Structural insights into understudied human cytochrome P450 enzymes

Human cytochrome P450 (CYP) enzymes are widely known for their pivotal role in the metabolism of drugs and other xenobiotics as well as of endogenous chemicals. In addition, CYPs are involved in numerous pathophysiological pathways and, hence, are therapeutically relevant. Remarkably, a portion of promising CYP targets is still understudied and, as a consequence, untargeted, despite their huge therapeutic potential. An increasing number of X-ray and cryo-electron microscopy (EM) structures for CYPs have recently provided new insights into the structural basis of CYP function and potential ligand binding. This structural knowledge of CYP functionality is essential for both understanding metabolism and exploiting understudied CYPs as drug targets. In this review, we summarize and highlight structural knowledge about this enzyme class, with a focus on understudied CYPs and resulting opportunities for structure-based drug design.Teaser: This review summarizes recent structural insights into understudied cytochrome P450 enzymes. We highlight the impact of molecular modeling for mechanistically explaining pathophysiological effects establishing understudied CYPs as promising drug targets.     

Cytochrome P450 inactivation by pharmaceuticals and phytochemicals: therapeutic relevance

One of the major clinical concerns is possible drug interactions that can be the result of abrogation of the P450 pathway(s) of metabolism causing toxicity due to elevated exposures of other drugs metabolized by these pathways. When the P450 substrate is catalytically activated to a reactive intermediate, this transient molecule may react with available nucleophilic residues from the enzyme - thereby resulting in the inactivation of the P450. The effects of CYP inactivation on the pharmacokinetics of co-administered drugs or on the inactivator itself depend on complex factors involving the molecular entities, the kinetics of inactivation (K(I), k(inact)), the partition ratio, the zero-order synthesis rate of new enzyme, multiple pathways of metabolism (competing pathways), the dose or exposure, and specific patient characteristics. This review summarizes the catalytic efficiencies of many inactivator drugs along with any consequent clinical relevance. The chemical agents described have been ranked for the kinetic efficiency of inactivation and contrasted with the known clinically relevant drug interactions. This will allow judicious consideration of the many factors that influence the importance of CYP inactivation and their relative contribution to systemic clearance of co-administered drugs. This study allows an improved characterization and dissection of potential physiological interactions with various drugs and nutrients. Knowing more about selective inactivation of cytochrome P450 by common xenobiotics, drugs and phytochemicals allows better understanding of expected interactions with chemotherapeutics and other xenobiotics.     

Possible involvement of singlet oxygen species as multiple oxidants in p450 catalytic reactions

Cytochrome P450 (P450) constitutes a superfamily of enzymes which activate dioxygen and carry out monooxygenation reactions of large numbers of endogenous and xenobiotic compounds. Drug metabolism is a particularly important P450 function, and, therefore, elucidating the metabolic products and pathways of drugs is essential for drug development. To explain the substrate selectivity of P450 reactions, it is necessary to understand the formation of multiple activated oxygen species to determine the type of catalyzed reactions, in addition to conducting structure analyses of P450s. Although an oxo-Fe(IV)-porphyrin-pi-cation radical is regarded as an activated oxygen species in P450 reactions, a nucleophilic Fe(III)-peroxo species has also been proposed as another oxidant. In the past decade, various studies indicated that P450-catalyzed oxygenations are complex, and that a single reaction pathway cannot explain all of the experimental results. In addition, the microsomal P450 system is known to generate reactive oxygen species (ROS). However, the contribution of ROS to P450 reactions remains unclear. We recently found that singlet oxygen (1O2) was involved in both several rat liver microsomal P450 reactions and four human CYP subfamily activities, as confirmed by the ESR spin-trapping method. In this review, we describe the studies that have been conducted on the detection and characterization of ROS in P450 reactions related to drug metabolism that involve the possibility of 1O2 in the P450 catalytic cycle. Gaining an understanding of the activated oxygen species that determine the type of drug metabolism will help us to predict the important metabolites formed.     

Cytochrome P450 Inactivation by Pharmaceuticals and Phytochemicals: Therapeutic Relevance

One of the major clinical concerns is possible drug interactions that can be the result of abrogation of the P450 pathway(s) of metabolism causing toxicity due to elevated exposures of other drugs metabolized by these pathways. When the P450 substrate is catalytically activated to a reactive intermediate, this transient molecule may react with available nucleophilic residues from the enzyme – thereby resulting in the inactivation of the P450. The effects of CYP inactivation on the pharmacokinetics of co-administered drugs or on the inactivator itself depend on complex factors involving the molecular entities, the kinetics of inactivation (K_I, k_inact), the partition ratio, the zero-order synthesis rate of new enzyme, multiple pathways of metabolism (competing pathways), the dose or exposure, and specific patient characteristics. This review summarizes the catalytic efficiencies of many inactivator drugs along with any consequent clinical relevance. The chemical agents described have been ranked for the kinetic efficiency of inactivation and contrasted with the known clinically relevant drug interactions. This will allow judicious consideration of the many factors that influence the importance of CYP inactivation and their relative contribution to systemic clearance of co-administered drugs. This study allows an improved characterization and dissection of potential physiological interactions with various drugs and nutrients. Knowing more about selective inactivation of cytochrome P450 by common xenobiotics, drugs and phytochemicals allows better understanding of expected interactions with chemotherapeutics and other xenobiotics.     

Substrate inhibition kinetics in drug metabolism reactions

Inhibition of enzyme activity at high substrate concentrations, so-called "substrate inhibition," is commonly observed and has been recognized in drug metabolism reactions since the last decade. Although the importance of such "atypical" kinetics in vivo remains poorly understood, a substrate with substrate inhibition kinetics has been shown to unconventionally alter the metabolism of other substrates. In recent years, it is becoming increasingly evident that the mechanisms for substrate inhibition are highly complex, which are possibly contributed by multiple (at least two) binding sites within the enzyme protein, the formation of a ternary dead-end enzyme complex, and/or the ligand-induced changes in enzyme conformation. This review primarily discusses the mechanisms for substrate inhibition displayed by the important drug-metabolizing enzymes, such as cytochrome p450s, UDP-glucuronyltransferases, and sulfotransferases. Kinetic modeling of substrate inhibition in the absence or presence of a modifier is another central issue in this review because of its importance in the determination of kinetic parameters and in vitro/in vivo predictions.     

Mechanism-based inactivators as probes of cytochrome P450 structure and function

The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.     

Thermodynamics of Ligand Binding to P450 2B4 and P450eryF Studied by Isothermal Titration Calorimetry

Structural plasticity and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. As more mammalian P450 crystal structures have emerged, computational modeling has become a major tool to predict drug metabolism and interactions. There is a need for real solution thermodynamic data to support modeling and crystallographic observations. Using isothermal titration calorimetry (ITC) we successfully evaluated the conformational flexibility of P450 2B4 in binding imidazole inhibitors of different size and chemistry and dissected the stoichiometry and energetics of ligand binding allostery in P450eryF. Thermodynamic signatures obtained by ITC nicely correlated with structural and modeling results. Thus, ITC is a powerful tool to study structure-function relationships in P450s.     

Thermodynamics of ligand binding to P450 2B4 and P450eryF studied by isothermal titration calorimetry

Structural plasticity and cooperativity in ligand recognition are two key aspects of the catalytic diversity of cytochrome P450 enzymes. As more mammalian P450 crystal structures have emerged, computational modeling has become a major tool to predict drug metabolism and interactions. There is a need for real solution thermodynamic data to support modeling and crystallographic observations. Using isothermal titration calorimetry (ITC) we successfully evaluated the conformational flexibility of P450 2B4 in binding imidazole inhibitors of different size and chemistry and dissected the stoichiometry and energetics of ligand binding allostery in P450eryF. Thermodynamic signatures obtained by ITC nicely correlated with structural and modeling results. Thus, ITC is a powerful tool to study structure-function relationships in P450s.     

Cytochrome p450 and chemical toxicology

The field of cytochrome P450 (P450) research has developed considerably over the past 20 years, and many important papers on the roles of P450s in chemical toxicology have appeared in Chemical Research in Toxicology. Today, our basic understanding of many of the human P450s is relatively well-established, in terms of the details of the individual genes, sequences, and basic catalytic mechanisms. Crystal structures of several of the major human P450s are now in hand. The animal P450s are still important in the context of metabolism and safety testing. Many well-defined examples exist for roles of P450s in decreasing the adverse effects of drugs through biotransformation, and an equally interesting field of investigation is the bioactivation of chemicals, including drugs. Unresolved problems include the characterization of the minor "orphan" P450s, ligand cooperativity and kinetic complexity of several P450s, the prediction of metabolism, the overall contribution of bioactivation to drug idiosyncratic problems, the extrapolation of animal test results to humans in drug development, and the contribution of genetic variation in human P450s to cancer incidence.     

Microsomal cytochrome P450 as a target for drug discovery and repurposing

Cytochrome P450 (P450) enzymes are ancient electron-transfer-chain system of remarkable biological importance. Microsomal P450 enzymes are the P450 attached to endoplasmic reticulum, which, in humans, are critical for body’s defenses against xenobiotics by mediating their metabolism, and cell signaling by mediating arachidonic acid (AA) transformation to several potent bioactive molecules. Only recently, modulating P450-mediated AA metabolism has risen as a promising new drug target. This review presents the therapeutic potential of finding effective, selective and safe treatments targeting P450-mediated AA metabolism, and the several approaches that have been used to find these treatments; among which, our focus was on modulators of P450 activities. We detailed the efforts done to develop new molecular entities designed to modulate P450, and the more recent efforts tried to employ our previous knowledge on drug metabolism to repurpose old drugs with the capacity to alter P450-mediated drug metabolism to target AA metabolism. Because of the long recognition of P450 role in xenobiotic metabolism, several clinically approved agents were identified to alter P450 activity. Repurposing old drugs as P450 modulators can facilitate bringing treatments targeting P450-mediated AA metabolism to clinical trials. However, the capacity of the modulation of P450-derived AA metabolites of clinically approved drugs has to be systematically investigated and validated for their new use in humans.     

The cytochrome P450 superfamily: biochemistry,evolution and drug metabolism in humans

Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.     

Advances in the interpretation and prediction of CYP2E1 metabolism from a biochemical perspective

BACKGROUND: Cytochrome P450 2E1 (CYP2E1) plays a central role in the metabolism and metabolic activation of a large number of small, mostly xenobiotic compounds. These qualities distinguish CYP2E1 from traditional enzymes and pose significant challenges to understanding the role and consequences of CYP2E1-mediated metabolism. OBJECTIVE: This review discusses recent advances in kinetic profiling, quantitative structure-activity relationships and structural studies that have furthered the development of tools to interpret and predict CYP2E1 metabolism. METHODS: Analysis of kinetic profiles by specific mechanisms produces important parameters describing specificity, stoichiometry and metabolism of molecules. Quantitative structure-activity relationships reveal a more specific basis for molecular recognition by CYP2E1. The corresponding protein structures imparting these interactions are the focus of chemical modifications, site-directed mutagenesis and homology modeling studies. RESULTS: Compilation of kinetic profiling for CYP2E1 substrates established the selectivity for small substrates, whose characteristics could be generalized in parameters for hydrophobicity and steric properties as determined by quantitative structure-activity relationships. The possibility of an effector site for monocyclic compounds added an interesting variable to these modeling efforts. Various structural studies identified important residues contributing to binding and catalysis as well as the volume and location of the active site relative to the heme moiety. Pressure and carbon monoxide-binding experiments also demonstrated the inherent conformational flexibility of CYP2E1 that may contribute to rate-limiting steps during catalytic turnover. CONCLUSION: Although combinations of these approaches have reinforced important observations, more work is needed to verify findings and seek broader impacts for various interpretative and predictive tools.     

Recent progress in cytochrome P450 enzyme electrochemistry

Cytochrome P450 (CYP) enzymes perform crucial functions in humans, including the metabolism of drugs and hormone synthesis. The catalytic reactions performed by these enzymes (typically monoxygenation) require the transfer of electrons. Thermodynamic and mechanistic detail of the electron transfer component of these catalytic processes has been obtained traditionally from potentiometric titrations. More recently, voltammetric approaches (that are inherently simpler and require less sample) have been used. This has been made possible by the creation of biocompatible electrode surfaces at which the P450 enzyme is confined and able to undergo physiologically relevant electron transfer processes. The continuing challenge has been to obtain an in vivo-like enzyme response, and to provide the basis for the creation of an artificial bioprocess in vitro. A powerful instrumental electrochemical method, employing Fourier-transformed large-amplitude ac voltammetry, offers the potential for greater insight and new opportunities to understand the nuances of the electron transfer process. This review highlights several recent advances in the electrochemistry of P450 enzymes rather than providing a comprehensive review of P450 electrochemistry.     

57 varieties: the human cytochromes P450

The human cytochrome P450 (CYP) complement of heme-thiolate enzymes is reviewed. Of the 57 individual P450s characterized in Homo sapiens thus far, it is apparent that approximately one-half are associated with the metabolism of drugs and other xenobiotics, whereas the other half have endogenous functions in steroid, prostanoid, eicosanoid and fatty acid metabolism. This review covers the extent of enzyme functionality for the known human P450s, focusing primarily on their role in the Phase I metabolism of foreign compounds, which involves the CYP1, CYP2 and CYP3 families.     

mRNA levels of drug-metabolizing enzymes in 11 brain regions of cynomolgus macaques

The cynomolgus macaque is an important nonhuman primate species in drug metabolism studies, in part because of its evolutionary closeness to humans. Cytochromes P450 (P450s) have been investigated in the major drug-metabolizing organs, i.e., the liver and small intestine, but have not been fully investigated in the brain. However, recent investigations have indicated possible important roles for P450s in the brain. In this study, by using the quantitative polymerase chain reaction, we measured the mRNA levels of 38 cynomolgus drug-metabolizing enzymes, including 19 P450s, 10 UDP-glycosyltransferases, and 9 other enzymes, in 11 brain regions. Among these drug-metabolizing enzymes, expression of 32 enzyme mRNAs were detected in one or more brain regions, indicating their possible roles in the brain. Further investigation of metabolic activities would facilitate better understanding of the importance of these enzymes in the brain.     

Mapping determinants of the substrate selectivities of P450 enzymes by site-directed mutagenesis.

Point-mutation studies in cytochrome P450s by site-directed mutagenesis have identified key residues that can confer the catalytic properties of one cytochrome P450 onto another. Most of these key residues cluster at sites that map to amino acids forming the substrate-binding site of P450cam, a distantly related enzyme. These sites are found on topological elements of P450cam, which by their surface location and lack of extensive secondary structure are likely to permit genetic variation without extensive disruption of the overall topology of the enzyme. If these topological features of P450cam are conserved in the mammalian enzymes, they are likely to accommodate the structural diversity seen for mammalian P450s in a manner that conserves a basic structure for P450 enzymes but that leads to the catalytic diversity seen for the mammalian enzymes.     

Cytochrome P450: Molecular Architecture,Mechanism, and Prospects for Rational Inhibitor Design

Cytochromes P450 catalyze the insertion of one O₂-derived oxygen atom into an aliphatic or aromatic molecule. P450s are best known for the metabolism of xenobiotic molecules, where hydroxylation renders insoluble hydrocarbons more soluble for easier elimination. In addition to this important catabolic function, P450s catalyze key steps in steroid and plant growth regulator metabolism. A variety of therapeutic, fungicidal, and agochemical agents that perturb these metabolic pathways very likely operate by binding in the lipophilic P450 active site and coordinating with the heme iron atom. Recent determination of a bacterial P450 crystal structure, P450cam from Pseudomonas putida, in addition to the crystal structure of four inhibited complexes, has provided some insight into the potential use of P450 as a model system for the rational design of therapeutic agents. The crystal structure has also shed light on the P450 catalytic mechanism. P450cam operates differently from peroxidase or catalase in cleaving the O–O bond, since unlike these other enzymes, P450 contains no acid–base catalytic groups near the oxygen binding site. Instead, the O₂ pocket is lined with aliphatic and aromatic residues. This strongly suggests that the catalytic push required to cleave the O–O bond resides with the ability of the Cys heme ligand to donate electron density to the heme–oxy system. A comparison of the substrate-free and -bound P450cam crystal structures has revealed some interesting aspects regarding the dynamics of substrate binding. The structures of both forms of P450cam are the same except that in the substrate-free enzyme, the active-site pocket fills with a network of water molecules, one of which coordinates with the iron atom. Despite this lack of any significant conformational rearrangement of protein groups, a careful analysis of crystallographic temperature factors shows dynamical differences. Segments of the protein that are separated in the sequence but that lie close to one another in the structure and that define a small entrance to the substrate pocket undergo significantly higher thermal motion in the substrate-free enzyme. This suggests that dynamical fluctuations at the molecular surface play an important role in controlling substrate binding.     

Investigation of the mechanisms underlying the differential effects of the K262R mutation of P450 2B6 on catalytic activity

Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-alpha-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.