Elucidating Substrate Promiscuity in the Human Cytochrome 3A4

Abstract

The human cytochrome P450 enzymes (CYPs) are heme-protein monooxygenases, which catalyze oxidative reactions of a broad spectrum of substrates. Consequently, they play a critical role in the metabolism of xenobiotics, such as drugs and carcinogens, and the catabolism of endogenous lipophilic factors. Bioavailability and toxicity, both of which can be related to CYPs, continue to pose problems in the development of new drugs. The isoform which metabolizes over one-third of drugs, CYP 3A4, was investigated employing ensemble-docking experiments of a 195-substrate library with induced fit and GOLD docking algorithms and a number of scoring functions. Enzyme conformations included three currently available CYP 3A4 crystal structures. All docking experiments were performed in duplicates with and without inclusion of crystallographic waters. Resultant poses were assessed based on accuracy of site of metabolism prediction. Analyses of the docked solutions pertaining to ranking efficacy, ligand molecular properties, stabilizing residues in the ligand-enzyme complexes, and metabolic reactions are discussed. Our analyses suggest that certain residues make favorable interactions with the bound substrates. Employing multiple receptor conformations enhances the accuracy of catalytic prediction, while ligand size and flexibility impact docking performance. The presence of waters observed in crystal complexes does not necessarily lead to improved performance.

Comprehensive assessment of Cytochrome P450 reactions: A multiplex approach using real-time ESI-MS

“…The contradiction between conversion rate and rate of reaction may have different causes such as the higher assay concentrations of enzyme and substrate in CYP3A4 experiments and as previously mentioned the lower specificity of CYP3A4 [10,11,41]. In this context, it should be noted that rate of reactions are more meaningful for enzymes with lower K M -values, i.e.…”

“…Since coumarin 7-hydroxylation is selective for CYP2A6, coumarin was used as substrate [47]. CYP3A4 is a more promiscuous enzyme and converts 10 times more substrates than CYP2A6 [10,11,41]. In most studies midazolam and testosterone are used for the assessment of enzymatic activity.…”

“…Prior studies described variable structural properties and binding features of different CYPs [11,49]. CYP3A4 contributes to the metabolism of approximately 30 % of all xenobiotics [9][10][11]. Moreover, it is able to metabolize large substrates and bind two or more ligands simultaneously [41].…”

“…Zanger and Schwab (2013) described ten single CYP isoforms that are predominantly involved in these reactions [9]. CYP2A6 has the highest substrate specificity and is involved in only 3.4 % of Cytochrome P450 drug metabolism, whereas CYP3A4/5 is the most promiscuous CYP that has the lowest substrate specificity and metabolizes about 30.2 % of the xenobiotics that are catalyzed by Cytochromes P450 [9][10][11].…”

Advances in Computational Prediction of Regioselective and Isoform-Specific Drug Metabolism Catalyzed by CYP450s.

“…Hayes et al have recently employed three crystal structures of CYP3A4 along with flexible docking using GOLD and Glide and different scoring functions to perform SOM predictions for 195 substrates. [97] They have tried to model the induced fit and conformational selection process by docking in protein with selected amino acids in the active sites trimmed and then redocking with restored residues respectively. Successful SOM prediction criteria was set to < 5 , prediction accuracy was 74-89 % depending on the protocol selected and 2J0D structure was found the best for SOM prediction.…”