ELSEWER SClENTWlC PUBLlSHERS lRELAND Toxicology 82 (1993) 39-52 Recombinant yeast in drug metabolism J.P. C. Renaud*a, M.A. Peyronneaua, P. Urbanb, G. Truanb, L Cullin b, D. Pompon ‘, Ph. Beaune’ and D. Mansuya zyxwvutsrqponmlkjihgf “Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS URAQOO, Paris France ‘Centre de GPnCtique MolPculaire, CNRS UPR2420, Gifsur- Yvette France ‘INSERM U75, Centre Hospitalo-Universitaire Necker-Enfants M alades, Paris France Abstract The usefulness of cDNA-directed expression of human hepatic P45Os in yeast for the in vitro study of drug metabolism is emphasized. The major advantages of yeast expression are: (i) relatively high yields of heterologous P450 (= 5- 10 nmol/l of culture medium) can be obtained; (ii) the expressed P45Os are directly active in yeast microsomes, allowing the deter- mination of specific catalytic activities of individual isoforms, which is a prerequisite for the prediction of metabolic pathways for new drug candidates; (iii) transformed yeast microsomes can also be used to study the specific affinity of individual P45Os for various substrates and the formation of P450-metabolite complexes by difference visible spectroscopy; such studies can help to predict drug interactions. The advantages of expression in yeast with respect to biochemical studies of drug metabolism are illustrated with data about P450 NF25 (P450 3A4), the major form of human liver. Expressed P450 NF25 is obtained in a functionally ac- tive state, and some specific catalytic activities observed in liver microsomes could be reproduced directly with transformed yeast microsomes. The use of genomically modified yeast strains coexpressing human cytochrome bS and/or overexpressing yeast P450-reductase allowed us to optimize these catalytic activities. In particular, this coexpression system was useful in the study of the in vitro formation of a P450 NF25 Fe(II)-RN0 complex. Such inhibitory complexes have been implied in numerous drug interactions involving P450 3A4. Key words: Drug interactions; Drug metabolism; NADPH-P450 reductase; P450 3A4; P450- metabolite complex; Yeast expressed * Corresponding author: UPR de Biologie Structurale du CNRS, IBMC, 15 Rue Descartes, 67084 Strasbourg Cedex, France. Abbreviations: cyt. b,, cytochrome bg; GALIO-CYCI promoter, hybrid promoter composed of the yeast GAL10 gene upstream activating sequence and the iso-I-cytochrome c gene transcription initiation sequence; NADPH-P450 reductase; P450 reductase, V8, YeDPI/8-2 plasmid; V60, YeDP60 plasmid.