Pharmacogenetics of cytochromes P450 in tropical medicine

Drug response is affected by genetic and non-genetic factors, such as dietary compounds, sex, disease status and multiple drug therapy. Inherited determinants of drug disposition remain, however, the major cause of inter-individual differences due to pharmacogenetic polymorphism in drug metabolizing enzymes and transporters, or drug targets. Differences on ethnicity may have a profound impact on drug clearance, affecting the safety, efficacy and dosing regimen. In the context of tropical regions, the situation may be even more serious due to endemic infectious diseases and multiple drug therapy, which may affect drug clearance. In this review, we focus on the pharmacogenetics of the Cytochrome P450 superfamily, responsible for the highest contribution for variability among drug metabolizing enzymes, among ethnic groups from tropical settings.     

Induction of cytochromes P450

The induction of cytochromes P450 (CYPs) has been appreciated for some time but an understanding of the mechanisms involved has been poorly understood until recently. The discovery of the role of nuclear receptors such as the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) has provided a major trigger for research in this area. This work has provided an explanation for species differences in hepatic induction. The production of a PXR crystal structure in the presence and absence of known high affinity ligands has offered the possibility of predicting structures which may bind to the receptor and hence act as inducing agents in man. An improvement in the technology of hepatocyte culture, access to good quality human hepatocytes and the miniaturisation of cultured preparations has meant that the potential of this technique to predict induction in man has been realised. Molecular biological techniques have also proved essential in both the science and the quantitation of CYP induction. The use of transient transfection cell based systems coupled with reporter gene assays have meant that dose response curves can be generated for many chemicals. Assays have been developed to measure the increase of the corresponding CYP mRNAs in primary hepatocytes and some cell lines with a high degree of sensitivity and specificity (allowing the quantitation of closely related CYPs). Although CYP induction is not usually considered as a major drawback in drug development, the aim should be to eliminate or reduce the inducing effects of a new drug to a minimum. Thus, it is essential to increase our understanding of the complex mechanisms that regulate induction and to pay attention to both the dose and the physicochemical and structural properties of CYP inducing agents.     

Induction of cytochromes P450

Humans and rodents are exposed to many foreign compounds in their diet (e.g., herbal supplements such as St. John's wart), in their environment (e.g., organochlorine pesticides and polychorinated biphenyls), and as clinically prescribed drugs (e.g., rifampin and phenobarbital). In response to these exposures mammals have evolved mechanisms to induce proteins involved in xenobiotic detoxification. Metabolism by Phase I enzymes, particularly the heme containing monooxygenases cytochromes P450 is frequently the first line of defense against such xenobiotics.     

Cholesterol-metabolizing cytochromes P450.

By catalyzing the first steps in different pathways of cholesterol degradation, cytochromes P450 (P450s) 7A1, 27A1, 11A1, and 46A1 play key roles in cholesterol homeostasis. CYP7A1 is a microsomal liver-specific enzyme that converts cholesterol to 7alpha-hydroxycholesterol. CYP27A1 is a ubiquitously expressed mitochondrial P450 that metabolizes cholesterol to 27-hydroxycholesterol. CYP11A1 also resides in mitochondria but is expressed mainly in steroidogenic tissues, where it catalyzes the conversion of cholesterol to pregnenolone. Finally, CYP46A1 is a brain-selective microsomal monooxygenase producing 24S-hydroxycholesterol from cholesterol. Catalytic efficiencies of cholesterol-metabolizing P450s vary significantly and probably reflect physiological requirements of different organs for the rate of cholesterol turnover. P450s 7A1, 27A1, 11A1, and 46A1 represent a unique system for elucidation of how different enzymes have adapted to fit their specific roles in cholesterol elimination. Studies of cholesterol-metabolizing P450s suggest that their activities could be modulated post-translationally and that they should also be considered as targets for regulation of cholesterol homeostasis.     

Comparative modelling of cytochromes P450

The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.     

Pharmacophore modeling of cytochromes P450

Understanding the binding of ligands in the active site of a membrane-bound protein is difficult in the absence of a crystal structure. When these proteins are the enzymes involved in drug metabolism, it leaves little option but to use site-directed mutagenesis and in vitro studies to provide critical information relating to determinants of binding affinity. Pharmacophore models and three-dimensional quantitative structure-activity relationships have been used either alone or in combination with protein homology models to provide this information for cytochrome P450s. At present, their application has been directed to the major enzymes but this may escalate in future as more in vitro data are generated for other P450s. The following review outlines the methodologies and models as well as future prospects for applying these technologies to P450s in the hope that future drugs will be selected with increased metabolic stability and fewer incidences of undesirable drug-drug interactions.     

Small intestinal cytochromes P450.

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.     

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.     

Characterization of orphan human cytochromes P450

Of the 57 human cytochromes P450 (P450) and 58 pseudogenes discovered to date, (http://drnelson.utmem.edu/CytochromeP450.html ), 1/4 still remain "orphans" in the sense that their function, expression sites, and regulation are still largely not elucidated. The post-human genome-sequencing project era has presented the research community with novel challenges. Despite many insights gathered about gene location and genetic variations in our human genome, we still lack important knowledge about these novel P450 enzymes and their functions in endogenous and exogenous metabolism, as well as their possible roles in the metabolism of toxicants and carcinogens. Our own list of such orphans currently consists of 13 members: P450 2A7, 2S1, 2U1, 2W1, 3A43, 4A22, 4F11, 4F22, 4V2, 4X1, 4Z1, 20A1, and 27C1. Some of the orphans, e.g. P450s 2W1 and 2U1, already have putative assigned functions in arachidonic acid metabolism and may activate carcinogens. However, at this point, for the majority of them more knowledge is available about their genes and single nucleotide polymorphisms than of their biological functions. It is noteworthy that most P450 orphans express high interspecies sequence conservation and have orthologs in rodents (e.g. CYP4X1/Cyp4x1, CYP4V2/Cyp4v3). This review summarizes recent knowledge about the P450 orphans and questions remaining about their specific roles in human metabolism.     

The human drug metabolizing cytochromes P450

The superfamily of heme-thiolate proteins known as the cytochromes P450 is responsible for the oxidative metabolism of the majority of drugs. Thus, the phenotypes of individuals with respect to their levels of catalytically active cytochromes P450 determines to a large part the substantial interindividual variation observed in the metabolic clearance of drugs. Over the past 10 years 15 different human cytochromes P450 involved in drug metabolism have been isolated and characterized to varying degrees. This brief review discusses the characterization of these cytochromes P450 and how this knowledge has been used by the pharmaceutical industry to aid in the development of new drugs.     

肠道细胞色素P450代谢酶研究进展

细胞色素P450(CYP450s)为肠道主要I相代谢酶,目前发现有CYP1A1、CYP2C9、CYP2C19、CYP2J2、CYP2D6、CYP3A4、CYP3A5 7种同工酶。肠道CYP在药物代谢及药物相互作用中发挥重要作用,与药物疗效及不良反应密切相关。基因多态性及个体差异均影响药物代谢,导致临床疗效差别。该文就肠道CYP各亚型相关研究进展做一综述。     

Endo-xenobiotic crosstalk and the regulation of cytochromes P450

Evolution has provided organisms with an elaborate defense system against foreign compounds and against the accumulation of potentially toxic endogenous molecules, e.g. bile acids. Cytochromes P450 represent an important group of enzymes in this system. This article describes experiments started in the 1970's in Dallas on the coordination of heme and cytochrome P450 synthesis and how these studies evolved over the years into a concept of molecular links between xenobiotic metabolism and endogenous pathways of sterol, lipid, bile acid and energy homeostasis.     

细胞色素P450酶在癌症研究中的应用

细胞色素P450酶是体内代谢转换的重要酶系.现从细胞色素P450酶在活性调节、代谢表型、致癌 物活化、抗癌药物代谢、肿瘤组织中特异表达和肿瘤基因治疗等方面综述细胞色素P450酶的特点及其在癌症研究中的应用潜力.     

Structural modelling of the human drug-metabolizing cytochromes P450

The structural and functional aspects of cytochrome P450 (CYP) enzymes are reviewed in the light of current developments in X-ray crystallography and other physical evidence, together with recent findings on the regulation of, and polymorphisms in, the human drug-metabolizing CYPs. It is emphasized that the crystal structures of eukaryotic CYPs are particuarly useful for constructing homology models of the human enzymes associated with drug metabolism, and that these models can aid in the high-throughput screening of novel compounds destined for human exposure.     

Crystallization of cytochromes P450 and substrate-enzyme interactions

Our understanding of structure-function relationships have made considerable advances owing to the increasing number of new P450 crystal structures. This is especially true with mammalian P450s. As always, the main bottleneck in a structure determination project is crystallization. While the crystallization techniques used for P450 crystal growth are not much different from that utilized for other proteins, special protein engineering strategies have been developed in order to generate soluble, homogeneous membrane-bound P450 samples amendable for crystallization. Newly determined P450 structures also provide convincing evidence that P450 enzymes are highly dynamic and flexible. Common structural elements found in all P450s have been identified that undergo large conformational changes to allow substrate access and product release. In addition, flexible regions may enable the active site to adapt to the binding of substrates of different size, shape, and polarity. This review will focus on the successful membrane P450 crystallization techniques and the new structural insights based on the growing P450 structure database.     

Mechanistic enzymology of oxygen activation by the cytochromes P450

The P450 cytochromes represent a universal class of heme-monooxygenases. The detailed mechanistic understanding of their oxidative prowess is a critical theme in the studies of metabolism of a wide range of organic compounds including xenobiotics. Integral to the O2 bond cleavage mechanism by P450 is the enzyme's concerted use of protein and solvent-mediated proton transfer events to transform reduced dioxygen to a species capable of oxidative chemistry. To this end, a wide range of kinetic, structural, and mutagenesis data has been accrued. A critical role of conserved acid-alcohol residues in the P450 distal pocket, as well as stabilized waters, enables the enzyme to catalyze effective monooxygenation chemistry. In this review, we discuss the detailed mechanism of P450 dioxygen scission utilizing the CYP101 hydroxylation of camphor as a model system. The application of low-temperature radiolytic techniques has enabled a structural and spectroscopic analysis of the nature of critical intermediate states in the reaction.     

Bioinformatic insight into the unity and diversity of cytochromes P450

For the past few decades, cytochromes P450 (CYPs) have been the subject of extensive research, owing to the ability of these enzymes to serve as drug targets as well as to their active participation in drug metabolism. Other varieties and functions of CYPs have been discovered and this superfamily currently comprises over 2000 different protein species. In the present study, the protein sequences of CYPs were submitted to computer analysis for elucidation of the structural basis of their pronounced functional diversity. The basic local alignment search tool (BLAST) was used to demonstrate that CYP protein sequences share a certain general similarity; at the same time, it was shown that the CYP superfamily may be split into a number of groups of intimately related proteins. These groups, the families, were revealed by means of cluster analysis, which demonstrated a strong hierarchy among the animal, bacterial and fungal P450s, and the lack of such a hierarchy for plant enzymes. Multiple alignment and consensus sequence analysis were the approaches taken to find out which structural peculiarities of P450s are responsible for the deviations from the random picture. Proteins within each family were aligned and collapsed to the corresponding consensus sequences, the alignment of which produced the consensus for the whole superfamily. The latter consensus yielded a number of unity motifs (most of which being related to the heme-fixing assembly), while the cross-family comparison of consensus sequences enabled the retrieval of some diversity motifs. Three consensus sequences (for the CYP51 and CYP2 families and for the superfamily) were compared, to line up the unity and diversity motifs with the appropriate X-ray data.