis. It was demonstrated that lindane reduced cortisol secretion, downregulated the expression of a subset of the genes encoding steroidogenic enzymes and repressed transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene promoter. Thus the H295R cell line provides a good in vitro system for the analysis of the human adrenal steroidogenic pathway at the level of hormone production and gene expression. This in vitro test can be used for the rapid detection of adrenal endocrine disruption and as a tool for mechanistic studies.

Comparative studies on the cytochrome p450-associated metabolism and interaction potential of selegiline between human liver-derived in vitro systems.

Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome p450 (p450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed p450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of p450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.