Revealing mammalian evolutionary relationships by comparative analysis of gene clusters

Many software tools for comparative analysis of genomic sequence data have been released in recent decades. Despite this, it remains challenging to determine evolutionary relationships in gene clusters due to their complex histories involving duplications, deletions, inversions, and conversions. One concept describing these relationships is orthology. Orthologs derive from a common ancestor by speciation, in contrast to paralogs, which derive from duplication. Discriminating orthologs from paralogs is a necessary step in most multispecies sequence analyses, but doing so accurately is impeded by the occurrence of gene conversion events. We propose a refined method of orthology assignment based on two paradigms for interpreting its definition: by genomic context or by sequence content. X-orthology (based on context) traces orthology resulting from speciation and duplication only, while N-orthology (based on content) includes the influence of conversion events

Benzylmorpholine Analogs as Selective Inhibitors of Lung Cytochrome P450 2A13 for the Chemoprevention of Lung Cancer in Tobacco Users

The original publication is available at www.springerlink.comPURPOSE 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. METHODS Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is the major challenge in compound design. RESULTS A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. CONCLUSIONS Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure

Cytochrome P450 CYP1B1 interacts with 8-<i>methoxypsoralen</i> (8-MOP) and influences psoralen-Ultraviolet A (PUVA) sensitivity

Background: There are unpredictable inter-individual differences in sensitivity to psoralen-UVA (PUVA) photochemotherapy, used to treat skin diseases including psoriasis. Psoralens are metabolised by cytochrome P450 enzymes (P450), and we hypothesised that variability in cutaneous P450 expression may influence PUVA sensitivity. We previously showed that P450 CYP1B1 was abundantly expressed in human skin and regulated by PUVA, and described marked inter-individual differences in cutaneous CYP1B1 expression.Objectives: We investigated whether CYP1B1 made a significant contribution to 8-methoxypsoralen (8-MOP) metabolism, and whether individuality in CYP1B1 activity influenced PUVA sensitivity.Methods: We used E. coli membranes co-expressing various P450s and cytochrome P450 reductase (CPR) to study 8-MOP metabolism and cytotoxicity assays in CYP1B1-expressing mammalian cells to assess PUVA sensitivity.Results: We showed that P450s CYP1A1, CYP1A2, CYP1B1, CYP2A6 and CYP2E1 influence 8-MOP metabolism. As CYP1B1 is the most abundant P450 in human skin, we further demonstrated that: (i) CYP1B1 interacts with 8-MOP (ii) metabolism of the CYP1B1 substrates 7-ethoxyresorufin and 17-b-estradiol showed concentration-dependent inhibition by 8-MOP and (iii) inhibition of 7-ethoxyresorufin metabolism by 8-MOP was influenced by CYP1B1 genotype. The influence of CYP1B1 on PUVA cytotoxicity was further investigated in a Chinese hamster ovary cell line, stably expressing CYP1B1 and CPR, which was more sensitive to PUVA than control cells, suggesting that CYP1B1 metabolises 8-MOP to a more phototoxicmetabolite(s).Conclusion: Our data therefore suggest that CYP1B1 significantly contributes to cutaneous 8-MOP metabolism, and that individuality in CYP1B1 expression may influence PUVA sensitivity

CYTOCHROMES P450 AS THERAPEUTIC TARGETS AND COUNTER-TARGETS FOR THE PREVENTION OF LUNG CANCER AND TREATMENT OF STEROIDOGENIC DISEASES

Cytochrome P450 (CYP) is a superfamily of heme-containing monooxygenase enzymes that metabolize a variety of endogenous and exogenous substrates. These transformations can be advantageous in the role of homeostasis or clearance of foreign compounds. However, aberrant CYP activity or biotransformations of procarcinogens can be detrimental to human health. Thus cytochrome P450 enzymes can be both therapeutic targets and counter-targets. In the process of drug discovery, in vitro evaluation of both the efficacy and selectivity of drug candidates is necessary before in vivo studies can be pursued. In the case of the xenobiotic-metabolizing cytochrome P450 2A13 (CYP2A13), in vitro analysis was used to identify and evaluate selective inhibitors for reducing the risk of lung cancer in tobacco users. Additionally, in vitro biochemical analysis of the steroidogenic cytochromes P450 21A2 (CYP21A2) and 11B1 (CYP11B1) is being pursued for counter-target evaluation in the development of selective CYP17A1 inhibitors for the treatment of prostate cancer and the rational design of selective CYP11B1 inhibitors for the treatment of cortisol-dependent diseases. Lung cancer is the leading cause of all cancer related deaths and results in 6 million annual deaths worldwide. Since 80% of all lung cancer incidence is attributed to tobacco use but tobacco cessation methods are unsuccessful in 95% of users, an increased emphasis has been placed on lung cancer chemoprevention. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most prevalent procarcinogens compounds in tobacco and is selectively activated by CYP2A13 metabolism in the respiratory tract. The resulting diazonium ions are able to form DNA adducts and initiate lung cancer. Therefore, the selective inhibition of CYP2A13 offers a novel therapeutic strategy in the chemoprevention of lung cancer. High throughput screening identified the benzylmorpholine scaffold, and a small library was evaluated for both binding (Kd) and inhibition (Ki) of CYP2A13 versus the 94% identical hepatic cytochrome P450 CYP2A6 (CYP2A6), which does not efficiently metabolize NNK. These investigations identified the structural features of benzylmorpholine analogs responsible for selective binding and inhibition of CYP2A13 versus CYP2A6, leading to the determination of structure-activity relationships for the benzylmorpholine scaffold. Docking and X-ray crystallography studies were further employed to identify the atomic-level interactions between benzylmorpholine analogs and CYP2A13 but were hampered by apparent binding in multiple orientations. Nevertheless, these results could be used to design additional selective and potent CYP2A13 inhibitors for reducing the risk of lung cancer in tobacco users who are unable, unwilling, or in the process of ceasing tobacco use. In a similar pursuit to identify inhibitors of CYP17A1 for the treatment of prostate cancer, it became important to evaluate the selectivity of potential drug candidates against obvious counter-targets. CYP21A2 is involved in the biosynthesis of glucocorticoids and mineralocorticoids and has overlapping substrates with CYP17A1. CYP11B1 follows CYP21A2 in the steroid biosynthetic pathway and is also a counter-target for the development of CYP17A1 inhibitors. Additionally, CYP11B1's crucial role in cortisol production also presents this enzyme as an independent therapeutic target for the treatment of Cushing's disease resulting from cortisol overproduction. However, biochemical studies for both human CYP21A2 and CYP11B1 have been limited by protein availability. Human CYP21A2 was successfully cloned, expressed, purified, and crystallized for the first time, which allows for structural and functional studies of the human enzyme. CYP11B1 was also successfully cloned and expressed, but more optimization is necessary for consistent large-scale expression and purification. This work provides the necessary groundwork for a biochemical and biophysical understanding of both CYP21A2 and CYP11B1 for the evaluation of these enzymes as counter-targets. In addition these studies could lead to the rational design of CYP11B1 inhibitors for the treatment of cortisol dependent diseases

Cytochromes P450: Inhibition of CYP2A Enzymes Involved in Xenobiotic Metabolism and Generation of CYP26 Enzymes Involved in Retinoic Acid Metabolism

The cytochrome P450 (P450) superfamily of mixed function oxidase enzymes catalyze the metabolism of a variety of endogenous and exogenous biochemicals, including steroids, fatty acids, vitamins, eicosanoids, drugs, pesticides, and toxins. P450-mediated oxidative metabolism often serves a beneficial role in the clearance of foreign compounds and the regulation of endogenous molecules, both of which are necessary for the maintenance of homeostasis. However, the reactions catalyzed by P450 enzymes also have the potential to promote disease and injury, whether it be in the activation of procarcinogens or by undermining the therapeutic efficacy of a drug. Altogether, these processes make P450 enzymes important subjects of interest for the prediction of chemical toxicology and the development of therapeutic agents. The focus of this thesis research is the characterization of the CYP26 and CYP2A families of P450 enzymes in an effort to contribute to an understanding of how substrates and inhibitors specifically interact with the individual enzymes within these families. The functional enzymes of the human CYP2A family include CYP2A6 and CYP2A13. CYP2A6 is primarily a hepatic enzyme, while CYP2A13 is mainly expressed within the respiratory tract. Though these two enzymes are primarily localized to different tissues within the body, they share many substrates in common as a result of their 94% amino acid sequence identity. These substrates include: nicotine, cotinine, para-nitrophenol, and coumarin. However, CYP2A13 appears to preferentially activate a number of procarcinogens, including the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), into reactive intermediates that can result in DNA adducts and the initiation or promotion of carcinogenesis. In order to evaluate the relative involvement of CYP2A6 activity versus that of CYP2A13 in the metabolism and/or activation of potentially harmful chemical agents in vivo, selective inhibitors are needed. A number of compounds were reported to selectively inhibit CYP2A6 before CYP2A13 was determined to be a functional member of the CYP2A family, or have simply not been tested against CYP2A13. This work examined such compounds including: phenethyl isothiocyante (PEITC), 4-dimethylaminobenzaldehyde (DMABA), 8-methoxypsoralen (8-MOP), tranylcypromine, tryptamine, pilocarpine, (S)-nicotine, (R)-(+)-menthofuran, and β-nicotyrine. The relative impact of these inhibitors on CYP2A6 and CYP2A13 function was evaluated by determining Ki values and modes of inhibition for each of the compounds against both enzymes, followed by the calculation of a selectivity factor. The results of these studies serve as the first explicit determination of the selectivity of these compounds for enzymes within the CYP2A family and indicate that only (R)-(+)-menthofuran and tranylcypromine demonstrate even a 10-fold preference for CYP2A6 inhibition over CYP2A13. This information can be used as a guide for the selection of inhibitors with the greatest potential for determining whether CYP2A6 or CYP2A13 is responsible for the metabolism or activation of procarcinogenic compounds, such as tobacco-derived NNK in the human respiratory tract. Collectively, the CYP26 family of P450 enzymes serve as important mediators of retinoic acid (RA) catabolism in the body. In humans, the CYP26 family of enzymes consists of three isoforms: CYP26A1, CYP26B1, and CYP26C1. As a result of RA's endogenous role in regulating cellular growth and differentiation, geometric isomers of RA such as all-trans-RA (atRA) and 13-cis-RA represent attractive targets for cancer therapy and the treatment of dermatological conditions. Unfortunately, RA resistance is often experienced in patients undergoing prolonged RA-based therapy. This failure has been suggested to be the result of up-regulation of P450 enzymes, particularly the enzymes of the CYP26 family, resulting in enhanced RA metabolism in vivo. As a result, a significant amount of work has been conducted for the development of chemical agents that inhibit P450-mediated metabolism of atRA, commonly referred to as retinoic acid metabolism blocking agents (RAMBAs). The development of safe and effective RAMBAs could be greatly facilitated by more detailed structural and functional characterizations of the CYP26 family of enzymes, since very little is known about these enzymes aside from their dominant role in atRA hydroxylation. However, the fact that the human CYP26 enzymes are membrane-bound proteins, a characteristic shared by all human P450 enzymes, represents a major challenge to the recombinant expression and purification of these enzymes in sufficient quantities to enable detailed biochemical and biophysical characterization studies. To address this challenge, a new method for the production of pure CYP26 enzymes was developed based on techniques that have proven successful in the expression and purification of other membrane-bound P450 enzymes in E. coli, while incorporating the use of detergents and stabilizing ligands specific to the CYP26 enzymes. The combination of these approaches led to the successful generation of mouse CYP26A1 and CYP26B1 proteins in yields of 200-400 nmol per 2.25 L of E. coli expression media and the ability to recover 20-50 nmol following a single chromatographic step. Unfortunately, this protein appeared to be inactive according to reduced carbon monoxide difference spectrum analysis and assays of RA metabolism. As a result, further optimization will be required to reach the original goal set forth to generate pure recombinant CYP26 enzymes for basic kinetic and structural determination studies

The human cytochrome P450 2A family: Comparisons and identification of amino acids essential for substrate recognition

The goal of this research was to identify the differential structure-activity relationships between cytochromes P450 (CYP) 2A13 and 2A6 and their substrates. Cytochromes P450 2A13 and 2A6 are very closely related, having 94% amino acid sequence identity. Both proteins metabolize drugs, toxins, and procarcinogens, including the nicotine derivative 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1. Yet CYP2A13 has a much higher rate of metabolism than CYP2A6 for both compounds. Combined with its increased affinity and expression in the respiratory tract, CYP2A13 is a candidate for breakdown of NNK into the reactive metabolites that can form DNA adducts in the lung, leading to the development of lung cancer (1). Thus, understanding differential metabolism is important. Due to the high identity of CYP2A13 and CYP2A6, we hypothesized that a few amino acids found within the active site are responsible for CYP2A substrate specificity. Of the 32 amino acids which differ between CYP2A13 and CYP2A6, ten are located in regions of the protein that often determine substrate recognition in cytochromes P450 (2). Crystallization of CYP2A13 by the Scott lab verified that these amino acids were in or near the active site. The goal of this project was to determine which of these ten amino acids were vital for substrate selectivity in the CYP2A enzymes. Several different methods were employed throughout this study including site-directed mutagenesis, ligand binding assays, metabolism assays, molecular modeling, and crystallography. Site-directed mutagenesis was used to construct ten single residue CYP2A13 mutants with each mutation substituting the residue found in CYP2A6 in that position. The consequences of these ten mutations to the binding of coumarin, 2&#8242;-methoxyacetophenone, phenyl isothiocyanate, and phenacetin were determined using spectral ligand binding assays. The amino acids in positions 117, 208, 300, 301, 365, and 369 caused significant changes in ligand affinity. To determine if these amino acids were also essential for substrate metabolism, phenacetin was used as a structural probe since CYP2A6 converts phenacetin to acetaminophen with decreased catalytic efficiency compared to CYP2A13 (3). Employing this assay, four mutations (S208I, F300I, A301G, and G369S) were identified that diminished CYP2A13 phenacetin O-deethylation to near CYP2A6 activity. Two mutants, CYP2A13 A117V and L366I, both increased enzyme activity more than four-fold. Construction of the reverse mutant series, a CYP2A6 protein incorporating the residues found at the corresponding active site positions in CYP2A13, confirmed that positions 208, 300, 301, and 369 were jointly responsible for phenacetin metabolism and binding. While all single 2A6 mutants had very low-level activity, the double, triple, and a quadruple mutant with changes at these four positions increasingly conferred phenacetin metabolism to CYP2A6. A structural basis for the effects of these four mutations on phenacetin binding and metabolism was explored with molecular docking studies using Surflex-Dock (4). The results suggested that the ability of CYP2A13 to bind and metabolize phenacetin was due to steric variations among key residues in the CYP2A13 and CYP2A6 active sites. This was confirmed with the crystal structure of the CYP2A6 I208S/I300F/G301A/G369S complexed with phenacetin. Finally, this study characterized the effects of naturally occurring CYP2A13 polymorphisms on the binding of the well-known CYP2A ligand coumarin (5). All of these polymorphisms were located on the exterior portions of the protein. None of the polymorphisms examined resulted in a significant change in ligand affinity, indicating that having one of these variants would not substantially alter an individual's ability to metabolize CYP2A13 substrates. Thus, the research presented in this thesis provides structural and functional insight into CYP2A13 and CYP2A6 substrate selectivity, which is largely modulated by the steric effects mediated by the differential amino acids at positions 208, 300, 301, and 369. References 1. Su, T., Bao, Z. P., Zhang, Q. Y., Smith, T. J., Hong, J. Y., and Ding, X. X. (2000) Cancer Res. 60(18), 5074-5079 2. Zhang, J. Y., Wang, Y., and Prakash, C. (2006) Curr Drug Metab 7(8), 939-948 3. Bieche, I., Narjoz, C., Asselah, T., Vacher, S., Marcellin, P., Lidereau, R., Beaune, P., and de Waziers, I. (2007) Pharmacogenetics and Genomics 17(9), 731-742 4. Nishimura M Fau - Yaguti, H., Yaguti H Fau - Yoshitsugu, H., Yoshitsugu H Fau - Naito, S., Naito S Fau - Satoh, T., and Satoh, T. (0031-6903 (Print)) 5. Schlicht, K. E., Michno, N., Smith, B. D., Scott, E. E., and Murphy, S. E. (2007) Xenobiotica, 1-1

Interference of Homologous Sequences on the SNP Study of CYP2A13 Gene

Background and objective It has been proven that cytochrome P450 enzyme 2A13 (CYP2A13) played an important role in the association between single nucleotide polymorphisms (SNP) and human diseases. Cytochrome P450 enzymes are a group of isoenzymes, whose sequence homology may interfere with the study for SNP. The aim of this study is to explore the interference on the SNP study of CYP2A13 caused by homologous sequences. Methods Taqman probe was applied to detect distribution of rs8192789 sites in 573 subjects, and BLAST method was used to analyze the amplified sequences. Partial sequences of CYP2A13 were emplified by PCR from 60 cases. The emplified sequences were TA cloned and sequenced. Results For rs8192789 loci in 573 cases, only 3 cases were TT, while the rest were CT heterozygotes, which was caused by homologous sequences. There are a large number of overlapping peaks in identical sequences of 60 cases, and the SNP of 101 amino acid site reported in the SNP database is not found. The cloned sequences are 247 bp, 235 bp fragments. Conclusion The homologous sequences may interfere the study for SNP of CYP2A13, and some SNP may not exist