RESUMEN
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.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas , Escherichia coli , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/aislamiento & purificación , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/química , Cromatografía de Afinidad , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesisRESUMEN
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.
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Antineoplásicos , Simulación del Acoplamiento Molecular , Humanos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Hidrazinas/farmacología , Hidrazinas/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Esteroides/química , Esteroides/farmacología , Semicarbazonas/farmacología , Semicarbazonas/química , Semicarbazonas/síntesis química , Tiosemicarbazonas/farmacología , Tiosemicarbazonas/química , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
Alchemilla vulgaris L., Trifolium pratense L. and Glycyrrhiza glabra L. are important remedies in traditional medicine, known for many usages, including treating gynecological diseases. Despite folkloric use of the plant materials, there is a lack of scientific data to support their therapeutic application. The aims of the present study were to evaluate the relative binding affinities (RBAs) of plant-derived phytoestrogens for estrogen receptor ß (ERß) using fluorescent biosensor in yeast and to apply this assay for the assessment of the potential of plant materials towards ERs and treatment of estrogen-related disorders. Ligand-binding domain of ERß fused with yellow fluorescent protein (ERß LBD-YFP) was expressed in S. cerevisiae and fluorescence was detected by fluorimetry and fluorescence microscopy. Structural basis for experimental results was explored by molecular docking. Yeast-based fluorescent assay was successfully optimized and applied for identification of natural phenolic compounds and phytoestrogen-rich plant extracts that interact with ERß-LBD, making this biosensor a valuable tool for screening estrogenic potential of a variety of plant extracts. This assay can be used for preliminary testing of plant-derived or fungal extracts, but also other sources of environmental substances with ER-modulating activity in order to assess their possible effects on the female reproductive system.
RESUMEN
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.
Asunto(s)
Antineoplásicos , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Simulación del Acoplamiento Molecular , Humanos , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/antagonistas & inhibidores , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Receptores Androgénicos/metabolismo , Aromatasa/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/antagonistas & inhibidoresRESUMEN
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.
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Androstanos , Antineoplásicos , Humanos , Ligandos , Androstanos/farmacología , Androstanos/química , Esteroides/metabolismo , Lactamas/farmacología , Relación Estructura-Actividad , Proliferación Celular , Estructura Molecular , Ensayos de Selección de Medicamentos Antitumorales , Línea Celular TumoralRESUMEN
The number of people diagnosed with diabetes continues to increase, especially among younger populations. Apart from genetic predisposition and lifestyle, there is increasing scientific and public concern that environmental agents may also contribute to diabetes. Food contamination by chemical substances that originate from packaging materials, or are the result of chemical reactions during food processing, is generally recognized as a worldwide problem with potential health hazards. Phthalates, bisphenol A (BPA) and acrylamide (AA) have been the focus of attention in recent years, due to the numerous adverse health effects associated with their exposure. This paper summarizes the available data about the association between phthalates, BPA and AA exposure and diabetes. Although their mechanism of action has not been fully clarified, in vitro, in vivo and epidemiological studies have made significant progress toward identifying the potential roles of phthalates, BPA and AA in diabetes development and progression. These chemicals interfere with multiple signaling pathways involved in glucose and lipid homeostasis and can aggravate the symptoms of diabetes. Especially concerning are the effects of exposure during early stages and the gestational period. Well-designed prospective studies are needed in order to better establish prevention strategies against the harmful effects of these food contaminants.
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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 â¼7 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.
RESUMEN
The major challenge in the fight against cancer is to design new drugs that will be more selective for cancer cells, with fewer side effects. Synthetic steroids such as cyproterone, fulvestrant, exemestane and abiraterone are approved powerful drugs for the treatment of hormone-dependent diseases such as breast and prostate cancers. Therefore, androstane derivatives in 17-substituted, 17a-homo lactone and 16,17-seco series, with potent anticancer activity, were selected for pharmacokinetic and druglike predictions from the absorption, distribution, metabolism and excretion (ADME) models. In silico determination of physico-chemical and ADMET properties was performed using SwissADME and ProTox-II web tools. The possibility of gastrointestinal absorption and brain penetration was analyzed using the BOILED-Egg model, while the in silico evaluation of the similarities between selected steroid derivatives and FDA-approved drugs was carried out using the SwissSimilarity tool. Of all tested, two compounds that showed good in silico ADMET results, in addition to promising cytotoxicity and molecular docking results, could potentially be evaluated in in vivo tests.
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Antineoplásicos , Neoplasias de la Próstata , Masculino , Humanos , Simulación del Acoplamiento Molecular , Androstanos/farmacología , Androstanos/química , Esteroides/química , Neoplasias de la Próstata/tratamiento farmacológico , Encéfalo , Antineoplásicos/químicaRESUMEN
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 â¼3.6â µM. Molecular dynamics simulations (20â 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.
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Productos Biológicos , Simulación de Dinámica Molecular , Aldo-Ceto Reductasas/metabolismo , Ácidos y Sales Biliares , Ésteres , Humanos , Simulación del Acoplamiento Molecular , Isoformas de Proteínas/metabolismo , Receptores de Glucocorticoides , Ácido UrsodesoxicólicoRESUMEN
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.
RESUMEN
Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum-localized channels that mediate Ca2+ release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca2+-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66-110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1-InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca2+ signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca2+ signaling and survival, further suggesting the importance of this residue in the NCS1-InsP3R1 interaction. In conclusion, we show that NCS1-InsP3R1 interaction enhances intracellular Ca2+ signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1-InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.
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Neoplasias de la Mama/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Sitios de Unión , Femenino , Humanos , Unión ProteicaRESUMEN
Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680 bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis-regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full-length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose-dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.
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Abejas/genética , Metalotioneína/genética , Animales , Abejas/efectos de los fármacos , Cadmio/toxicidad , Cobre/toxicidad , Himenópteros/efectos de los fármacos , Himenópteros/genética , Plomo/toxicidadRESUMEN
Two novel rhodium(III) complexes, namely, [RhIII(X)Cl3] (X = 2 2,6-bis((4 S,7 R)-7,8,8-trimethyl-4,5,6,7-tetrahydro-1 H-4,7-methanoindazol-3-yl)pyridine or 2,6-bis((4 S,7 R)-1,7,8,8-tetramethyl-4,5,6,7-tetrahydro-1 H-4,7-methanoindazol-3-yl)pyridine), were synthesized from camphor derivatives of a bis(pyrazolylpyridine), tridentate nitrogen-donor chelate system, giving [RhIII(H2L*)Cl3] (1a) and [RhIII(Me2L*)Cl3] (1b). A rhodium(III) terpyridine (terpy) ligand complex, [RhIII(terpy)Cl3] (1c), was also synthesized. By single-crystal X-ray analysis, 1b crystallizes in an orthorhombic P212121 system, with two molecules in the asymmetric unit. Tridentate coordination by the N,N,N-donor localizes the central nitrogen atom close to the rhodium(III) center. Compounds 1a and 1b were reactive toward l-methionine (l-Met), guanosine-5'-monophosphate (5'-GMP), and glutathione (GSH), with an order of reactivity of 5'-GMP > GSH > l-Met. The order of reactivity of the RhIII complexes was: 1b> 1a > 1c. The RhIII complexes showed affinity for calf thymus DNA and bovine serum albumin by UV-vis and emission spectral studies. Furthermore, 1b showed significant in vitro cytotoxicity against human epithelial colorectal carcinoma cells. Since the RhIII complexes have similar coordination modes, stability differences were evaluated by density functional theory (DFT) calculations (B3LYP(CPCM)/LANL2DZp). With (H2L*) and (terpy) as model ligands, DFT calculations suggest that both tridentate ligand systems have similar stability. In addition, molecular docking suggests that all test compounds have affinity for the minor groove of DNA, while 1b and 1c have potential for DNA intercalation.
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Alcanfor/análogos & derivados , Alcanfor/farmacología , Complejos de Coordinación/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Rodio/química , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Alcanfor/síntesis química , Alcanfor/química , Bovinos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , ADN/química , Teoría Funcional de la Densidad , Células HCT116 , Humanos , Sustancias Intercalantes/síntesis química , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Cinética , Ligandos , Modelos Químicos , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Piridinas/síntesis química , Piridinas/química , Albúmina Sérica Bovina/químicaRESUMEN
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.
RESUMEN
Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.
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Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/química , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/metabolismo , Histidina , Trombina/metabolismo , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas/genética , Secuencia de Aminoácidos , Cristalización/métodos , Cristalografía por Rayos X/métodos , Histidina/genética , Humanos , Proteolisis , Difracción de Rayos X/métodosRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/metabolismo , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas Biosensibles , Femenino , Humanos , Células MCF-7 , Microscopía Fluorescente , Estructura Molecular , Esteroides/metabolismoRESUMEN
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.
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Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Nitrilos/química , Nitrilos/farmacología , Esteroides/química , Esteroides/farmacología , Antineoplásicos/síntesis química , Aromatasa/metabolismo , Mama/efectos de los fármacos , Mama/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Receptor alfa de Estrógeno/metabolismo , Femenino , Células HeLa , Humanos , Masculino , Simulación del Acoplamiento Molecular , Nitrilos/síntesis química , Próstata/efectos de los fármacos , Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Esteroides/síntesis químicaRESUMEN
Since many estrogen derivatives exhibit anti-hormone or enzyme inhibition potential, a large number of steroidal derivatives have been synthesised from appropriate precursors, in order to obtain potential therapeutics for the treatment of hormone-dependent cancers. In molecular docking studies, based on X-ray crystallographic analysis, selected D-homo and D-seco estratriene derivatives were predicted to bind strongly to estrogen receptor α (ERα), aromatase and 17,20 lyase, suggesting they could be good starting compounds for antihormonal studies. Test results in vivo suggest that these compounds do not possess estrogenic activity, while some of them showed weak anti-estrogenic properties. In vitro anti-aromatase and anti-lyase assays showed partial inhibition of these two enzymes, while some compounds activated aromatase. Aromatase activators are capable of promoting estrogen synthesis for treatment of pathological conditions caused by estrogen depletion, e.g. osteopenia or osteoporosis.
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Aromatasa/metabolismo , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Homoesteroides/farmacología , Antagonistas de Hormonas/farmacología , Secoesteroides/farmacología , Esteroide 17-alfa-Hidroxilasa/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Estrenos/síntesis química , Estrenos/química , Estrógenos/biosíntesis , Femenino , Homoesteroides/síntesis química , Homoesteroides/química , Antagonistas de Hormonas/síntesis química , Antagonistas de Hormonas/química , Modelos Moleculares , Conformación Molecular , Ratas , Ratas Wistar , Secoesteroides/síntesis química , Secoesteroides/química , Estereoisomerismo , Esteroide 17-alfa-Hidroxilasa/metabolismo , Relación Estructura-ActividadRESUMEN
Polycystin 2 (PC2) is a calcium-dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C-terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single-channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild-type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium-binding EF hand is intact. These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function.
Asunto(s)
Motivos EF Hand , Canales Catiónicos TRPP/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Canales de Calcio/metabolismo , Línea Celular , Biología Computacional , Humanos , Riñón/metabolismo , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Filogenia , Plásmidos/metabolismo , Unión Proteica , Erizos de Mar , Homología de Secuencia de Aminoácido , Transducción de Señal , PorcinosRESUMEN
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.