Structure-activity relationship for human cytochrome P450 substrates and inhibitors

Criteria governing the avidity of substrate binding to human hepatic cytochromes P450 (CYP) associated with Phase 1 metabolism of drugs are described. The results of extensive quantitative structure-activity relationship (QSAR) analyses are reported for substrates of human P450s: CYPIA2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, representing the enzymes exhibiting major involvement in the metabolism of drug substrates in Homo sapiens. In particular, it is shown that hydrogen bond properties in each class of enzyme-substrate complex are especially important factors in determining substrate binding affinity towards those human P450s which are involved in drug metabolism.     

细胞色素P450 CYP2E1酶构型特征及其表达调控机制的研究进展

细胞色素P450CYP2E1酶参与代谢活化及失活多种前毒物、前致癌物和少数药物。在细胞色素P450超家族中,CYP2E1具有易介导自由基生成引发氧化应激反应的特征。CYP2E1表达水平可能是机体对环境和工业毒物或致癌物敏感程度的重要因素。研究表明,CYP2E1可被多种内、外源性物质所调控,并且CYP2E1的药理和毒理学功能与其以蛋白构型为基础的代谢行为密切相关。本文综述了CYP2E1基因多态性、酶构型特征与其代谢活性间的关系,并分析了其区别于其他细胞色素P450亚型的表达调控机制。     

Negatively Cooperative Binding Properties of Human Cytochrome P450 2E1 with Monocyclic Substrates

Human CYP2E1 accounts for almost 2% of total CYP enzymes in the liver cells, and plays a crucial role in the metabolism of small molecular weight compounds. This enzyme is associated with the nearly 6% metabolisms of the currently clinical drugs. However, it is found that CYP2E1 has a non-hyperbolic kinetic profile that can not be explained by the common Michaelis-Menten mechanism. Further studies show that the non-hyperbolic kinetic behaviors are associated with multiple substrate binding, which is also known as the cooperative binding properties. However, the detailed mechanism for the cooperative binding is not clear by now. In this paper, we summarized the experimental and theoretical studies on the cooperative binding mechanism. Based on the structural analysis, a second substrate binding site is confirmed in human CYP2E1, which is located neither in the region near Leu103, Leu210 and Phe478, nor far from the active site. Additionally, two important residues Thr303 and Phe478 are also identified to be the key factors in the cooperative binding on the short-range and long-range effects, respectively. The former plays a crucial role in the positioning of substrates and in proton delivery to the active site; the latter is located between the substrate access channel and the active site, and exhibits directly effects on substrate access or on substrate positioning in the active site. All these points can provide useful information for the cooperative binding in human CYP2E1, revealing the detailed mechanism for the non-hyperbolic kinetic behaviors.     

A reaction network model for CYP2E1-mediated metabolism of toxicant mixtures

In this paper, we describe a modeling approach to predict the interlinked pathways and kinetics resulting from CYP2E1-mediated metabolism of both pure species and chemical mixtures. This approach is based on the concept of chemical reaction networks, an idea that has formed the basis for simulation tools that have shown good predictive capabilities in the petroleum industry, but also an idea that has heretofore seen minimal application in the biomedical research arena. Although the initial target for developing this reaction network approach was cytochrome P450 2E1 (CYP2E1) and its over 200 substrates, this technology has been used for other families of CYP enzymes and their substrates in our laboratory. Utilizing this approach, we have produced a modular 'predictive metabolomics' simulation framework comprising interdependent software components that perform such tasks as testing of substrate binding feasibility, performing virtual chemistry, formulating reaction-rate equations, computing reaction kinetics and predicting time-dependent species concentrations. As an illustrative example, we outline the application of this framework to the prediction of the reaction networks resulting from the Phase I metabolism of two compounds of important toxicological interest. The potential of this modeling technology is immense in providing a computer simulation platform for complex-chemical mixtures and complex-biological systems. It is possible that this technology will play an important role in formulating a 'Virtual Human'.     

Extrahepatic metabolism by CYP2E1 in PBPK modeling of lipophilic volatile organic chemicals: impacts on metabolic parameter estimation and prediction of dose metrics

Physiologically based pharmacokinetic (PBPK) models are increasingly available for environmental chemicals and applied in risk assessments. Volatile organic compounds (VOCs) are important pollutants in air, soil, and water. CYP2E1 metabolically activates many VOCs in animals and humans. Despite its presence in extrahepatic tissues, the metabolism by CYP2E1 is often described as restricted to the liver in PBPK models, unless target tissue dose metrics in extrahepatic tissues are needed for the model application, including risk assessment. The impact of accounting for extrahepatic metabolism by CYP2E1 on the estimation of metabolic parameters and the prediction of dose metrics was evaluated for three lipophilic VOCs: vinyl chloride, trichloroethylene, and carbon tetrachloride. Metabolic parameters estimated from fitting gas uptake data with and without extrahepatic metabolism were similar. The impact of extrahepatic metabolism on PBPK predictions was evaluated using inhalation exposure scenarios relevant for animal toxicity studies and human risk assessment. Although small, the relative role of extrahepatic metabolism and the differences in the predicted dose metrics were greater at low exposure concentrations. The impact was species dependent and influenced by Km for CYP2E1. The current study indicates that inhalation modeling for several representative VOCs that are CYP2E1 substrates is not affected by the inclusion of extrahepatic metabolism, implying that liver-only metabolism may be a reasonable simplification for PBPK modeling of lipophilic VOCs. The PBPK predictions using this assumption can be applied confidently for risk assessment, but this conclusion should not necessarily be applied to VOCs that are metabolized by other enzymes.     

Insights into the molecular requirements for the anti-obesity activity of a series of CB1 ligands

Two-dimensional and 3D quantitative structure-activity relationships studies were performed on a series of diarylpyridines that acts as cannabinoid receptor ligands by means of hologram quantitative structure-activity relationships and comparative molecular field analysis methods. The quantitative structure-activity relationships models were built using a data set of 52 CB1 ligands that can be used as anti-obesity agents. Significant correlation coefficients (hologram quantitative structure-activity relationships: r2 = 0.91, q2 = 0.78; comparative molecular field analysis: r2 = 0.98, q2 = 0.77) were obtained, indicating the potential of these 2D and 3D models for untested compounds. The models were then used to predict the potency of an external test set, and the predicted (calculated) values are in good agreement with the experimental results. The final quantitative structure-activity relationships models, along with the information obtained from 2D contribution maps and 3D contour maps, obtained in this study are useful tools for the design of novel CB1 ligands with improved anti-obesity potency.     

Homology modelling of human CYP2E1 based on the CYP2C5 crystal structure: investigation of enzyme-substrate and enzyme-inhibitor interactions.

The construction of a homology model of human cytochrome P450 2E1 (CYP2E1) is reported, based on the CYP2C5 crystallographic template. A relatively high degree of primary sequence homology (identity=59%), as expected for proteins of the same CYP family, ensured a straightforward generation of the 3-dimensional model due to relatively few deletions and insertions of amino acid residues with respect to the CYP2C5 crystal structure. Probing the CYP2E1 model with typical substrates of the enzyme showed a good agreement with experimental information in the form of positions of metabolism for substrates, and with site-directed mutagenesis data on certain residues. Furthermore, quantitative relationships between substrate binding affinity and various structural parameters associated with the substrate molecules facilitated the formulation of a procedure for estimating relative binding energy and, consequently, K(m) or K(D) values towards the CYP2E1 enzyme. This method has been based on a consideration of the active site interactions between substrates and key amino acid residues lining the haem pocket, together with compound lipophilicity data from partition coefficients.     

CYP2E1 hydroxylation of aniline involves negative cooperativity

CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n = 0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K_s = 30 μM) followed by a second weaker binding event (K_ss = 1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis–Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.     

Species differences in coumarin metabolism: a molecular modelling evaluation of CYP2A interactions

1. An account of the differences in coumarin metabolism between several mammalian species, including man, is reported. 2. The metabolism of coumarin via 7-hydroxylation in the human (CYP2A6) and mouse (CYP2A5) enzymes is explained in terms of molecular modelling of the active site interactions, whereas the rat orthologue (CYP2A1) exhibits 3,4-epoxidation of coumarin, which is also consistent with the modelled interaction between enzyme and substrate. 3. In addition, quantitative structure-activity relationships (QSARs) for coumarin 7-hydroxylation in wild-type and mutant CYP2A5 show the importance of amino acid residue properties for substrate binding, whereas QSARs for CYP2A6 substrates indicate the importance of hydrogen bonding and lipophilicity for favourable interactions with the enzyme.     

Species differences in coumarin metabolism: a molecular modelling evaluation of CYP2A interactions

1. An account of the differences in coumarin metabolism between several mammalian species, including man, is reported. 2. The metabolism of coumarin via 7-hydroxylation in the human (CYP2A6) and mouse (CYP2A5) enzymes is explained in terms of molecular modelling of the active site interactions, whereas the rat orthologue (CYP2A1) exhibits 3,4-epoxidation of coumarin, which is also consistent with the modelled interaction between enzyme and substrate. 3. In addition, quantitative structure-activity relationships (QSARs) for coumarin 7-hydroxylation in wild-type and mutant CYP2A5 show the importance of amino acid residue properties for substrate binding, whereas QSARs for CYP2A6 substrates indicate the importance of hydrogen bonding and lipophilicity for favourable interactions with the enzyme.     

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L. CYP2C9*1 (wild-type) metabolized each of the three compounds with a distinctive profile reflective of typical hyperbolic (flurbiprofen), biphasic (naproxen), and substrate inhibition (piroxicam) kinetics. CYP2C9*3 metabolism was again hyperbolic for flurbiprofen, of a linear form for naproxen (no saturation noted), and exhibited substrate inhibition with piroxicam. CYP2C9*5-mediated metabolism was hyperbolic for flurbiprofen and piroxicam but linear with respect to naproxen turnover. The F114L mutant exhibited a hyperbolic kinetic profile for flurbiprofen metabolism, a linear profile for naproxen metabolism, and a substrate inhibition kinetic profile for piroxicam metabolism. In all cases except F114L-mediated piroxicam metabolism, turnover decreased and the K(m) generally increased for each allelic variant compared with wild-type enzyme. It seems that the kinetic profile of CYP2C9-mediated metabolism is dependent on both substrate and the CYP2C9 allelic variant, thus having potential ramifications on drug disposition predictions made during the development process.     

Molecular modeling of human cytochrome P450-substrate interactions

The results of homology modeling of 10 human cytochrome P450 (CYP) enzymes involved in the Phase 1 metabolism of drugs and other foreign compounds are reported. The models have been constructed from the CYP102 hemoprotein domain template for which the substrate-bound crystallographic coordinates are available. Selective substrates of individual human P450s: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11 are all shown to fit within the corresponding enzymes' active sites in such a manner that is consistent with reported experimental data for both known pathways of substrate metabolism and from the results of site-directed mutagenesis, either in those particular human P450 enzymes concerned or for ones within the same subfamily. The self-consistency of these homology models indicates that they may have potential utility for the pre-screening of novel drug structures.     

Human cytochromes P450 associated with the phase 1 metabolism of drugs and other xenobiotics: a compilation of substrates and inhibitors of the CYP1, CYP2 and CYP3 families

This review represents a compilation of typical substrates and inhibitors for human cytochrome P450 (CYP) enzymes that are involved in drug metabolism, specifically those from the CYP1, CYP2 and CYP3 families. Relatively recent literature on substrates and inhibitors has been collected and the relevant K(m) and K(i) values, respectively, are tabulated. Furthermore, physicochemical properties in the form of lipophilicity (log P and log D(7.4) values) and acidity/basicity (pK(a) values) are also tabulated for a significant number of substrates, together with some information on inhibitors, although only key inhibitors have been selected as the main focus is on substrates. The collated information indicates that there are certain commonalities between substrates for the same enzyme, especially with respect to their positions of metabolism and likely interactions with the relevant enzyme active site regions. The compilation therefore assists in establishing substrate structure-activity relationships (SSARs) within human drug-metabolizing P450s.     

Quantitative structure-activity relationships (QSARs) within cytochromes P450 2B (CYP2B) subfamily enzymes: the importance of lipophilicity for binding and metabolism

The results of qualitative structure-activity relationship (QSAR) analysis are reported for several series of compounds which act as substrates for mammalian CYP2B subfamily enzymes, together with a homologous series of aliphatic primary amines which are known to inhibit CYP2B enzymes. It is found that the compound lipophilicity in the form of the log P value (where P is the octanol/water partition coefficient) is related, either linearly or quadratically, to equilibrium constants of inhibition (Ki), binding (Ks) or metabolism (Km) depending on the series of compounds in question. In some instances, the difference between frontier orbital energy levels (deltaE) also features in several of the log P expressions with biological activity. Also present in a small number of correlations are parameters which are likely to be related to logP: namely, Rm, which is the partitioning factor derived from thin layer chromatography (TLC) retention times, and also the compound molecular weight (Mr). All of these three parameters ((log P, Rm and Mr) are thought to be related to the compound's ability to desolvate the P450 active site when they bind to the enzyme. Although the linear relationships between lipophilicity and CYP2B-related activity point to a major role for desolvation of the enzyme binding site in the overall interaction, it is noted that there may be an optimal log P value displayed by preferred substrates as shown by parabolic relationships with this lipophilic parameter. In addition, there is a remarkable similarity in the coefficients for the log P term of any QSAR expression, which suggests that the hydrophobicity of CYP2B active sites may be broadly equivalent between the various mammalian species.     

Molecular modeling-guided site-directed mutagenesis of cytochrome P450 2D6

Cytochrome P450 (CYP) 2D6 is one of the most important drug metabolizing enzymes and the rationalization and prediction of potential CYP2D6 substrates is therefore advantageous in the discovery and development of new drugs. Experimentally, the active site of CYP2D6 can be probed by site directed mutagenesis studies. Such studies can be designed from structural models of enzyme-substrate complexes. Modeling approaches can subsequently be used to rationalize the observed effect of mutations on metabolism and inhibition. The current paper will present the construction, refinement and validation of the CYP2D6 homology model used in our laboratory for the prediction and rationalisation of CYP2D6 substrate metabolism and CYP2D6-ligand interactions. The model could explain reported site-directed mutagenesis data (for example, mutation of E216 and D301). Furthermore, based on the model, new CYP2D6 mutants were constructed and studied in our lab, and also for these mutants a rationalization of experimentally observed characteristics could be achieved (I106E, F120A, T309V, F483A). CYP2D6-substrate interaction fingerprint analysis of docked substrates in our homology model suggests that several other active site residues are probably interacting with ligands as well, opening the way for further mutagenesis studies. Our homology model was found to agree with most of the details of the recently solved substrate-free CYP2D6 crystal structure [Rowland et al. J. Biol. Chem. 2006, 281, 7614-7622]. Structural differences between the homology model and crystal structure were the same differences observed between substrate-free and substrate-bound structures of other CYPs, suggesting that these conformational changes are required upon substrate binding. The CYP2D6 crystal structure further validates our homology modeling approach and shows that computational chemistry is a useful and valuable tool to provide models for substrate-bound complexes of CYPs which give insight into CYP-ligand interactions. This information is essential for successful pre-experimental virtual screening, as well as accurate hypothesis generation for in vitro studies in drug discovery and development.     

In vitro assessment of the allelic variants of cytochrome P450

The cytochrome P450 (CYP) superfamily is one of the most important groups of enzymes involved in drug metabolism. It is responsible for the metabolism of a large number of drugs. Many CYP isoforms are expressed polymorphically, and catalytic alterations of allelic variant proteins can affect the metabolic activities of many drugs. The CYP2D6, CYP2C9, CYP2C19, and CYP2B6 genes are particularly polymorphic, whereas CYP1A1, CYP1A2, CYP2E1, and CYP3A4 are relatively well conserved without common functional polymorphisms. In vitro studies using cDNA expression systems are useful tools for evaluating functional alterations of the allelic variants of CYP, particularly for low-frequency alleles. Recombinant CYPs have been successfully expressed in bacteria, yeast, baculoviruses, and several mammalian cells. Determination of CYP variant-mediated kinetic parameters (Km and Vmax) in vitro can be useful for predicting drug dosing and clearance in humans. This review focuses on the advantages and disadvantages of the various cDNA-expression systems used to determine the kinetic parameters for CYP allelic variants, the methods for determining the kinetic parameters, and the findings of in vitro studies on highly polymorphic CYPs, including CYP2D6, CYP2C9, CYP2C19, and CYP2B6.     

Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform

1. The construction of a three-dimensional model of human CYP2E1 is reported. It is based on homology with the haemoprotein domain of the unusual bacterial P450, CYP102, which is of known crystal structure. 2. Interactive docking of a number of human CYP2E1 substrates is consistent with their known positions of CYP2E1-mediated metabolism, where specific interactions with key active site amino acid side-chains appear to rationalize the binding and orientation of substrate molecules. 3. Amino acid residues within the putative active site of human CYP2E1, including those associated with the binding of substrates and inhibitors, are shown to correspond with those identified by site-directed mutagenesis experiments conducted on CYP2 family isoforms, and they are known to affect substrate metabolism regioselectivity. 4. Consequently, it was found that the CYP2E1 active site exhibits complementarity with the structural characteristics of known substrates and inhibitors of this enzyme, including their relatively low molecular weights and disposition of hydrogen bond-forming groups.     

Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform

1. The construction of a three-dimensional model of human CYP2E1 is reported. It is based on homology with the haemoprotein domain of the unusual bacterial P450, CYP102, which is of known crystal structure. 2. Interactive docking of a number of human CYP2E1 substrates is consistent with their known positions of CYP2E1-mediated metabolism, where specific interactions with key active site amino acid side-chains appear to rationalize the binding and orientation of substrate molecules. 3. Amino acid residues within the putative active site of human CYP2E1, including those associated with the binding of substrates and inhibitors, are shown to correspond with those identified by site-directed mutagenesis experiments conducted on CYP2 family isoforms, and they are known to affect substrate metabolism regioselectivity. 4. Consequently, it was found that the CYP2E1 active site exhibits complementarity with the structural characteristics of known substrates and inhibitors of this enzyme, including their relatively low molecular weights and disposition of hydrogen bond-forming groups.