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Characterisation of cytochrome P450s

Cytochrome P450s - biological significance

The CYPs are a superfamily of mixed-fucntion oxidases, members of which are present in virtually all living organisms.  CYPs are characterised into families and sub-families by their sequence similarities.  There are 103 isoforms identified in mouse and 57 in humans, predominantly expressed in liver, but also specific forms occurring in other tissues, including tumours.  Each member can also be characterised by their substrate (endogenous and foreign metabolites) specificity (http://medicine.iupui.edu/flockhart/table.htm). CYPs are thought to have evolved, in part, as a protective adaptive response against the toxic effects of environmental chemicals [1]. They are the most important drug metabolizing enzymes in mammals, and, in humans, are responsible for the phase I metabolism of 70-80% of all clinically used drugs [2]. Essentially, by monitoring the changes in protein expression profile of CYPs, the effect of drugs on target tissues can be determined, and hypotheses proposed as to the likely mechanisms of drug action. In addition to their detoxification role, CYPs can also be responsible for the conversion of chemical toxins and procarcinogens to their toxic or carcinogenic forms [3]. The ability of CYPs to activate chemical toxins has been exploited in cancer chemotherapy, where several anticancer drugs, including cyclophosphamide, tamoxifen and banoxantrone, are known to require metabolic CYP activation in order to exert their cytotoxic effects [4].

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Cytochrome P450s - commercial significance

Because of the ability to metabolise drugs, CYPs have huge significance in pharmaceutical research [5].  The action of CYPs is one of the major causes of adverse drug reactions to many marketed drugs and drug-combination therapies and many failures of novel drugs during their development have been attributed to their interactions with this class of enzymes.  For example, drugs may be metabolized too rapidly by CYPs before they have had time to be effective, or they may be metabolized into smaller molecules which are toxic beyond their site of proposed action.  Certain drugs may also inhibit the activity of a CYP enzyme which is involved in the metabolism of another drug that, given at the same time, can become elevated in the patient to levels which can cause side effects or become dangerous.

There is now the potential to use a subjects' CYP profile to develop personalised medicines.  A subjects' genotype may impact on the pharmacodynamics (drug concentration versus time versus pharmacological effect), pharmacokinetics (absorption, distribution, metabolism and excretion) and/or the incidence of adverse events.  The use of pharmacogenetic tests to determine this genetic variation can facilitate correct drug selection for treatment efficacy and minimise adverse effects.  Up to 10 per cent of the population, for example, do not experience any pain relief from codeine due to the presence of one specific form (CYP2D6) in their livers.  Different forms of  the same enzyme are believed to be responsible for the very variable response to antidepressants such as Prozac.

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Cytochrome P450s - biochemical properties 

Cytochrome P450s have proven difficult to handle by traditional proteomics strategies, such as 2D gels [6] - the proteins are membrane associated, have pls between 8.5 and 9.5 and intact molecular weights between 55,000 and 61,000 Da.  Traditional approaches to the analysis of CYP enzymes rely on immunoblotting, activity assays and the detection of CYP mRNA.  These techniques have significant limitations: not only do they require pre-selection of the CYP enzymes to be investigated, so that if an unknown or unexpected CYP were present in a sample it would be overlooked, but they are unable to provide reliable quantitative information.  Immunoblotting, whilst being very sensitive, relies on the availability of enzyme-specific antibodies; this specificity can be confounded by the high degree of sequence homology between members of the same subfamily.  After pre-selection of the CYPs to be investigated, each enzyme must be identified in turn.  Since different CYP enzymes exhibit a broad, and often overlapping substrate specificity, activity assays that are designed to interrogate the activity a CYP enzyme may not be totally enzyme-specific in addition, multiple analysis techniques are invariably required, since different assays must be developed for different target substrates.  Measurement at the expression level is fraught with uncertainty since the presence and abundance of a particular type of mRNA does not necessarily infer the presence and abundance of the corresponding protein. [7-9].

A method that was able to non-selectively identify the CYP enzymes present in diseased tissue and could be developed to quantify expression levels relative to that of surrounding normal tissue, would provide invaluable information regarding the metabolic fate or potential therapeutic efficacy of the selected drugs and hence the outcome of therapy.

As part of an investigation of methods for profiling CYPs, we used SDS PAGE to enrich the enzymes, all having electrophoretic mobilities between 46 and 52 kDa, from mouse liver microsomes and subsequently identified 26 isoforms using in-gel digestion, nanoHPLC, MALDI MS and database searching [10].

References

[1] Lewis, D. F., Watson, E., Lake, B. G., Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics. Mutat Res 1998, 410, 245-270.
[2] Evans, W. E., Relling, M. V., Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999, 286, 487-491.
[3] Wolf, C. R., Metabolic factors in cancer susceptibility. Cancer Surv 1990, 9, 437-474.
[4] Patterson, L. H., Murray, G. I., Tumour cytochrome P450 and drug activation. Curr Pharm Des 2002, 8, 1335-1347.
[5] Yuan, R., Madani, S., Wei, X. X., Reynolds, K., Huang, S. M., Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos 2002, 30, 1311-1319.
[6] Galeva, N., Altermann, M., Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes: cytochromes P450 and other membrane proteins. Proteomics 2002, 2, 713-722.
[7] McFadyen, M. C., Rooney, P. H., Melvin, W. T., Murray, G. I., Quantitative analysis of the Ah receptor/cytochrome P450 CYP1B1/CYP1A1 signalling pathway. Biochem Pharmacol 2003, 65, 1663-1674.
[8] Gygi, S. P., Rochon, Y., Franza, B. R., Aebersold, R., Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999, 19, 1720-1730.
[9] Scharf, M. E., Neal, J. J., Marcus, C. B., Bennett, G. W., Cytochrome P450 purification and immunological detection in an insecticide resistant strain of German cockroach (Blattella germanica, L.). Insect Biochem Mol Biol 1998, 28, 1-9.
[10] Sutton, C. W., Sutherland, M., Shnyder, S., Patterson, L. H., Improved preparation and detection of cytochrome P450 isoforms using mass spectrometric methods. Proteomics 2009.
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