Investigating human P450s involved in drug metabolism via homology with high-resolution P450 crystal structures of the CYP2C subfamily

The important role of high-resolution crystal structures of cytochrome P450 (CYP) enzymes for the generation of P450 models by homology is discussed. The main focus is on human P450 enzymes involved in drug metabolism, where the role of homology modelling has been emphasized in the recent literature. Report of the first human P450 crystal structure has provided an opportunity for comparison between those modelled from other crystallographic templates, and the recent substrate-bound rabbit CYP2C5 structure exemplifies the relevance of high-resolution template structures to generating 3-D models of P450s where the homology is relatively high. In particular, the homology models of human CYP1 and CYP2 family enzymes are presented, where good agreement with experiment findings are apparent.     

Structural analysis of CYP2C9 and CYP2C5 and an evaluation of commonly used molecular modeling techniques.

This work had two separate aims: to evaluate different modeling techniques and to make a detailed structural characterization of CYP2C9. To achieve these goals, the consensus principal component analysis (CPCA) technique and distance measurements were used to explore available crystal structures, newly built homology models, and repeated molecular dynamics simulations. The CPCA was based on molecular interaction fields focused on the active site regions of the proteins and include detailed amino acid analysis. The comparison of the CYP2C9 and CYP2C5 crystal structures revealed differences in the flexible regions such as the B-C and F-G loop and the N and C termini. Cross homology models of CYP2C9 and CYP2C5, using their respective crystal structures as templates, indicated that such models were more similar to their templates than to their target proteins. Inclusion of multiple templates slightly improved the similarity to the crystal target in some cases and could be recommended even though it requires a careful manual alignment process. The application of molecular dynamics simulations to highly flexible proteins such as cytochromes P450 is also explored and the information is extracted by the CPCA. Advantages and drawbacks are presented for the different modeling techniques. Despite the varying modeling success, the models give insight and understanding by the mutual forming and discarding of hypotheses. This is a dynamic process since the crystal structures are improving with time and, therefore, the answers to the models are also changing accordingly.     

Dissecting the function of cytochrome P450

1 The role that P450 plays in the metabolism of drugs and other chemicals is often pivotal in determining the duration and magnitude of their biological effects, so that it is important to identify the specific forms of the enzyme involved.
2 The similarity between different forms of P450 is such that polyclonal antibodies raised by conventional means, even against a homogeneous preparation of the enzyme will often react with several different P450s.
3 Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to their use.
4 The use of short sequences of amino acids (4–7 residues) predicted to occur in surface loop regions of the protein as immunogens, affords a means of targeting antibodies to specific forms of P450.
5 The terminal amino acid of a peptide, here the C-terminal residue, is very immunodominant in determining the specificity of the resultant antibody. Hence, antibodies against the C-terminal residues of P450 have a high affinity and, in most cases, will be specific.
6 Application of anti-peptide antibodies to the major forms of P450 in human liver revealed the anticipated interindividual variation. However, evidence was found that the polymorphism in CYP3A5 expression is quantitative rather than qualitative.
7 A putative pro-inhibitory region on the surface of mammalian P450, possibly involved in intramolecular electron transport, has been identified, using a combination of directed anti-peptide antibodies and sequence alignment based on secondary structure.
8 Antibodies directed against defined regions of P450 enzymes are proving invaluable in exploring the regulation, specificity and function of these key enzymes.     

A natural variant of the heme-binding signature (R441C) resulting in complete loss of function of CYP2D6.

A new variant allele CYP2D6*62 (g.4044C>T; R441C) of the drug-metabolizing cytochrome P450 (P450) CYP2D6 was identified in a person with reduced sparteine oxidation phenotype, which was unexpected based on a genetic CYP2D6*1A/*41 background. The recombinantly expressed variant protein had no activity toward propafenone as a result of missing heme incorporation. Sequence alignment revealed that the positively charged R441 residue is part of the heme-binding signature but not strictly conserved among all the P450s. A compilation of described P450 monooxygenase variants revealed that other enzymes can functionally tolerate even nonconservative amino acid changes at the corresponding position (i.e., the invariant cysteine 2). This suggests that heme binding in mammalian P450s depends not only on the integrity of the heme-binding signature but also on other family- and subfamily-specific sequence determinants.     

Peptide-based in vitro assay for the detection of reactive metabolites

We describe a novel peptide-based in vitro method for the detection of reactive metabolites that is amenable for use with microsomal or purified enzyme systems. Covalently bound adducts are detected by mass spectrometry using a surface-enhanced laser desorption ionizationtime of flight detector. The trapping molecule is an 11 amino acid peptide (ECGHDRKAHYK) that contains cysteine and other nucleophilic amino acid residues, as well as charged residues to enhance binding to a weak cation exchange chip surface used with the detection system. The assay concept was initially tested using rat or human liver microsomes with a series of benzodioxolanes. The assay was refined using human recombinant cytochrome P450 3A4 as the bioactivation system and validated with a series of positive and negative reference compounds. Alternative individual human recombinant P450 enzymes (e.g., 1A1, 2C9, or 2D6) may be used in place of 3A4 as the bioactivation system, or several P450 enzymes can be combined together into a single bioactivation system. We found that a mixture of P450s 3A4, 2C9, and 2D6 was suitable as a rapid general screen for the detection of reactive metabolites that covalently bind to proteins. Combining results from assays of individual P450 enzymes with microsomal systems allows the rapid profiling of metabolic pathways involved in reactive metabolite generation and provides valuable information that can be used to guide structural modifications to minimize the potential for metabolic bioactivation. In addition, non-P450 enzymes may be used as activation systems, such as peroxidases or alcohol dehydrogenase. In summary, this peptide-based assay system is able to detect reactive metabolites generated from a structurally diverse set of drugs and xenobiotics using a variety of microsomal or purified enzyme activation systems.     

The CYP2 family: models,mutants and interactions

1. The construction of three-dimensional models of mammalian cytochromes P450 from the CYP2 family is reported based on protein sequence alignment with CYP102, a bacterial P450 of known crystal structure. 2. The homology models of CYP2 family enzymes appear to show self-consistency with the currently accumulated information from site-directed mutagenesis and chemical modification of amino acid residues known to affect redox partner interactions. 3. The generation of these models from the recently reported crystal structure of substrate-bound CYP102 enables the exploration of likely active site contacts with specific substrates of CYP2 family isozymes.     

Functional expression of human cytochrome P450 enzymes in Escherichia coli

Knowledge regarding cytochrome P450 (P450) is crucial to the fields of drug therapy and drug development, as well as in our understanding of the mechanisms underlying the metabolic activation of potentially toxic and carcinogenic compounds. Escherichia coli is the most extensively utilized host in the production of recombinant human P450 enzymes. However, the recovery of substantial yields of functionally active P450 proteins remains problematic. Mammalian P450 protein was first expressed in 1991, via the modification of the N-terminal amino acid sequences in E. coli cells. Since that time, a variety of strategies have been established for the functional expression of recombinant P450s in E. coli, including N-terminal modification, the use of molecular chaperones, and culturing at lower temperatures. In all cases, human P450 expressed in E. coli cells has been shown to efficiently catalyze the oxidation of representative substrates at efficient rates. These recombinant P450s are applicable to studies which estimate the kinetic parameters of drug oxidation, and have also been used to determine the metabolic pathways of drugs and carcinogens exploited by human P450s. Despite the potential of P450s in various pharmaceutical and biotechnological fields, however, a host of substantial challenges must be overcome before these enzymes can be routinely utilized. Intrinsically, these enzymes are not very active, and exhibit poor stability. In this review, we have described current developments in the heterologous expression of human P450 enzymes.     

Insights into drug metabolism by cytochromes P450 from modelling studies of CYP2D6-drug interactions

The cytochromes P450 (CYPs) comprise a vast superfamily of enzymes found in virtually all life forms. In mammals, xenobiotic metabolizing CYPs provide crucial protection from the effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. Ideally, the information on the possible metabolism by CYPs required during drug development would be obtained from crystal structures of all the CYPs of interest. For some years only crystal structures of distantly related bacterial CYPs were available and homology modelling techniques were used to bridge the gap and produce structural models of human CYPs, and thereby obtain useful functional information. A significant step forward in the reliability of these models came seven years ago with the first crystal structure of a mammalian CYP, rabbit CYP2C5, followed by the structures of six human enzymes, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6 and CYP3A4, and a second rabbit enzyme, CYP2B4. In this review we describe as a case study the evolution of a CYP2D6 model, leading to the validation of the model as an in silico tool for predicting binding and metabolism. This work has led directly to the successful design of CYP2D6 mutants with novel activity-including creating a testosterone hydroxylase, converting quinidine from inhibitor to substrate, creating a diclofenac hydroxylase and creating a dextromethorphan O-demethylase. Our modelling-derived hypothesis-driven integrated interdisciplinary studies have given key insight into the molecular determinants of CYP2D6 and other important drug metabolizing enzymes.     

Plasticity of CYP2B enzymes: structural and solution biophysical methods

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 Arg(262) 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.     

Molecular modelling of CYP3A4 from an alignment with CYP102: Identification of key interactions between putative active site residues and CYP3A-specific chemicals

1. A structural model of CYP3A4 is reported on the basis of a novel amino acid sequence alignment between the CYP3 family and CYP102, a bacterial P450 of known crystal structure.

2. Construction of the CYP3A4 model from CYP102 is facilitated by the relatively high sequence homology between the two proteins (52% homology; 27% identity) with many conservative amino acid changes, yielding a structure of low internal energy.

3. A considerable number of specific substrates, and some specific inhibitors, are shown to occupy the putative CYP3A4 active site via interactions with the same amino acid residues in almost all cases investigated.

4. The CYP3A4 model rationalizes the known positions of metabolism for many substrates of this major human P450 such that the route of metabolism in novel development compounds can be predicted.     

Designing better drugs: predicting cytochrome P450 metabolism

Many 3D ligand-based and structure-based computational approaches have been used to predict, and thus help explain, the metabolism catalyzed by the enzymes of the cytochrome P450 superfamily (P450s). P450s are responsible for >90% of the metabolism of all drugs, so the computational prediction of metabolism can help to design out drug-drug interactions in the early phases of the drug discovery process. Computational methodologies have focused on a few P450s that are directly involved in drug metabolism. The recently derived crystal structures for human P450s enable better 3D modelling of these important metabolizing enzymes. Models derived for P450s have evolved from simple comparisons of known substrates to more-elaborate experiments that require considerable computer power involving 3D overlaps and docking experiments. These models help to explain and, more importantly, predict the involvement of P450s in the metabolism of specific compounds and guide the drug-design process.     

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.     

Cloning CYP2D21 and CYP3A22 cDNAs from liver of miniature pigs.

To compare the identity of the primary structure of drug-metabolizing cytochrome P450 between miniature pigs and humans, two cDNA clones, coding for miniature pig CYP2D21 and CYP3A22, were isolated. The deduced amino acid sequences of CYP2D21 and CYP3A22 were 78.3 and 75.0% identical to human CYP2D6 and CYP3A4, respectively. These values were nearly the same as those of bovine, dog, and some rodent isoforms, and 12.2 to 18.4% lower than those of nonhuman primates such as cynomolgus monkeys, Japanese monkey, and marmosets. These data indicate that miniature pig P450s are genetically not so close as monkey P450s to human P450s as previously expected. The recombinant CYP2D21 enzyme, however, showed bufuralol 1'-hydroxylase activity, suggesting that miniature pig CYP2D21 is capable of metabolizing some of the same substrates associated with human CYP2D6 despite its low identity to human counterparts.     

Inhibition of cytochrome p450 enzymes by quinones and anthraquinones

In silico docking studies and quantitative structure-activity relationship analysis of a number of in-house cytochrome P450 inhibitors have revealed important structural characteristics that are required for a molecule to function as a good inhibitor of P450 enzymes 1A1, 1A2, 2B1, and/or 2A6. These insights were incorporated into the design of pharmacophores used for a 2D search of the Chinese medicine database. Emodin, a natural anthraquinone isolated from Rheum emodi and known to be metabolized by cytochrome P450 enzymes, was one of the hits and was used as the lead compound. Emodin was found to inhibit P450s 1A1, 1A2, and 2B1 with IC(50) values of 12.25, 3.73, and 14.89 μM, respectively. On the basis of the emodin molecular structure, further similarity searches of the PubChem and ZINC chemical databases were conducted resulting in the identification of 12 emodin analogues for testing against P450s 1A1-, 1A2-, 2B1-, and 2A6-dependent activities. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) showed the best inhibition potency for P450 1A1 with an IC(50) value of 0.40 μM. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) and 1-amino-4-hydroxyanthracene-9,10-dione (compound 2) both inhibited P450 1A2 with the same IC(50) value of 0.53 μM. In addition, compound 1 acted as a mechanism-based inhibitor of cytochrome P450s 1A1 and 1A2 with K(I) and K(inactivation) values of 5.38 μM and 1.57 min(-1) for P450 1A1 and 0.50 μM and 0.08 min(-1) for P450 1A2. 2,6-Di-tert-butyl-5-hydroxynaphthalene-1,4-dione (compound 8) directly inhibited P450 2B1 with good selectivity and inhibition potency (IC(50) = 5.66 μM). Docking studies using the 3D structures of the enzymes were carried out on all of the compounds. The binding modes of these compounds revealed the structural characteristics responsible for their potency and selectivity. Compound 1, which is structurally similar to compound 2 with the presence of an amino group at position 1, showed a difference in the mechanism of inhibition toward P450s 1A1 and 1A2. The mechanism-based inhibition seen for compound 1 may be attributed to the presence of the methyl group at the 2-position, in close proximity to the amino group. Compound 2, which is otherwise similar, lacks that methyl moiety and did not show mechanism-based inhibition.     

On the recognition of mammalian microsomal cytochrome P450 substrates and their characteristics: towards the prediction of human p450 substrate specificity and metabolism

The characteristics of mammalian microsomal P450 xenobiotic substrates are described, particularly with reference to the major P450 isoforms associated with drug metabolism in humans. It is further reported that a relatively small number of molecular, electronic, and physico-chemical properties are required to discriminate between chemicals that exhibit specificity for human P450 isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Molecular templates of superimposed substrates are shown to be complementary with the putative active sites of the relevant enzymes, thus enabling a possible prediction of P450 specificity from structure. Factors contributing to metabolic clearance and binding affinity are also discussed, and methods for their calculation are described.     

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.     

Human P450s involved in drug metabolism and the use of structural modelling for understanding substrate selectivity and binding affinity

1. The human cytochrome P450 enzymes and their substrates are reviewed, together with current knowledge on the three-dimensional structures of P450s obtained from X-ray crystallographic studies and from homology modelling based on mammalian P450 template crystal structures.

2. There is a particular focus on human Phase 1 drug metabolism mediated by P450s, and a rationalization of their substrate selectivities and binding strengths in terms of lipophilicity and active site interactions.

3. The combination of molecular modelling and quantitative structure–activity relationship (QSAR) studies facilitates understanding of the factors which determine substrate selectivity and binding to the human drug-metabolizing P450s.

     

Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates,metoprolol, bufuralol and dextromethorphan

Abstract

• 1.?Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient.

• 2.?A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83–86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat.

• 3.?Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting.

• 4.?By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1′-hydroxylation.

• 5.?These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6.