Synthesis of new inhibitors of aldosterone biosynthesis

Viger, Antoinette; Coustal, Suzy; Pérard, Serge; Marquet, Andrée

doi:10.1016/s0040-4020(01)85892-0

Cited by 18 publications

(10 citation statements)

References 16 publications

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confidence: 99%

“…We developed a new approach namely by focusing on the inhibition of cytochrome P-4501,, (P-450,,,), the last enzyme in aldosterone biosynthesis. For this purpose, progesterone derivatives designed as mechanism-based inhibitors of P-4501,, were synthesized in our laboratory (Viger et al, 1988).The most potent inhibitors, tested with rat adrenal cell-free extracts, were 18-vinylprogesterone (1 8-VP) and 18-ethynylprogesterone (18-EP) (Fig. 1) which completely inhibit aldosterone production at 0.8 pM and 8 pM, respectively (Viger et al, 1989).…”

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Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Delorme¹,

Piffeteau²,

Viger³

et al. 1995

European Journal of Biochemistry

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The last step of aldosterone biosynthesis, an 1 1p-hydroxylation followed by two 18-hydroxyIations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-45Oll, (deoxycorticosterone (DOC) -corticosterone -18-hydroxycorticosterone -aldosterone). The 1 lp-and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 1 l-de~xy Steroid Biochem. 33, 119-1241, were characterized for both activities (llp-and 18-hydroxylase). The value of reversible K, for the 18-hydroxylation (Ki = 5 pM for 18-vinylprogesterone and 30 pM for 18-ethynylprogesterone) is lower than that for the 1lp-hydroxylation (30 pM and 100-150 pM, respectively); the former inhibitor is stronger than the latter for both steps.The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18-Vinylprogesterone gave rise to a type I spectrum with a K, value of 35 pM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher K, value (140 pM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.Keywords: cytochrome ; P-45OI1,; mechanism-based inhibitors ; aldosterone biosynthesis ; 18-vinylprogesterone.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 a new approach namely by focusing on the inhibition of cytochrome P-4501,, (P-450,,,), the last enzyme in aldosterone biosynthesis. For this purpose, progesterone derivatives designed as mechanism-based inhibitors of P-4501,, were synthesized in our laboratory (Viger et al., 1988).The most potent inhibitors, tested with rat adrenal cell-free extracts, were 18-vinylprogesterone (1 8-VP) and 18-ethynylprogesterone (18-EP) (Fig. 1) which completely inhibit aldosterone production at 0.8 pM and 8 pM, respectively (Viger et al., 1989). In this study the inhibitions by 18-VP and 18-EP were studied more accurately using a bovine reconstituted enzymic P-450,,,, P-450,,, and P-450,,,,, cytochromes P-450 1 lP-18-hydroxylase, P-450 cholesterol side-chain cleavage, P-450 17a-hydro~ylase/C,,,~, lyase and P-450 aromatase.Enzyme. Steroid 1 ID-monoxygenase; cytochrome P-450, ,8 (EC 1.14.15.4). system including P-45011,, adrenodoxin and adrenodoxin reductase.P-450,,, purified from bovine adrenal cortex can catalyze the 1 lp-hydroxylation of 11 -deoxycorticosterone, the 18-hydroxylation of corticosterone as well as aldosterone formation (Fig. 2) (Wada et al., 1985;Yanagibashi et al., 1986). However two distinct forms of P-45Ol,,, encoded by two different genes exist in the bovine adrenal cortex (for a review, see Muller, 1993). Indeed two bovine P-45011, cDNAs, pc P-450 118-3 and pc P-450 llp-2 were cloned (Moroashi et al., 1987;Kirita et al., 1988). Expression into COS-7 cells revealed that both enzymes were capable of catalyzin...

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Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Delorme¹,

Piffeteau²,

Viger³

et al. 1995

European Journal of Biochemistry

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“…18-Hydroxycorticosterone and 18-hydroxy-1 l-deoxycorticosterone were obtained from Sigma. 18-VP and 18-EP were synthesized as described previously (Viger et al, 1988). 18-Hydroxy-18-vinylprogesterone (18-OHVP) and were synthesized in our laboratory (Coustal et al, 1995).…”

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confidence: 99%

“…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).…”

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

Delorme

Piffeteau

Sobrio³

et al. 1997

European Journal of Biochemistry

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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...

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ChemInform Abstract: Synthesis of New Inhibitors of Aldosterone Biosynthesis.

et al. 1988

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Synthesis of new inhibitors of aldosterone biosynthesis

Cited by 18 publications

References 16 publications

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

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

ChemInform Abstract: Synthesis of New Inhibitors of Aldosterone Biosynthesis.

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Synthesis of new inhibitors of aldosterone biosynthesis

Cited by 18 publications

References 16 publications

Inhibition of Bovine Cytochrome P‐45011β by 18‐Unsaturated Progesterone Derivatives

Inhibition of Bovine Cytochrome P‐45011β by 18‐Unsaturated Progesterone Derivatives

Mechanism‐Based Inactivation of Bovine Cytochrome P‐450U11β by 18‐Unsaturated Progesterone Derivatives

ChemInform Abstract: Synthesis of New Inhibitors of Aldosterone Biosynthesis.

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Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

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