Inhibitory effects of sex steroids on chimeric aldosterone synthase and wild type enzyme in vitro

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 355-388-Sex Hormone Receptor Action & Reaction
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-385
Andrea Vecchiola*1, Carlos F Lagos1, Cristobal A Fuentes1, Fidel Allende1, Carmen Campino1, Carolina Valdivia1, Alejandra Tapia-Castillo1, Gareth Ivor Owen2, Sandra Solari1, Cristian A Carvajal1 and Carlos E Fardella1
1Pontificia Universidad Catolica de Chile, Santiago, Chile, 2Pontificia Universidad Catolica de Chile, Santiago De Chile, Chile
Background. Familial hyperaldosteronism type I (FH-I) is caused by the unequal recombination between the 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) genes resulting in the generation of a CYP11B1/B2 chimeric gene (CG) and increased aldosterone production. We recently reported a four-generation pedigree for FH-I in which chimeric gene positive patients exhibited a gradual normalization of the aldosterone-to-renin ratio (ARR) between infants and adults. We speculate that the inverse association between the ARR and age could be due in part to the appearance of sex steroids in puberty. Aim. To investigate whether sex steroids modulate the activity of wild-type aldosterone synthase (ASWT) and chimeric enzyme (ASCE). Methods. We designed an in vitro assay using the HEK-293 cell line transiently transfected with vectors containing the full ASWT or the ASCE cDNAs. To evaluate the steroids effect on AS enzymes activity, the transfected cells were incubated with Deoxycorticosterone (DOC, 1.5μmol/L) alone or co-incubated with increasing doses of progesterone, testosterone or estradiol (0.6-10.0μmol/L). In addition, we constructed and validated 3-D models of both enzymes to investigate the putative binding mode of the steroids. Results. We observed that in vitro,ASWT and ASCE displayed similar aldosterone synthase enzymatic parameters. Inhibition studies showed that progesterone and testosterone inhibited differentially the activity of ASWT and the ASCE. Progesterone inhibited ASWT with higher potency but similar efficacy compared to ASCE (IC50 ASWT=2.240 μmol/L and IC50ASCE=3.907 μmol/L respectively).Testosterone exerted a similar effect as progesterone, inhibiting the ASWT with more potency and efficiency than the ASCE (IC50ASWT=1.690 μmol/L, and IC50ASCE=3.176 μmol/L, respectively). On the other hand, estradiol showed no effect. Molecular modeling studies and binding affinity estimations indicated that progesterone and testosterone might bind and fit to the substrate site in the wild type and chimeric enzymes (for ASWTΔGbind= -6.52 and -6.61 kcal/mol respectively and for ASCE ΔGbind= -6.90 and -5.97 kcal/mol respectively), while estradiol is not able to accommodate within the site properly. Conclusion. Our in vitro and in silico results suggests that progesterone and testosterone can modulate the enzymes activities, and might contribute for the decay of aldosterone synthase activity in chimeric gene positive patients.

Nothing to Disclose: AV, CFL, CAF, FA, CC, CV, AT, GIO, SS, CAC, CEF

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Sources of Research Support: Acknowledgments. Supported by FONDEF D08i1087, FONDEF IDeA Nº CA12i10150, FONDECYT 1100356, FONDECYT 1130427 & IMII P09/016-F grants. The authors thank OpenEye Software for academic license of their products (FILTER, OMEGA, vROCS& FRED), and the DTP/NCI for kindly providing the compounds screened in this study. CFL and CAC are CONICYT PhD fellows.