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.
Mitchell
Michael D
MD
Drug Safety Research and Development, Pfizer Global Research and Development, 700 Chesterfield Parkway West T1A, Chesterfield, Missouri 63017, USA.
Elrick
Mollisa M
MM
Walgren
Jennie L
JL
Mueller
Richard A
RA
Morris
Dale L
DL
Thompson
David C
DC
D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001711:Biotransformation; D051544:Cytochrome P-450 CYP3A / Q000235:genetics / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D011994:Recombinant Proteins; D006735:Horseradish Peroxidase; D009195:Peroxidase; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D000444:Aldehyde Dehydrogenase