18-Vinyldeoxycorticosterone: a potent inhibitor of the bovine cytochrome P-450(11) beta.

18-Vinylprogesterone (18-VP) and 18-ethynylprogesterone (18-EP) have proved to be potent suicide inhibitors of P-450(11) beta, the last enzyme of aldosterone biosynthesis (Delorme, C.; Piffeteau, A.; Viger, A.; Marquet, A. Eur. J. Biochem. 1995, 232, 247; Delorme, C.; Piffeteau, A.; Sobrio, F.; Marquet, A. Eur. J. Biochem. 1997, 248, 252). This paper describes the synthesis of 18-vinyldeoxycorticosterone (18-VDOC), an analogue of deoxycorticosterone (DOC), the physiological substrate of the enzyme, and the evaluation of its reversible inhibiting properties for deoxycorticosterone and corticosterone oxidation by the bovine enzyme. 18-VDOC has been obtained by hydroxylation at C-21 of a 18-VP precursor. Its reversible Ki values are, respectively, 0.3 microM for the 11 beta-hydroxylation and 0.8 microM for the 18-hydroxylation. Hence, 18-VDOC is the strongest competitive inhibitor of bovine P-450(11) beta described so far, but in contrast with 18-VP, it does not inhibit more efficiently the 18-hydroxylation than the 11-hydroxylation.

Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21.

The ability of a glucocorticoid (triamcinolone acetonide: TA) and three progesterone derivatives with photoreactive groups at different positions (promegestone: R5020; 18-oxo-18-vinylprogesterone: 18OVP; 21-diazoprogesterone: 21DP) to bind covalently to the human mineralocorticoid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimentation gradient analysis and exchange assays with aldosterone showed that [3H]TA, a partial mineralocorticoid agonist, and [3H]R5020, a pure antimineralocorticoid, were covalently bound to hMR after UV irradiation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradiated [3H]TA- and [3H]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band labelled by [3H]TA or [3H]R5020, having a molecular mass of 120 kDa. This band was not detected in the presence of an excess of the corresponding unlabelled steroid or when the cytosol was recovered from non-infected Sf9 cells. Electrophoresis of a truncated hMR (hMRDelta(1-351)) photolabelled with [3H]TA revealed a 80 kDa band, compatible with the molecular mass of the truncated hMR. Limited chymotrypsin proteolysis of the [3H]TA photolabelled hMR generated a 30 kDa fragment covalently associated with [3H]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S. Jalaguier, M. Lombès, A. Souque, M.E. Rafestin-Oblin, Biochem. J. 315 (1996) 421-427), the present experiments provide evidence that [3H]TA is covalently bound to the ligand binding domain of the hMR. Exchange assays with [3H]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are covalently bound to hMR with an approx. 30-35% labelling efficiency.

New steroidal diazo ketones as potential photoaffinity labeling reagents for the mineralocorticoid receptor: synthesis and biological activities.

Three diazo ketones in the progesterone series were synthesized as potential photoaffinity reagents. The diazo ketone group was introduced at the C17 (21-diazopregn-4-ene-3,20-dione, 1) or C13 (18-(diazomethyl)-20-hydroxypregn-4-ene-3,18-dione, 2, 18-(diazomethyl)pregn-4-ene-3, 18,20-trione, 3) position of the pregnene skeleton. Whereas compound 1 could be easily obtained from the corresponding acid chloride, preparation of 2 and 3 required a less straightforward route involving reaction of tosyl azide on the formyl derivative of methyl ketone 5. The affinity of the diazo ketones for the human mineralocorticoid receptor (hMR), expressed in Sf9 insect cells using the Baculovirus system, was estimated by competition experiments using [3H]aldosterone as specific ligand. The affinity of 1 for hMR was almost identical with that of aldosterone. The affinities of 2 and 3 were 1, order of magnitude lower than that of aldosterone. The mineralocorticoid activity of the diazo ketones was measured in cis-trans cotransfection assays in CV-1 cells with the mouse mammary tumor virus as DNA target sequence. Compound 1 exhibits an agonist activity (ED50 = 6 x 10(-9) M) with no antagonist activity. In contrast 2 and 3 behave as antagonists, displaying an IC50 of approximately 10(-6) M whether the substituent at the C20 position is a hydroxy (2) or an oxo (3) group.

The mineralocorticoid activity of progesterone derivatives depends on the nature of the C18 substituent.

To investigate the role of the C18 substituents in the agonist/antagonist properties of mineralocorticoids, the activities of certain C18-substituted progesterone (P) derivatives were examined. These compounds were characterized by an unsaturated side-chain in the case of 18-vinylprogesterone (18VP) and 18-ethynylprogesterone (18EP) and by an enone group in the case of 18-oxo-18-vinylprogesterone (18OVP). P and its 18-substituted derivatives bind to the recombinant human MR (hMR) overexpressed in Sf9 cells with the following hierarchy of affinity: P > aldosterone > 18VP > 18EP >> 18OVP. Functional cotransfection assays in CV-1 cells, using mouse mammary tumor virus promoter as a steroid receptor-inducible DNA target sequence, indicated that the mineralocorticoid activity depends on the nature of the C18 substituent. 18VP and 18EP retained the antimineralocorticoid feature of P, with the following order of activity: P = 18VP > 18EP. The antagonist potency of 18VP was higher (IC50, approximately 10(-8) M) than that of spironolactone (IC50, approximately 7 x 10(-8) M), the most widely used aldosterone antagonist. Interestingly, introducing an oxo function at C18 conferred agonist mineralocorticoid properties; 18OVP behaves as a full agonist (ED50, approximately 10(-7) M) with no antagonist activity. In contrast to what was observed when the three 18-substituted P derivatives acted through hMR, they retained the agonist feature of P through the human P receptor, with the following order of potency: P > 18VP = 18OVP > 18EP. The activity of the 18-substituted P derivatives through the human glucocorticoid receptor was only detected at concentrations higher than 10(-6) M; P and 18VP displayed a partial antagonist activity, whereas 18OVP had a full agonist activity (ED50, approximately 2 x 10(-6) M). Thus, the presence of an oxo group at C18(18OVP) does not change the agonist feature of P through human P receptor, but confers to the ligand an agonist activity through hMR, suggesting that the C18 carbonyl group of aldosterone plays a crucial role in its agonist activity.

An improved synthesis of 18-norandrost-4-ene-3,17-dione.

We describe the synthesis of 13 beta- and 13 alpha-H-18-nor-androst-4-ene-3,17-dione (1a and 1b) from 18-hydroxyprogesterone (18-->20) hemiketal, via the 18-acetoxy-17 beta-hydroxyandrost-4-en-3-one formed by a modified Baeyer-Villiger reaction. Saponification of 18-acetoxyandrost-4-ene-3,17-dione with sonication, then retroaldolization in the presence of a formaldehyde trap, methone, afforded the mixture of 1a and 1b with 80% yield in a "one-pot" procedure and at room temperature. This yield was greatly improved, compared with the already published procedure.

High Sensitivity to Auxin is a Common Feature of Hairy Root.

The responses to auxin of Lycopersicon esculentum roots transformed by (T(l)+T(r))-DNA of the Ri plasmid of agropine-type Agrobacterium rhizogenes strain 15834 and Catharanthus trichophyllus roots transformed by the (T(l)+T(r))-DNA, and by T(l)- or T(r)- DNA alone of the same bacterial strain were compared to that of their normal counterparts. The transmembrane electrical potential difference of root protoplasts was measured as a function of the concentration of exogenous naphthalene acetic acid. The sensitivity to auxin expressed by this response was shown to be independent of the measurement conditions and of the basal polarization of isolated protoplasts. According to this electrical response, as well as to the modulation by auxin of proton excretion by root tips and root tip elongation, roots transformed by (T(l)+T(r)) DNA are 100 to 1000 times more sensitive to exogenous auxin than normal roots, as is the case with normal and transformed roots from Lotus corniculatus (WH Shen, A Petit, J Guern, J Tempé [1988] Proc Natl Acad Sci USA 85: 3417-3421). Further-more, transformed roots of C. trichophyllus are not modified in their sensitivity to fusicoccin, illustrating the specificity of the modification of the auxin sensitivity. Roots transformed by the T(r)-DNA alone showed the same sensitivity to auxin as normal roots, whereas the roots transformed by the T(l)-DNA alone exhibited an auxin sensitivity as high as the roots transformed by (T(l)+T(r))-DNA. It was concluded that the high sensitivity to auxin is controlled by the T(l)-DNA in agropine type Ri plasmids.