Exploration and biological evaluation of 20-vinyl pregnenes: A step forward toward selective modulators of the estrogen receptor α signaling for breast cancer treatment.

A series of D-ring modified steroids bearing a vinyl ketone pendant were synthesized and evaluated for antiproliferative activity against breast cancer cell line and cytochromes P450. The lead compound, 21-vinyl 20-keto-pregnene (2f) (IC50 = 2.4 µM), was shown to be a promising candidate for future anticancer drug design, particularly against estrogen receptor α (ERα)-positive breast cancer. The lead compound was found to have a significant effect on the signaling pathways in parental and 4-hydroxytamoxifen-resistant cells. Compound 2f modulated the ERK, cyclin D1, and CDK4 pathways and blocked the expression of ERα, the main driver of breast cancer growth. Compound 2f significantly reduced 17ß-estradiol-induced progesterone receptor expression. Accumulation of cleaved poly(ADP-ribose) polymerase in cells treated with compound 2f indicated induction of apoptosis. The selectivity analysis showed that lead compound 2f produces no significant effects on cytochromes P450, CYP19A1, CYP21A2, and CYP7B1.

New insights into the substrate specificity of cholesterol oxidases for more aware application.

Cholesterol oxidases (ChOxes) are enzymes that catalyze the oxidation of cholesterol to cholest-4-en-3-one. These enzymes find wide applications across various diagnostic and industrial settings. In addition, as a pathogenic factor of several bacteria, they have significant clinical implications. The current classification system for ChOxes is based on the type of bond connecting FAD to the apoenzyme, which does not adequately illustrate the enzymatic and structural characteristics of these proteins. In this study, we have adopted an integrative approach, combining evolutionary analysis, classic enzymatic techniques and computational approaches, to elucidate the distinct features of four various ChOxes from Rhodococcus sp. (RCO), Cromobacterium sp. (CCO), Pseudomonas aeruginosa (PCO) and Burkhoderia cepacia (BCO). Comparative and evolutionary analysis of substrate-binding domain (SBD) and FAD-binding domain (FBD) helped to reveal the origin of ChOxes. We discovered that all forms of ChOxes had a common ancestor and that the structural differences evolved later during divergence. Further examination of amino acid variations revealed SBD as a more variable compared to FBD independently of FAD coupling mechanism. Revealed differences in amino acid positions turned out to be critical in determining common for ChOxes properties and those that account for the individual differences in substrate specificity. A novel look with the help of chemical descriptors on found distinct features were sufficient to attempt an alternative classification system aimed at application approach. While univocal characteristics necessary to establish such a system remain elusive, we were able to demonstrate the substrate and protein features that explain the differences in substrate profile.

Discovery of highly potent proapoptotic antiestrogens in a series of androst-5,16-dienes D-modified with imidazole-annulated pendants.

Heterocyclic derivatives of steroid hormones are potent anticancer agents, which are used in the chemotherapy of breast and prostate cancers. Here, we describe a novel series of androstenes, D-modified with imidazole-annulated pendants, with significant anticancer activity. Novel C17-linked imidazole-annulated heterocyclic derivatives of dehydropregnenolone acetate were synthesized by the cyclocondensation with amidines using 3ß-acetoxy-21-bromopregna-5,16-dien-20-one as the substrate. The antiproliferative potency of all the synthesized compounds was evaluated against human prostate (22Rv1) and human breast (MCF7) cancer cell lines and cytochromes P450. The lead compound, imidazo[1,2-a]pyridine derivative 3h, was revealed to be a promising candidate for future anticancer drug design, particularly against ERα-positive breast cancer. Lead compound 3h was found to be selective against MCF7 cells with IC50 of 0.1 µM and to act as both a potent selective agent blocking estrogen receptor α, which is involved in the stimulation of breast cancer growth, and an effective apoptosis inducer. The potential ability of compound 3h to bind to ERα was studded using molecular docking and molecular dynamics simulation. The selectivity analysis showed that lead steroid 3h produces no effects on cytochromes P450 CYP17A1, CYP7A1, and CYP21A2.

5'-DMT-protected double-stranded DNA: Synthesis and competence to enzymatic reactions.

The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4'-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5'-DMT-protected ONs could allow producing both symmetric and asymmetric 5'-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5'-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5'-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5'-DMT modification of dsDNA does not affect activity of T5 5',3'-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5'-DMT-modification or search of ways to separate long 5'-DMT-ssDNA and 5'-OH-ssDNA could allow finding application of 5'-DMT-modified oligo- and polynucleotides.

Novel alkylaminoethyl derivatives of androstane 3-oximes as anticancer candidates: synthesis and evaluation of cytotoxic effects.

Steroid anticancer drugs are the focus of numerous scientific research efforts. Due to their high cytotoxic effects against tumor cells, some natural or synthetic steroid compounds seem to be promising for the treatment of different classes of cancer. In the present study, fourteen novel O-alkylated oxyimino androst-4-ene derivatives were synthesized from isomerically pure 3E-oximes, using different alkylaminoethyl chlorides. Their in vitro cytotoxic activity was evaluated against eight human cancer cell lines, as well as against normal fetal lung (MRC-5) and human foreskin (BJ) fibroblasts, to test the efficiency and selectivity of the compounds. Most derivatives displayed strong activity against malignant melanoma (G-361), lung adenocarcinoma (A549) and colon adenocarcinoma (HT-29) cell lines. Angiogenesis was assessed in vitro using migration scratch and tube formation assays on HUVEC cells, where partial inhibition of endothelial cell migration was observed for the 17α-(pyridin-2-yl)methyl 2-(morpholin-4-yl)ethyl derivative. Among the compounds that most impaired the growth of lung cancer A549 cells, the (17E)-(pyridin-2-yl)methylidene derivative bearing a 2-(pyrrolidin-1-yl)ethyl substituent induced significant apoptosis in these cells. In combination with low cytotoxicity toward normal MRC-5 cells, this molecule stands out as a good candidate for further anticancer studies. In addition, in vitro investigations against cytochrome P450 enzymes revealed that certain compounds can bind selectively in the active sites of human steroid hydroxylases CYP7, CYP17A1, CYP19A1 or CYP21A2, which could be important for the development of novel activity modulators of these enzymes and identification of possible side effects.

Modified bile acids and androstanes-Novel promising inhibitors of human cytochrome P450 17A1.

Cytochromes P450 are key enzymes for steroid hormone biosynthesis in human body. They are considered as targets for the screening of novel high efficient drugs. The results of screening of bile acids and androstane derivatives toward human recombinant steroid 17α-hydroxylase/17,20-lyase (CYP17A1) are presented in this paper. A group of steroids, binding with micromolar or submicromolar affinity (in a range from 9 µM - less than 0.1 µM), was identified. Results presented here showed that these steroidal compounds are able to decrease rate of hydroxylation of essential CYP17A1 substrate - progesterone, while some compounds completely inhibited enzyme activity. Structure-activity relationship (SAR) analysis based on in vitro and in silico studies showed that high affinity of the enzyme to bile acids derivatives is correlated with side chain hydrophobicity and presence of hydroxyl or keto group at C3 position. From the other side, bile acid-derived compounds with more polar side chain or substituents at C7 and C12 positions possess higher Kd values. Among androstane-derived steroids couple of Δ5-steroids with hydroxyl group at C3 position, as well as 16,17-secosteroids, were found to be high affinity ligands of this enzyme. The data obtained could be useful for the design of novel highly efficient inhibitors of CYP17A1, since the bile acids-derived compounds are for first time recognized as effective CYP17A1 inhibitors.

Fe(iii)-Catalyzed synthesis of steroidal imidazoheterocycles as potent antiproliferative agents.

An efficient and practical method has been developed for the synthesis of steroidal imidazoheterocycles via cost-effective and environmentally benign FeCl3-catalyzed oxidative amination. A library of steroidal imidazo[1,2-a]pyridines was directly synthesized from readily available 2-aminopyridines and steroidal ketones in aerobic conditions. The synthesized compounds were screened for activity on human microsomal cytochrome P450s CYP7, CYP17 and CYP21. Antiproliferative activity of two lead compounds 3ia and 3la was additionally evaluated against the human MCF-7 (breast cancer), SKOV3 (ovarian cancer), and 22Rv1 (prostate cancer) cell lines. Steroidal imidazo[1,2-a]pyridine 3la which is a substrate molecule for CYP17A1 with IC50 = 1.7 µM (MCF-7), 3.0 (SKOV3), and 6.0 µM (22Rv1) has proved to be more active than reference drug cisplatin.

Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells.

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6' [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1',3',4']thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16ß,17ß-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1',3',4']thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC50 = 2.1-6.6 µM) than the antiandrogen bicalutamide. Compounds 7d with IC50 = 3.0 µM and 7j with IC50 = 2.1 µM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.