A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4

Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy.

[1]  David E. Gloriam,et al.  SMARTCyp: A 2D Method for Prediction of Cytochrome P450-Mediated Drug Metabolism. , 2010, ACS medicinal chemistry letters.

[2]  F. Guengerich,et al.  Rate-limiting steps in oxidations catalyzed by rabbit cytochrome P450 1A2. , 2004, Biochemistry.

[3]  David Brown,et al.  Unfinished business: target-based drug discovery. , 2007, Drug discovery today.

[4]  Tatiana Nikolskaya,et al.  Modeling of human cytochrome p450-mediated drug metabolism using unsupervised machine learning approach. , 2003, Journal of medicinal chemistry.

[5]  Chris de Graaf,et al.  Cytochrome p450 in silico: an integrative modeling approach. , 2005, Journal of medicinal chemistry.

[6]  Andreas Bender,et al.  Computational Prediction of Metabolism: Sites, Products, SAR, P450 Enzyme Dynamics, and Mechanisms , 2012, J. Chem. Inf. Model..

[7]  Guangbo Ge,et al.  A highly selective probe for human cytochrome P450 3A4: isoform selectivity, kinetic characterization and its applications. , 2013, Chemical communications.

[8]  Slobodan Petar Rendic,et al.  Metabolism of anabolic steroids by recombinant human cytochrome P450 enzymes. Gas chromatographic-mass spectrometric determination of metabolites. , 1999, Journal of chromatography. B, Biomedical sciences and applications.

[9]  Dennis A. Smith,et al.  Properties of cytochrome P450 isoenzymes and their substrates Part 2: properties of cytochrome P450 substrates , 1997 .

[10]  Sason Shaik,et al.  What factors affect the regioselectivity of oxidation by cytochrome p450? A DFT study of allylic hydroxylation and double bond epoxidation in a model reaction. , 2002, Journal of the American Chemical Society.

[11]  Vladimir Poroikov,et al.  A New Statistical Approach to Predicting Aromatic Hydroxylation Sites. Comparison with Model-Based Approaches , 2004, J. Chem. Inf. Model..

[12]  Doo Nam Kim,et al.  Regioselectivity Prediction of CYP1A2-Mediated Phase I Metabolism , 2008, J. Chem. Inf. Model..

[13]  Keli Han,et al.  Structural determinants of steroids for cytochrome P450 3A4-mediated metabolism , 2004 .

[14]  Olivier Taboureau,et al.  Virtual Screening and Prediction of Site of Metabolism for Cytochrome P450 1A2 Ligands , 2009, J. Chem. Inf. Model..

[15]  Guangbo Ge,et al.  Characterization of Phase I Metabolism of Resibufogenin and Evaluation of the Metabolic Effects on Its Antitumor Activity and Toxicity , 2015, Drug Metabolism and Disposition.

[16]  Guangbo Ge,et al.  Comparative Metabolism of Cinobufagin in Liver Microsomes from Mouse, Rat, Dog, Minipig, Monkey, and Human , 2011, Drug Metabolism and Disposition.

[17]  B. Potter,et al.  Journal of Steroid Biochemistry and Molecular Biology the Structural Biology of Oestrogen Metabolism , 2022 .

[18]  I. Blair.  Analysis of estrogens in serum and plasma from postmenopausal women: Past present, and future , 2010, Steroids.

[19]  D. F. V. Lewis,et al.  Molecular modelling of the human glucocorticoid receptor (hGR) ligand-binding domain (LBD) by homology with the human estrogen receptor α (hERα) LBD: quantitative structure–activity relationships within a series of CYP3A4 inducers where induction is mediated via hGR involvement , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[20]  Gilles Klopman,et al.  META. 1. A Program for the Evaluation of Metabolic Transformation of Chemicals , 1994, J. Chem. Inf. Comput. Sci..

[21]  F. Guengerich,et al.  Evidence for specific base catalysis in N-dealkylation reactions catalyzed by cytochrome P450 and chloroperoxidase. Differences in rates of deprotonation of aminium radicals as an explanation for high kinetic hydrogen isotope effects observed with peroxidases. , 1993, The Journal of biological chemistry.

[22]  D. Lewis,et al.  On the recognition of mammalian microsomal cytochrome P450 substrates and their characteristics: towards the prediction of human p450 substrate specificity and metabolism. , 2000, Biochemical pharmacology.

[23]  A. Hofman,et al.  Cytochrome P450 3A gene variation, steroid hormone serum levels and prostate cancer––The Rotterdam Study , 2010, Steroids.

[24]  Barry C. Jones,et al.  Properties of cytochrome P450 isoenzymes and their substrates Part 1: active site characteristics , 1997 .

[25]  H. Yamazaki,et al.  Progesterone and testosterone hydroxylation by cytochromes P450 2C19, 2C9, and 3A4 in human liver microsomes. , 1997, Archives of biochemistry and biophysics.

[26]  Slobodan Petar Rendic.  Summary of information on human CYP enzymes: human P450 metabolism data , 2002, Drug metabolism reviews.

[27]  Arnout Ceulemans,et al.  Structure-based site of metabolism prediction for cytochrome P450 2D6. , 2011, Journal of medicinal chemistry.

[28]  Ronald N. Hines,et al.  Developmental Expression of the Major Human Hepatic CYP3A Enzymes , 2003, Journal of Pharmacology and Experimental Therapeutics.

[29]  Jose Cosme,et al.  Crystal Structures of Human Cytochrome P450 3A4 Bound to Metyrapone and Progesterone , 2004, Science.

[30]  T. Poulos,et al.  Interaction of human cytochrome P4503A4 with ritonavir analogs. , 2012, Archives of biochemistry and biophysics.

[31]  R. Sheridan,et al.  Empirical regioselectivity models for human cytochromes P450 3A4, 2D6, and 2C9. , 2007, Journal of medicinal chemistry.

[32]  Jin Liu,et al.  2D SMARTCyp Reactivity-Based Site of Metabolism Prediction for Major Drug-Metabolizing Cytochrome P450 Enzymes , 2012, J. Chem. Inf. Model..

[33]  R. Sheridan,et al.  A model for predicting likely sites of CYP3A4-mediated metabolism on drug-like molecules. , 2003, Journal of medicinal chemistry.

[34]  D A Smith,et al.  Putative active site template model for cytochrome P4502C9 (tolbutamide hydroxylase). , 1996, Drug metabolism and disposition: the biological fate of chemicals.

[35]  Maurice Dickins,et al.  Compound lipophilicity for substrate binding to human P450s in drug metabolism. , 2004, Drug discovery today.

[36]  M. J. Coon,et al.  Aliphatic hydroxylation by highly purified liver microsomal cytochrome P-450. Evidence for a carbon radical intermediate. , 1978, Biochemical and biophysical research communications.

[37]  Baojian Wu,et al.  Substrate selectivity of drug-metabolizing cytochrome P450s predicted from crystal structures and in silico modeling , 2012, Drug metabolism reviews.

[38]  Thomas Lengauer,et al.  A fast flexible docking method using an incremental construction algorithm. , 1996, Journal of molecular biology.

[39]  H-Z Bu,et al.  A literature review of enzyme kinetic parameters for CYP3A4-mediated metabolic reactions of 113 drugs in human liver microsomes: structure-kinetics relationship assessment. , 2006, Current drug metabolism.

[40]  Chris Oostenbrink,et al.  Computational prediction of drug binding and rationalisation of selectivity towards cytochromes P450. , 2008, Expert opinion on drug metabolism & toxicology.

[41]  Eric F. Johnson,et al.  The Structure of Human Microsomal Cytochrome P450 3A4 Determined by X-ray Crystallography to 2.05-Å Resolution* , 2004, Journal of Biological Chemistry.

[42]  T. Igarashi,et al.  Effects of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes. , 2002, Drug metabolism and disposition: the biological fate of chemicals.

[43]  S. Narasimhulu.  Differential behavior of the sub-sites of cytochrome 450 active site in binding of substrates, and products (implications for coupling/uncoupling). , 2007, Biochimica et biophysica acta.

[44]  Kristin P. Bennett,et al.  RS-Predictor: A New Tool for Predicting Sites of Cytochrome P450-Mediated Metabolism Applied to CYP 3A4 , 2011, J. Chem. Inf. Model..

[45]  J. Dawson,et al.  Heme-Containing Oxygenases. , 1996, Chemical reviews.

[46]  Markus A Lill,et al.  Prediction of Small‐Molecule Binding to Cytochrome P450 3A4: Flexible Docking Combined with Multidimensional QSAR , 2006, ChemMedChem.

[47]  Lars Olsen,et al.  Prediction of activation energies for hydrogen abstraction by cytochrome p450. , 2006, Journal of medicinal chemistry.

[48]  Yan-Yan Zhang,et al.  Metabolic Profiling and Cytochrome P450 Reaction Phenotyping of Medroxyprogesterone Acetate , 2008, Drug Metabolism and Disposition.

[49]  V. Luu‐The.  Assessment of steroidogenesis and steroidogenic enzyme functions , 2013, The Journal of Steroid Biochemistry and Molecular Biology.

[50]  Bu Hz,et al.  A literature review of enzyme kinetic parameters for CYP3A4-mediated metabolic reactions of 113 drugs in human liver microsomes: structure-kinetics relationship assessment. , 2006 .

[51]  Keli Han,et al.  Theoretical study of N-dealkylation of N-cyclopropyl-N-methylaniline catalyzed by cytochrome P450: insight into the origin of the regioselectivity. , 2009, Dalton transactions.