OR02-5 Novel Intermediates Are Catalysed By 11βHSD And CYP17A1 In The C11-oxy Backdoor Pathway Leading To The Production Of 11-ketodihydrotestosterone

Adrenal C(19) steroids, testosterone and androstenedione, have long been the only androgens considered contributing to clinical conditions characterised by androgen excess. More recently, we showed that adrenal 11β-hydroxyandrostenedione is converted to 11-ketotestosterone and 11-ketodihydrotestosterone (11KDHT), both activating the androgen receptor comparable to dihydrotestosterone (1). The adrenal, however, also produces C11-oxy C(21) steroids, 21-deoxycortisol (21dF) and low levels of 11β-hydroxyprogesterone (11OHP4), which are both metabolised to 11KDHT. While 11βHSD2 efficiently converts 11OHP4 and 21dF to 11-ketoprogesterone (11KP4) and 21-deoxycortisone (21dE) respectively, 11OHP4 and 11KP4 are more readily reduced by SRD5A than 21dF and 21dE in the backdoor pathway (2,3). To study the contribution of 11OHP4 and 21dF to the metabolic shunt towards active androgens, 11βHSD and CYP17A1 were expressed in HEK-293 cells and the conversion of novel intermediates (1μM) analysed using UPC(2)-MS/MS. Once reduced at C3 and the C4-C5 double bond, the 11OHP4 and 21dF intermediates can be further metabolised by 11βHSD2 and CYP17A1 in the C11-oxy backdoor pathway. Conversion by 11βHSD2 yielded alfaxalone (±0.47 μM) and 11-ketoPdiol (±0.75 μM) with 3,11diOH-dihydroprogesterone (3,11diOHDHP4) (±0.52 μM) and 11OHPdiol (±0.24 μM) remaining after 24 hrs. In the conversion of alfaxalone and 11-ketoPdiol by 11βHSD1 most of the substrate was still detected after 24 hrs, ±0.9 μM. Conversion of 1 μM 11OHPdiol and 11-ketoPdiol by CYP17 yielded the lyase products: ±0.18 μM 11β-hydroxyandrosterone (11OHAST) and ±0.25 μM 11ketoandrosterone (11KAST) after 12 hrs. Conversion of 1 μM 3,11diOHDHP4 and alfaxalone yielded the hydroxylase products 11OHPdiol (±0.15 μM) and 11-ketoPdiol (±0.05 μM) and the lyase products, 11OHAST (±0.06 μM) and 11KAST (±0.14 μM). It was only 3,11diOHDHP4 that was further converted to 0.33 μM 11OHPdiol and ±0.16 μM11OHAST after 24hrs. Both adrenal 11OHP4 and 21dF would, via 11OHAST which is also converted to 11KAST by 11βHSD2, readily lead to the production of 11KDHT catalysed by the backdoor steroidogenic enzymes. Data show that the C17 hydroxyl group favours the conversion of intermediates catalysed by 11βHSD2 (75% vs 50 % substrate conversion) with the oxidation reaction by 11βHSD2 being the preferred reaction compared to 11βHSD1. CYP17A1 catalyses both the hydroxylase and 17,21 lyase reactions with the conversion of 3,11diOHDHP4 being the most favourable. Overall, this study highlights the contribution of the C11-oxy C(21) steroids to the active androgen pool and relates their physiological relevance to adrenal-linked endocrine diseases characterised by androgen excess. (1) Bloem et al., J. Steroid Biochem. Mol. Biol. 2015 153:80-92 (2) Barnard et al., J. Steroid Biochem. Mol. Biol. 2017 174:86-95 (3) van Rooyen et al., J. Steroid Biochem. Mol. Biol. 178:203-212