Expression and purification of active human 17ß-Hydroxysteroid dehydrogenase type 1 from Escherichia coli.

Breast cancer cell growth is often dependent on the presence of steroidal hormones. The 17ß-hydroxysteroid dehydrogenase type 1 isoform (17ßHSD1) catalyzes NADPH-dependent conversion of estrone to estradiol, a more potent estrogen, and represents potential drug target for breast cancer treatment.  To provide active enzyme for inhibitor screening, 17ßHSD1 is usually expressed in insect or mammalian cells, or isolated from human placenta. In the present study we describe a simple protocol for expression and purification of active human 17ßHSD1 from BL21(DE3) Escherichia coli cells. Soluble human 17ßHSD1 was expressed using a pET28a(+)-based plasmid, which encodes a hexahistidine tag fused to the N-terminus of the protein, and purified by nickel affinity chromatography. The enzyme activity of purified 17ßHSD1 was verified by three methods: thin-layer chromatography, an alkali assay and a spectroscopic assay. These non-radioactive enzyme assays require only standard laboratory equipment, and can be used for screening compounds that modulate 17ßHSD1 activity.

Development of new steroid-based hydrazide and (thio)semicarbazone compounds with anticancer properties.

Most breast and prostate cancers are caused by abnormal production or action of steroidal hormones. Hormonal drugs based on steroid scaffolds represent a significant class of chemotherapeutics that are routinely used in chemotherapy. In this study, the synthesis of new 17a-homo lactone and 17α-(pyridine-2-ylmethyl) androstane derivatives with hydrazide and semicarbazone motifs is presented. All compounds were screened for their effect on cell viability against a panel of five cancer cell lines and one healthy cell line. Two compounds showed significant cytotoxicity against cancer cells, with low toxicity against healthy cells. The relative binding affinities of compounds for the ligand-binding domains of estrogen receptor α, estrogen receptor ß, androgen receptor and glucocorticoid receptor were tested using a fluorescence screen in yeast. Potential for inhibition of aldo-keto reductase 1C3 and 1C4 activity was measured in vitro. Experimental results are analyzed in the context of molecular docking simulations. Our results could help guide design of steroid compounds with improved anticancer properties against androgen- and estrogen-dependent cancers.

Synthesis, in vitro and in silico anticancer evaluation of novel pyridin-2-yl estra-1,3,5(10)-triene derivatives.

Aim: The aim of this study was the synthesis of steroid compounds with heterocyclic rings and good anticancer properties. Materials & methods: The synthesis, in silico and in vitro anticancer testing of novel pyridin-2-yl estra-1,3,5(10)-triene derivatives was performed. Results: All synthesized compounds have shown promising results for, antiproliferative activity, relative binding affinities for the ligand binding domains of estrogen receptors α, ß and androgen receptor, aromatase binding potential, and inhibition of AKR1C3 enzyme. Conclusion: 3-Benzyloxy (17E)-pycolinilidene derivative 9 showed the best antitumor potential against MDA-MB-231 cell line, an activity that can be explained by its moderate inhibition of AKR1C3. Molecular docking simulation indicates that it binds to AKR1C3 in a very similar orientation and geometry as steroidal inhibitor EM1404.

The series of pyridine-containing estra-1,3,5(10)-triene derivatives was synthesized. One novel derivative stood out by its excellent activity against the MDA-MB-231 cell line. This activity can be explained by its moderate inhibition of the AKR1C3 enzyme.

Novel D-modified heterocyclic androstane derivatives as potential anticancer agents: Synthesis, characterization, in vitro and in silico studies.

Cancer remains a major health concern worldwide. The most frequently diagnosed types of cancer are caused by abnormal production or action of steroid hormones. In the present study, the synthesis and structural characterization of new heterocyclic androstane derivatives with D-homo lactone, 17α-(pyridine-2''-ylmethyl) or 17(E)-(pyridine-2''-ylmethylidene) moiety are presented. All compounds were evaluated for their anti-proliferative activity against HeLa cervical cancer cell line and non-cancerous kidney MDCK cells, where A-homo lactam compound 9A showed the greatest selectivity. Based on in vitro binding assays, N-formyl lactam compound 18 appeared to be the strong and isoform-selective ligand for ERα, while compound 9A displayed binding affinity for the GR-LBD, but also inhibited aldo-keto reductase 1C4 enzyme. Out of four selected compounds, methylpyrazolo derivative 13 showed potential for aromatase binding, while in silico studies provided insight into experimentally confirmed protein-ligand interactions.

X-ray structure of human aldo-keto reductase 1C3 in complex with a bile acid fused tetrazole inhibitor: experimental validation, molecular docking and structural analysis.

Aldo-keto reductase 1C3 (AKR1C3) catalyzes the reduction of androstenedione to testosterone and reduces the effectiveness of chemotherapeutics. AKR1C3 is a target for treatment of breast and prostate cancer and AKR1C3 inhibition could be an effective adjuvant therapy in the context of leukemia and other cancers. In the present study, steroidal bile acid fused tetrazoles were screened for their ability to inhibit AKR1C3. Four C24 bile acids with C-ring fused tetrazoles were moderate to strong AKR1C3 inhibitors (37-88% inhibition), while B-ring fused tetrazoles had no effect on AKR1C3 activity. Based on a fluorescence assay in yeast cells, these four compounds displayed no affinity for estrogen receptor-α, or the androgen receptor, suggesting a lack of estrogenic or androgenic effects. A top inhibitor showed specificity for AKR1C3 over AKR1C2, and inhibited AKR1C3 with an IC50 of ∼7 µM. The structure of AKR1C3·NADP+ in complex with this C-ring fused bile acid tetrazole was determined by X-ray crystallography at 1.4 Å resolution, revealing that the C24 carboxylate is anchored to the catalytic oxyanion site (H117, Y55); meanwhile the tetrazole interacts with a tryptophan (W227) important for steroid recognition. Molecular docking predicts that all four top AKR1C3 inhibitors bind with nearly identical geometry, suggesting that C-ring bile acid fused tetrazoles represent a new class of AKR1C3 inhibitors.

Hormone receptor binding, selectivity and cytotoxicity of steroid D-homo lactone loaded chitosan nanoparticles for the treatment of breast and prostate cancer cells.

Chemically modified steroids have a long history as anti-neoplastic drugs. Incorporation of a lactone moiety in the steroid nucleus, as in previously obtained 3ß-acetoxy-17-oxa-17a-homoandrost-5-en-16-one (A) and 3ß-hidroxy-17-oxa-17a-homoandrost-5-en-16-one (B), often results in enhanced anticancer properties. In this work, chitosan-based (Ch) nanoparticles were created and loaded with potent anticancer steroidal compounds, A and B. Changes to hormone receptor binding and cytotoxicity were then measured. In agreement with our previous results for A and B, A- and B-loaded Ch displayed cytotoxic properties against cancer cell lines. Both A-Ch and B-Ch showed activity toward estrogen negative breast cancer (MDA-MB-231) and androgen negative prostate cancer cell lines (PC-3). Greater selectivity toward cancer cells versus healthy lung fibroblast (MRC-5) was observed for B-Ch particles. Cell viability and cytotoxicity measurements after a recovery period indicate more robust recovery of healthy cells versus malignant cells. Compounds A and B or their Ch-encapsulated forms were shown to have negligible affinity for the ligand binding domain of estrogen receptor ß or the androgen receptor in a fluorescent yeast screen, suggesting a lack of estrogenicity and androgenicity. Steroid-loaded chitosan nanoparticles display strong cytotoxicity towards MDA-MB-231 and PC-3 with a lack of hormone activity, indicating their safety and efficacy.

Investigation of the Potential of Bile Acid Methyl Esters as Inhibitors of Aldo-keto Reductase 1C2: Insight from Molecular Docking, Virtual Screening, Experimental Assays and Molecular Dynamics.

Human aldo-keto reductase 1C isoforms (AKR1C1-C4) catalyze reduction of endogenous and exogenous compounds, including therapeutic drugs, and are associated with chemotherapy resistance. AKR1C2 is involved in metastatic processes and is a target for the treatment of various cancers. Here we used molecular docking to explore the potential of a series of eleven bile acid methyl esters as AKR1C2 inhibitors. Autodock 4.2 ranked 10 of the 11 test compounds above a decoy set generated based on ursodeoxycholic acid, a known AKR1C2 inhibitor, while 5 of these 10 ranked above 94 % of decoys in Autodock Vina. Seven inactives reported in the literature not to inhibit AKR1C2 ranked below the decoy threshold: 5 of these are specific inhibitors of AKR1C3, a related isoform. Using the same parameters, Autodock Vina identified steroidal analogs of AKR1C substrates, bile acids, and AKR1C inhibitors in the top 5 % of a virtual screen of a natural product library. In experimental assays, 6 out of 11 of the tested bile acid methyl esters inhibited >50 % of AKR1C2 activity, while 2 compounds were strong AKR1C3 inhibitors. Potential off-target interactions with the glucocorticoid receptor were measured using a yeast-based fluorescence assay, where results suggest that the methyl ester could interfere with binding. The top ranking compound based on docking and experimental results showed dose-dependent inhibition of AKR1C2 with an IC50 of ∼3.6 µM. Molecular dynamics simulations (20 ns) were used to explore potential interactions between a bile acid methyl ester and residues in the AKR1C2 active site. Our molecular docking results identify AKR1C2 as a target for bile acid methyl esters, which combined with virtual screening results could provide new directions for researchers interested in synthesis of AKR1C inhibitors.

Microwave-assisted green synthesis of bile acid derivatives and evaluation of glucocorticoid receptor binding.

Herein, we present microwave-assisted AlCl3 catalyzed oxidation of bile acid hydroxyl groups in the presence of Oxone® in water media. Significant rate enhancements were observed for Wolff-Kishner reduction of synthesized bile acids oxo derivatives to the 5ß-cholanic acid. Reaction of amidation of the simplest bile acid and aminolysis of the deoxycholic acid was accomplished in the absence of solvent and catalysts under sealed vessel microwave conditions. Because 5ß-cholanic acid reportedly modulates glucocorticoid receptor signaling in cell models of Parkinson's disease, we tested the affinity of 5ß-cholanic acid and deoxycholic acid derivatives for the glucocorticoid receptor in vitro using a yeast-based fluorescent screen. Treatment of GR-expressing yeast with prednisolone resulted in a dose-dependent increase in fluorescence; whereas 5ß-cholanic acid binds to the glucocorticoid receptor with more moderate affinity. Similarly, molecular docking also suggests that 5ß-cholanic acid can bind to the glucocorticoid receptor, with similar geometry to known GR ligands.

Evaluation of A-ring fused pyridine d-modified androstane derivatives for antiproliferative and aldo-keto reductase 1C3 inhibitory activity.

New A-ring pyridine fused androstanes in 17a-homo-17-oxa (d-homo lactone), 17α-picolyl or 17(E)-picolinylidene series were synthesized and validated by X-ray crystallography, HRMS, IR and NMR spectroscopy. Novel compounds 3, 5, 8 and 12 were prepared by treatment of 4-en-3-one or 4-ene-3,6-dione d-modified androstane derivatives with propargylamine catalyzed by Cu(ii), and evaluated for potential anticancer activity in vitro using human cancer cell lines and recombinant targets of steroidal anti-cancer drugs. Pyridine fusion to position 3,4 of the A-ring may dramatically enhance affinity of 17α-picolyl compounds for CYP17 while conferring selective antiproliferative activity against PC-3 cells. Similarly, pyridine fusion to the A-ring of steroidal d-homo lactones led to identification of new inhibitors of aldo-keto reductase 1C3, an enzyme targeted in acute myeloid leukemia, breast and prostate cancers. One A-pyridine d-lactone steroid 5 also has selective submicromolar antiproliferative activity against HT-29 colon cancer cells. None of the new derivatives have affinity for estrogen or androgen receptors in a yeast screen, suggesting negligible estrogenicity and androgenicity. Combined, our results suggest that A-ring pyridine fusions have potential in modulating the anticancer activity of steroidal compounds.

Identification of d-seco modified steroid derivatives with affinity for estrogen receptor α and ß isoforms using a non-transcriptional fluorescent cell assay in yeast.

Synthesis and biological evaluation of steroidal derivatives with anticancer properties is an active area of drug discovery. Here we measured the relative affinities of d-seco modified steroidal derivatives for estrogen receptor α, estrogen receptor ß or androgen receptor ligand binding domains using an optimized non-transcriptional fluorescent cell assay in yeast. Ligand binding domains of steroid receptors were expressed in-frame with yellow fluorescent protein in the yeast Saccharomyces cerevisiae. Addition of known steroid ligands to yeast expressing the appropriate cognate receptor results in increased fluorescence intensity, enabling estimation of receptor binding affinities in a dose-response and time-dependent manner. Relative binding affinities of d-seco modified steroidal derivatives 1-4 were then evaluated using this yeast system by live cell fluorimetry and fluorescence microscopy, coupled with in vitro cytotoxicity and in silico molecular docking studies. d-Seco estratriene derivative 2displayed strong affinity for both estrogen receptor α and ß ligand binding domains and negligible affinity for the androgen receptor ligand binding domain. Compound 2 also showed moderate cytotoxicity against estrogen receptor positive MCF-7 breast adenocarcinoma cells. In addition to identification of new ligands for steroid receptors, this assay could also be used to filter out compounds with potential for off-target interactions with steroid receptors during the early stages of compound screening.

Synthesis and anticancer cell potential of steroidal 16,17-seco-16,17a-dinitriles: identification of a selective inhibitor of hormone-independent breast cancer cells.

We report the synthesis of steroidal 16,17-seco-16,17a-dinitriles and investigate their antitumor cell properties. Compounds were evaluated for anticancer potential by in vitro antiproliferation studies, molecular docking and virtual screening. Several compounds inhibit the growth of breast and prostate cancer cell lines (MCF-7, MDA-MB-231 and PC3), and/or cervical cancer cells (HeLa). Supporting this, molecular docking predicts that steroidal 16,17-seco-16,17a-dinitriles could bind with high affinity to multiple molecular targets of breast and prostate cancer treatment (aromatase, estrogen receptor α, androgen receptor and 17α-hydroxylase) facilitated by D-seco flexibility and nitrile-mediated contacts. Thus, 16,17-seco-16,17a-dinitriles may be useful for the design of inhibitors of multiple steroidogenesis pathways. Strikingly, 10, a 1,4-dien-3-on derivative, displayed selective submicromolar antiproliferative activity against hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cells (IC50 0.52, 0.11µM, respectively). Ligand-based 3D similarity searches suggest AKR1C, 17ß-HSD and/or 3ß-HSD subfamilies as responsible for this antiproliferative activity, while fast molecular docking identified AKR1C and ERß as potential binders-both targets in the treatment of hormone-independent breast cancers.

17(E)-picolinylidene androstane derivatives as potential inhibitors of prostate cancer cell growth: antiproliferative activity and molecular docking studies.

We report a rapid and efficient synthesis of A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives from dehydroepiandrosterone. Compounds were validated spectroscopically and structurally characterized by X-ray crystallography. Virtual screening by molecular docking against clinical targets of steroidal anticancer drugs (ERα, AR, Aromatase and CYP17A1) suggests that 17(E)-picolinylidene, but not 17α-picolyl androstanes could specifically interact with CYP17A1 (17α-hydroxylase) with similar geometry and affinity as Abiraterone, a 17-pyridinyl androstane drug clinically used in the treatment of prostate cancer. In addition, several 17(E)-picolinylidene androstanes demonstrated selective antiproliferative activity against PC3 prostate cancer cells, which correlates with Abiraterone antiproliferative activity and predicted CYP17A1 binding affinities. Based on these preliminary results, 17(E)-picolinylidene androstane derivatives could be a promising starting point for the development of new compounds for the treatment of prostate cancer.

Calcium-induced conformational changes in C-terminal tail of polycystin-2 are necessary for channel gating.

Polycystin-2 (PC2) is a Ca(2+)-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca(2+). PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca(2+)-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca(2+) remains unknown. We propose that the PC2 EF-hand is a Ca(2+) sensor required for channel gating. Consistent with this, Ca(2+) binding causes a dramatic decrease in the radius of gyration (R(g)) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca(2+) concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Šdecrease in maximum interatomic distance (D(max)) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca(2+) via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca(2+) release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca(2+) levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.