Current Drug Metabolism - Volume 13, Issue 2, 2012

Cytochromes P450 represent a class of hemoproteins participating in reactions leading to drug metabolism as well as in biotransformations of endogenous substrates. Variability of drugs is reflected in structures of human liver microsomal drug metabolizing cytochromes P450. Effective biotransformation is conditioned by the ability of both partners, of the substrate as well as of the enzyme, to bind in a correct manner; in other words, by the ability of a substrate to reach the active site, to bind there and undergo the chemical reaction and to leave the protein after successful biotransformation. Recent results of experimental as well as theoretical work on structure of cytochromes P450 document that the flexibility and plasticity of the enzyme is one of the most important properties which (together with the amino acid web of the active site and of the lining of the access/egress channels) determine substrate specificity of individual cytochrome P450 enzymes. Seven contributions of this Special issue named “Cytochromes P450: Flexibility and Plasticity - Properties Determining Substrate Preferences” discuss the structural properties of the respective cytochromes P450 as well as of substrates, structural adaptation of structures of cytochromes P450 determining their function [1, 2], the plasticity/malleability of the protein structure [3], flexible regions of individual enzymes, flexibility and solvation of their active sites [4], access/egress of the substrates/metabolites [5], as well as the structural adaptation and recognition of substrates in the respective active sites [6, 7]. Both approaches, theoretical and/or experimental, are jointly applied to contribute to solution of the questions raised. REFERENCES [1] Seifert, A.; Pleiss, J., Identification of Selectivity Determinants in CYP Monooxygenases by Modelling and Systematic Analysis of Sequence and Structure by Seifert and Pleiss. Curr. Drug Metab., 2012, 13(2): 197-202. [2] Otyepka, M.; Berka, K.; Anzenbacher, P., Is There a Relationship Between the Substrate Preferences and Structural Flexibility of Cytochromes P450? Curr. Drug Metab., 2012, 13(2): 130-142. [3] Wilderman, P., R.; Halpert, J., R., Plasticity of CYP2B Enzymes: Structural and Solution Biophysical Methods. Curr. Drug Metab., 2012, 13(2): 167- 176. [4] Hendrychova, T.; Berka, K.; Navratilova, V.; Anzenbacher, P.; Otyepka, M., Dynamics and Hydration of the Active Sites of Mammalian Cytochromes P450 Probed by Molecular Dynamics Simulations. Curr. Drug Metab., 2012, 13(2): 177-189. [5] Cojocaru, V.; Winn, P.J.; Wade, R., C., Multiple, Ligand-dependent Routes from the Active Site of Cytochrome P450 2C9. Curr. Drug Metab., 2012, 13(2): 143-154. [6] Oostenbrink, Ch.; de Ruiter, A.; Hritz, J.; Vermeulen, N., P., E., Malleability and Versatility of Cytochrome P450 Active Sites Studied by Molecular Simulations. Curr. Drug Metab., 2012, 13(2): 190-196. [7] de Beer, S., B., A.; van Bergen, L., A., H.; Keijzer, K.; Rea, V.; Venkataraman, H.; Fonseca Guerra, C.; Bickelhaupt, F., M.; Vermeulen, N., P., E., Commandeur, J., N., M.; Geerke, D., P., The Role of Protein Plasticity in Computational Rationalization Studies on Regioselectivity in Testosterone Hydroxylation by Cytochrome P450 BM3 mutants. Curr. Drug Metab., 2012, 13(2): 155-166.

In the last decades, the structural flexibility of cytochromes P450 has been extensively studied by spectroscopic and in silico methods. Here, both approaches are reviewed and compared. Comparison of both methods indicates that the individual cytochromes P450 differ significantly in the flexibilities of their substrate-binding active sites. This finding probably accounts for the large number of isoforms of these enzymes (there are fifty-seven known cytochrome P450 genes in the human genome) and their functional versatility. On the other hand, most of the known cytochrome P450s have a set of common structural features, with an overall structure consisting of a relatively flexible domain (the distal side), a more rigid domain (the heme-binding core) and a domain on the proximal side of the hemoprotein with intermediate flexibility. Substrate access and product egress channels of CYP enzymes are also important structural elements as the majority of these channels are located in the flexible distal side; the location, flexibility, and function of these channels are discussed.

The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYPmembrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

Recently, it was found that mutations in the binding cavity of drug-metabolizing Cytochrome P450 BM3 mutants can result in major changes in regioselectivity in testosterone (TES) hydroxylation. In the current work, we report the intrinsic reactivity of TES' C-H bonds and our attempts to rationalize experimentally observed changes in TES hydroxylation using a protein structure-based in silico approach, by setting up and employing a combined Molecular Dynamics (MD) and ligand docking approach to account for the flexibility and plasticity of BM3 mutants. Using this approach, about 100,000 TES binding poses were obtained per mutant. The predicted regioselectivity in TES hydroxylation by the mutants was found to be in disagreement with experiment. As revealed in a detailed structural analysis of the obtained docking poses, this disagreement is due to limitations in correctly scoring hydrogen-bonding and steric interactions with specific active-site residues, which could explain the experimentally observed trends in regioselectivity in TES hydroxylation.

In the past three years, major advances in understanding cytochrome P450 2B (CYP2B) structure-function relationships have been made through determination of multiple ligand-bound and one ligand-free X-ray crystal structure of CYP2B4 and one ligand-bound X-ray crystal structure of CYP2B6. These structures have provided insight into the features that provide the high degree of plasticity of the enzymes. A combination of a phenylalanine cluster that allows for concerted movement of helices F through G and a conserved set of electrostatic interactions involving Arg262 facilitates movement of this region to accommodate binding of ligands of various sizes without perturbing most of the P450 fold. Integrating solution based techniques such as NMR or deuterium exchange mass spectrometry (DXMS) with computational methods including molecular docking has provided further insight into enzyme behavior upon ligand binding. In addition, extended molecular dynamics simulations have provided a link between an open and a closed conformation of ligand-free CYP2B4 found in crystal structures. Other studies revealed the utility of rational engineering in improving stability of P450s to facilitate structural studies. The solution and computational results combined with the X-ray crystal structures yield a comprehensive picture of how these enzymes adopt different conformations to bind various ligands.

The flexibility, active site volume, solvation, and access path dynamics of six metabolically active mammalian cytochromes P450 (human 2A6, 2C9, 2D6, 2E1, 3A4 and rabbit 2B4) are extensively studied using molecular dynamics (MD) simulations. On average, the enzymes' overall structures equilibrate on a 50+ ns timescale. The very open CYP2B4 structure closes slowly over the course of the simulation. The volumes of the active sites fluctuate by more than 50% during the MD runs; these fluctuations are mainly due to movements of the main chains, with only a handful of amino acid residues in CYP2B4, CYP2D6, CYP2A6 and CYP2C9 showing significant independent side chain movement. The volume of the active site of CYP2E1 fluctuates heavily, ranging from 220 to 1310 A3, due to the opening and closing of gates to two adjacent cavities. CYP2E1 has the least hydrated active site of the studied CYPs; this is consistent with its preference for non-polar substrates. The CYP2A6 and CYP2E1 active sites are deeply buried, with access paths that are narrower than the radius of a water molecule. However, waters are still able to access these active sites due to local adaptations of the channel to accommodate their passage. This finding may imply that the access paths of the CYPs never fully open prior to contact with the substrate; instead, the substrate may induce adaptive conformational changes during its passage to the active site. This may also explain why some substrate recognition sites are localized along individual enzymes' access paths.

As the most important phase I drug metabolizing enzymes, the human Cytochromes P450 display an enormous versatility in the molecular structures of possible substrates. Individual isoforms may preferentially bind specific classes of molecules, but also within these classes, some isoforms show remarkable levels of promiscuity. In this work, we try to link this promiscuity to the versatility and malleability of the active site at the hand of examples from our own work. Mainly focusing on the flexibility of protein structures and the presence or absence of water molecules, we establish molecular reasons for observed promiscuity, determine the relevant factors to take into account when predicting binding poses and rationalize the role of individual interactions in the process of ligand binding. A high level of active site flexibility does not only allow for the binding of a large variety of substrates and inhibitors, but also appears to be important to facilitate ligand binding and unbinding.

Cytochrome P450 monooxygenases (CYPs) form a large, ubiquitous enzyme family and are of great interest in red and white biotechnology. To investigate the effect of protein structure on selectivity, the binding of substrate molecules near to the active site was modelled by molecular dynamics simulations. From a comprehensive and systematic comparison of more than 6300 CYP sequences and 31 structures using the Cytochrome P450 Engineering Database (CYPED), residues were identified which are predicted to point close to the heme centre and thus restrict accessibility for substrates. As a result, sequence-structure-function relationships are described that can be used to predict selectivity-determining positions from CYP sequences and structures. Based on this analysis, a minimal library consisting of bacterial CYP102A1 (P450BM3) and 24 variants was constructed. All variants were functionally expressed in E. coli, and the library was screened with four terpene substrates. Only 3 variants showed no activity towards all 4 terpenes, while 11 variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type. The minimal library also contains variants that show interesting side products which are not generated by the wild type enzyme. By two additional rounds of molecular modelling, diversification, and screening, the selectivity of one of these variants for a new product was optimised with a minimal screening effort. We propose this as a generic approach for other CYP substrates.

The Wnt/β-catenin pathway plays an important role in liver homeostasis, as well as during prenatal liver development, liver regeneration, and hepatocarcinogenesis. The connection of hepatic β-catenin activation and expression of drug-metabolizing enzymes has been established in the past few years: on the whole, a generally positive-regulatory effect of β-catenin on the expression and inducibility of many enzymes involved in phase I and phase II of drug metabolism has been described by different groups. The mechanisms underlying these processes are still not fully understood. However, there is accumulating evidence for a complex interaction of β-catenin with different xenobiotic-sensing receptors, which act as transcription factors after ligand activation, for example the aryl hydrocarbon receptor or the constitutive androstane receptor. Among others, these crosstalk mechanisms might explain the manifold effects of β-catenin on hepatic drug metabolism. In this review, the current knowledge regarding the role of Wnt/β-catenin signaling in the regulation of hepatic expression of glutathione S-transferases is presented. In addition, the crosstalk of β-catenin signaling with nuclear receptors involved in the regulation of glutathione S-transferases will be discussed.

Early understanding of the metabolic pathway and potential interaction of new drug candidates with other drugs is one of the goals of preclinical studies in the drug discovery process. Although other body organs are involved in drug biotransformation, the liver is the predominant organ of metabolism for a wide range of endogenous compounds and xenobiotics. The set of enzymes contained in the cytochrome P450 superfamily present predominantly in the liver have been identified as the single most important agent of drug metabolism and have formed the bedrock of most matured technologies for in vitro drug biotransformation studies. With the development of a number of liver-based technologies, in vitro metabolism has gained significant popularity in the past three decades. This has come in response to several demanding factors including the questionable relevance of data from animal studies; the high cost and stringent regulatory and ethical requirement, as well as safety issues involved with studies using human subjects; and the need for high throughput due to the wide range of chemical entities for routine investigations. These technologies which vary from whole liver to subcellular fractions have found ready application in generating the desired information on the substrate and inhibitor specificity of most metabolic enzymes. This paper reviews such technologies as isolated fresh liver; liver slices; primary, cultured and cryopreserved hepatocytes; microsomes; cytosolic fractions; and purified or heterologously expressed drug-metabolizing enzymes. It highlights the general principles of in vitro enzyme kinetics and the factors that determine the choice of each in vitro technology for biotransformation studies.

Platelet activation and aggregation have been established as pivotal elements in the pathogenesis of atherosclerotic and ischemic diseases, including acute coronary syndromes. The difficulty of achieving optimal platelet inhibition remains a major constraint following dual-antiplatelet therapy, which can lead to a diminished response following initiation of clopidogrel therapy. Though the absolute mechanisms underlying clopidogrel resistance are controversial, a variety of responsible factors are recognized. Clopidogrel, being a prodrug, requires conversion to an active metabolite for its activity. This metabolism involves various cytochrome P450 (CYP) enzymes at different steps, and it is hypothesized that competitive inhibition of CYPs may contribute to clopidogrel resistance. Proton pump inhibitors (PPIs) are competitive inhibitors of CYPs that can attenuate the antiplatelet activity of clopidogrel, and this can lead to clopidogrel resistance. Available data from different clinical studies have postulated the possibility of a drug-drug interaction between clopidogrel and PPIs. PPIs differ somewhat in their pharmacokinetic properties like bioavaibility and affinity for CYP2C19. However it is not clear whether the proposed drug interaction of PPI with clopidogrel is same with all PPIs (i.e., a class effect) or it is limited to a subset of PPIs (i.e., a drug effect). This interaction needs further assessment with well designed prospective clinical trials, before any change in clinical practice should be considered. In this review, we attempt to evaluate the available evidence exploring drug interactions with PPIs as the underlying mechanism for the reduced antiplatelet effect of clopidogrel.