Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2.
First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively. Using a statistic experimental design, only 36 assays were needed to be exhaustive. Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent. Two factors had no significant effect: total ionic concentration and catalase/reduced glutathione. Six factors influenced CYP1A2-mediated MROD rates: buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol. Four factors had no significant effect: total ionic concentration, cytochrome b5, reductase, and NAD+. Secondly, the combined effects of ionic strength and Mg concentration on NIF/CYP3A4 were studied and easily modeled using another statistic experimental design. The effect of Mg was strong at a constant ionic strength of 100 mM and negligible at a constant ionic strength of 500 mM. Thirdly, the effects of influencing factors and their interactions on MROD/CYP1A2 were modeled after 40 assays using a third statistic experimental design. Later experiments confirmed the predictivity of the models and the efficiency of optimized conditions. This approach can be applied to other biochemistry areas.
Bournique
B
B
Rhône-Poulenc Rorer, Drug Metabolism and Pharmacokinetics, and Pharmaceutical Sciences, 13 Quai Jules Guesdes, Vitry s/Seine Cedex, 94403, France. bruno.bournique@rp-rorer.fr
Petry
M
M
Gousset
G
G
D001699:Biometry; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D008274:Magnesium