Steroids with a nitrogen-containing heterocycle in the side chain are known as effective inhibitors of androgen signaling and/or testosterone biosynthesis, thus showing beneficial effects for the treatment of prostate cancer. In this work, a series of 3β-hydroxy-5-ene steroids, containing an isoxazole fragment in their side chain, was synthesized. The key steps included the preparation of Weinreb amide, its conversion to acetylenic ketones, and the 1,2- or 1,4-addition of hydroxylamine, depending on the solvent used. The biological activity of the obtained compounds was studied in a number of tests, including their effects on 17α-hydroxylase and 17,20-lyase activity of human CYP17A1 and the ability of selected compounds to affect the downstream androgen receptor signaling. Three derivatives diminished the transcriptional activity of androgen receptor and displayed reasonable antiproliferative activity. The candidate compound, 24j (17R)-17-((3-(2-hydroxypropan-2-yl)isoxazol-5-yl)methyl)-androst-5-en-3β-ol, suppressed the androgen receptor signaling and decreased its protein level in two prostate cancer cell lines, LNCaP and LAPC-4. Interaction of compounds with CYP17A1 and the androgen receptor was confirmed and described by molecular docking.

Dehydroepiandrosterone (DHEA), a precursor of steroid sex hormones, is synthesized by steroid 17-alpha-hydroxylase/17,20-lyase (CYP17A1) with the participation of microsomal cytochrome b5 (CYB5A) and cytochrome P450 reductase (CPR), followed by sulfation by two cytosolic sulfotransferases, SULT1E1 and SULT2A1, for storage and transport to tissues in which its synthesis is not available. The involvement of CYP17A1 and SULTs in these successive reactions led us to consider the possible interaction of SULTs with DHEA-producing CYP17A1 and its redox partners. Text mining analysis, protein–protein network analysis, and gene co-expression analysis were performed to determine the relationships between SULTs and microsomal CYP isoforms. For the first time, using surface plasmon resonance, we detected interactions between CYP17A1 and SULT2A1 or SULT1E1. SULTs also interacted with CYB5A and CPR. The interaction parameters of SULT2A1/CYP17A1 and SULT2A1/CYB5A complexes seemed to be modulated by 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Affinity purification, combined with mass spectrometry (AP-MS), allowed us to identify a spectrum of SULT1E1 potential protein partners, including CYB5A. We showed that the enzymatic activity of SULTs increased in the presence of only CYP17A1 or CYP17A1 and CYB5A mixture. The structures of CYP17A1/SULT1E1 and CYB5A/SULT1E1 complexes were predicted. Our data provide novel fundamental information about the organization of microsomal CYP-dependent macromolecular complexes.

С целью определения оптимальных редокс-партнеров CYP124 M. tuberculosis в in vitro реконструированной системе осуществлена гетерологическая экспрессия, выделение и очистка рекомбинантных цитохрома CYP124 M. tuberculosis и потенциальных редокс-партнеров: Fdx, FdR, FprA, Adx, AdR, CPR, Arh1_A18G, Etpfd. Показано, что CYP124 проявляет наибольшую каталитическую активность в системе с митохондриальными электрон-транспортными белками S. pombe: Etp1fd и Arh1_A18G.

The high physiological activity of steroid hormones requires multilevel regulation of their biosynthesis. Cytosolic sulfotransferases, specifically SULT2A1 and SULT1E1, convert steroid precursors into mobile sulfated depot forms, diverting them from key steroidogenic cytochromes P450. Given the previously demonstrated CYP17A1–SULT interactions, this study aims to verify the hypothesis that CYP21A2 also interacts with SULTs to co-regulate steroid biosynthesis. Using surface plasmon resonance (SPR) biosensing, in vitro enzymatic activity assays, and in silico molecular modeling, we investigated the interplay between CYP21A2 and SULT2A1/1E1. SPR analysis revealed direct, high-affinity protein–protein interactions in the nanomolar range. Thermodynamic assessments indicated that the CYP21A2–SULT1E1 complex is entropy-driven, contrasting with the enthalpy-driven CYP17A1–SULT1E1 interaction. Enzymatic assays demonstrated reciprocal modulatory effects: CYP21A2 exerted a stimulatory effect on pregnenolone sulfation by SULTs, whereas SULT2A1 moderately inhibited CYP21A2 activity. Molecular modeling of binary complexes predicted structural interfaces and continuous intraprotein channels connecting the enzymes' active sites. These findings suggest that CYP–SULT interactions contribute to the fine-tuning of steroid hormone biosynthesis. We propose that these enzymes participate in a highly organized multiprotein network, acting as metabolic switches that dictate the balance between downstream corticosteroid pathways and structural sequestration into sulfated pools.

The high physiological activity of steroid hormones requires multilevel regulation of their biosynthesis. Cytosolic sulfotransferases, specifically SULT2A1 and SULT1E1, convert steroid precursors into mobile sulfated depot forms, diverting them from key steroidogenic cytochromes P450. Given the previously demonstrated CYP17A1–SULT interactions, this study aims to verify the hypothesis that CYP21A2 can interact with SULTs and thereby potentially contribute to the regulation of steroid metabolism. Using surface plasmon resonance (SPR) biosensing, in vitro enzymatic activity assays, and in silico molecular modeling, we investigated the interplay between CYP21A2 and SULT2A1/1E1. SPR analysis revealed high-affinity binding between purified recombinant CYP21A2 and SULT2A1/SULT1E1 in vitro in the nanomolar range (9.5 × 10⁻⁹ M for SULT2A1 and 1.15 × 10⁻⁸ M for SULT1E1). Thermodynamic assessments indicated that the CYP21A2–SULT1E1 complex is entropy-driven, contrasting with the enthalpy-driven CYP17A1–SULT1E1 interaction. Enzymatic assays demonstrated reciprocal modulatory effects: CYP21A2 exerted a stimulatory effect on pregnenolone sulfation by SULTs, whereas SULT2A1 moderately inhibited CYP21A2 activity. Molecular modeling of binary complexes predicted structural interfaces and continuous intraprotein channels connecting the enzymes’ active sites. These findings suggest that CYP–SULT interactions may contribute to the fine-tuning of steroid hormone biosynthesis. These findings raise the possibility that CYP–SULT interactions may form part of a broader regulatory network, acting as metabolic switches that dictate the balance between downstream corticosteroid pathways and structural sequestration into sulfated pools.