Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors.
Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11beta-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC(50) values of 1.6+/-0.1 and 9.9+/-0.9 nM (mean+/-SEM, n=3-6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K(i) values of 0.8+/-0.04 and 2.2+/-0.2 nM (n=3-4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.
LaSala
Daniel
D
Cardiovascular & Metabolism Disease Area Research, Novartis Institutes for BioMedical Research, East Hanover, NJ 07936, USA.
Shibanaka
Yasuhiko
Y
Jeng
Arco Y
AY
D000325:Adrenodoxin / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019405:Cytochrome P-450 CYP11B2 / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D015203:Reproducibility of Results; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000325:Adrenodoxin; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D005287:Ferredoxin-NADP Reductase