Mechanism‐Based Inactivation of Bovine Cytochrome P‐450U_11β by 18‐Unsaturated Progesterone Derivatives

Abstract

Two 18-unsaturated progesterone derivatives, 18-vinylprogesterone (18-VP) and 1 8-ethynylprogesterone (1 8-EP) have proved to be potent inhibitors of the bovine cytochrome P-450, ,/!, the enzyme involved in the last steps of aldosterone biosynthesis [Delorme, C., Piffeteau, A., Viger, A. & Marquet, A. (1995) E m J. Biochem. 232, 247-2561. In the present study, we demonstrate that these two compounds exhibit the characteristics of mechanism-based inactivators of this enzyme. Inactivation followed pseudo-firstorder and saturation kinetics. The kinetic parameters of inactivation were k, = 0.11 min-' and K, = 4 pM for 18-VP, and k, =0.12 min-' and 22 pM for 18-EP. Inactivation of P-450,,, activity was strictly dependent on the presence of NADPH. Protection by the substrate deoxycorticosterone was observed, demonstrating a selective modification at the substrate-binding site. With radiolabeled 18-VP, inactivation was shown to be irreversible with a stoichiometry of 1.4 mol bound ['H]18-VP/mol inactivated cytochrome P-45OlIp. SDS/PAGE analysis of the [3H] 18-VP-inactivated enzyme showed that, under conditions preventing heme dissociation, the P-450,,, band was labeled, while no labeling of the apoprotein was observed under denaturating conditions. Furthermore, the loss of catalytic activity could be correlated with the destruction of the P-450 chromophore evaluated by the Fe"-CO versus Fe" difference spectra. These arguments led us to propose that 18-vinylprogesterone inactivates cytochrome P-450, , , by heme destruction rather than by protein modification.Keywords: cytochrome P-450,,,; mechanism-based inactivator; aldosterone biosynthesis ; 18-vinylprogesterone; 18-ethynylprogesterone.Aldosterone overproduction leads to oedematous diseases and hypertension (Corvol et al., 1990). These disorders are clinically treated with spirolactones, which act as antagonists at the receptor level. We developed an approach focusing on the inhibition of cytochrome P-450, (P-450,,,), an enzyme implied in the last steps of aldosterone biosynthesis. For this purpose, several progesterone analogs, designed as mechanism-based inhibitors of P-450,,,, were synthesized in our laboratory (Viger et al., 1988(Viger et al., , 1989.These compounds could potentially belong to the three wellknown classes of inactivators of cytochrome P-450, i.e. those binding covalently to the prosthetic, heme group, those binding to the protein, and those coordinating quasi-irreversibly the iron atom (Ortiz de Montellano, 1984). Among them, two 18-unsaturated progesterone derivatives, 18-vinylprogesterone (1 8-VP) and 18-ethynylprogesterone (18-EP; Fig. 1) were the most potent inhibitors tested on a rat adrenal cell-free extract since they completely inhibited aldosterone production at concentrations of 0.8 pM and 8 pM, respectively (Viger et al., 1989 Enzyme. Steroid 1 ID-monooxygenase (EC 1.14.15.4).ined the inhibition of P-450 11P-hydroxylase and 18-hydroxylase activities with a reconstituted enzyme system with a purified bovine P-450,,,) preparation containin...

Overcoming Undesirable CYP1A2 Inhibition of Pyridylnaphthalene-Type Aldosterone Synthase Inhibitors: Influence of Heteroaryl Derivatization on Potency and Selectivity

“…Spironolactone, however, showed severe side effects, presumably due to its steroidal structure. , Although the development of nonsteroidal aldosterone receptor antagonists has been reported recently, several issues associated with the unaffected and pathophysiologically elevated plasma aldosterone levels remain unsolved by this therapeutic strategy such as the up-regulation of the mineralocorticoid receptor expression and nongenomic aldosterone effects . A novel approach for the treatment of diseases affected by elevated aldosterone levels is the blockade of aldosterone biosynthesis by inhibition of CYP11B2. , Aldosterone synthase has previously been proposed as a potential pharmacological target, and preliminary work focused on the development of steroidal inhibitors, i.e., progesterone and deoxycorticosterone derivatives with unsaturated C 18 -substituents. These compounds were found to be mechanism-based inhibitors binding covalently to the active site of bovine CYP11B, however, data on inhibitory action toward human enzyme are essentially absent in these studies.…”

Acetylenes: cytochrome P450 oxidation and mechanism-based enzyme inactivation

“…Blakey and White 1986White 1978, 1981Ortiz de Montellano et al 1979;Blakey and White 1986White 1978Guengerich 1990Delorme et al 1997 Ortiz de Montellano Page 43 Table 3.…”