Inhibition Of Aromatase Activity Research Articles

Novel triazole-tetrahydroisoquinoline hybrids as human aromatase inhibitors

Novel thirteen triazole-tetrahydroisoquinoline derivatives (2a-m) were synthesized and evaluated for their aromatase inhibitory activities. Seven triazoles showed significant aromatase inhibitory activity (IC50 = 0.07–1.9 μM). Interestingly, the analog bearing naphthalenyloxymethyl substituent at position 4 of the triazole ring (2i) displayed the most potent aromatase inhibitory activity (IC50 = 70 nM) without significant cytotoxicity to a normal cell. Molecular docking also suggested that the direct H-bonding interaction with residue Thr310 may be responsible for a striking inhibitory effect of the most potent compound 2i.

Anandamide targets aromatase: A breakthrough on human decidualization

In each menstrual cycle endometrial stromal cells (hESC) proliferate and differentiate into specialized decidual cells, a process termed decidualization, which regulates endometrial receptivity. Decidualization is mainly controlled by sex ovarian hormones, estradiol (E2) and progesterone. E2 plays an important role in the expression of the progesterone receptor and promotes the endometrial stromal cells differentiation. Our group previously reported that anandamide (AEA) impairs decidualization through cannabinoid receptor 1 (CB1). In this study, we hypothesized whether AEA inhibitory effect on cell decidualization could be mediated through interaction with aromatase and consequent interference in estradiol production/signaling. We used an immortalized human endometrial stromal cell line (St-T1b) and human decidual fibroblasts (HdF) derived from human term placenta. In cells exposed to a differentiation stimulus, AEA-treatment prevents the increase of the expression of CYP19A1 gene encoding aromatase, E2 levels and of estradiol receptor expression, that are observed in differentiating cells. Regarding CYP19A1 mRNA levels, the effect was partially reverted by a CB1 receptor antagonist and by a COX2 inhibitor. In addition, we report that AEA presents anti-aromatase activity in placental microsomes, the nature of the inhibition being the uncommon mixed type as revealed by the kinetic studies. Structural analysis of the AEA-Aromatase complexes determined that AEA may bind to the active site pocket of the enzyme. In overall we report that AEA inhibits aromatase activity and may affect E2 signaling crucial for the decidualization process, indicating that a deregulation of the endocannabinoid system may be implicated in endometrial dysfunction and in fertility/infertility disorders.

A novel GC-MS methodology to evaluate aromatase activity in human placental microsomes: a comparative study with the standard radiometric assay.

Estrogens are key factors in the development of the estrogen receptor-positive (ER+) breast cancer. Estrogens, estrone (E1), and estradiol (E2) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER+ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E1 (ratio E1/AD) using isotopic analogues as internal standards. The method showed good linearity (r2 = 0.9908 for AD and 0.9944 for E1), high accuracy (more than 74% for AD and more than 66% for E1), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC50 valuesof the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from theradiometric assay are also compared, and similar IC50 values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs. Graphical abstract.

Aromatase (CYP19) inhibition by biflavonoids obtained from the branches of Garcinia gardneriana (Clusiaceae).

Overexpression of aromatase in breast cancer cells may substantially influence its progression and maintenance. In this sense, the inhibition of aromatase is a key target for the treatment of breast cancer in postmenopausal women. Although several flavonoids had already demonstrated the capacity of inhibiting aromatase activity, the role of biflavonoids as aromatase inhibitors is poorly studied. In this work, the biflavonoids isolated from Garcinia gardneriana, morelloflavone (1), Gb-2a (2) and Gb-2a-7-O-glucose (3) were submitted to in vitro assay to evaluate the aromatase modulatory effect. As results, it was demonstrated that all biflavonoids were able to inhibit the enzyme, with IC50 values ranging from 1.35 to 7.67 μM. This demonstrates that biflavonoids are an important source of scaffolds for the development of new aromatase inhibitors, focusing on the development of new anticancer agents.

Synthesis, molecular docking, and QSAR study of bis-sulfonamide derivatives as potential aromatase inhibitors

A library of bis-sulfonamides (9–26) were synthesized and tested for their aromatase inhibitory activities. Interestingly, all bis-sulfonamide derivatives inhibited the aromatase with IC50 range of 0.05–11.6 μM except for compound 23. The analogs 15 and 16 bearing hydrophobic chloro and bromo groups exhibited the potent aromatase inhibitory activity in sub-micromolar IC50 values (i.e., 50 and 60 nM, respectively) with high safety index. Molecular docking revealed that the chloro and bromo benzenesulfonamides (15 and 16) may play role in the hydrophobic interaction with Leu477 of the aromatase to mimic steroidal backbone of the natural substrate, androstenedione. QSAR study also revealed that the most potent activity of compounds was governed by van der Waals volume (GATS6v) and mass (Mor03m) descriptors. Finally, the two compounds (15 and 16) were highlighted as promising compounds to be further developed as novel aromatase inhibitors.

Pubertal exposure to tebuconazole increases testosterone production via inhibiting testicular aromatase activity in rats

Tebuconazole is a triazole compound used agriculturally to treat plant pathogenic fungi. However, whether pubertal exposure to tebuconazole affects Leydig cell development remains unknown. Here, we exposed male Sprague-Dawley rats at 35 days of age to 0, 25, 50, or 100 mg kg−1 day−1 tebuconazole for 21 days. Tebuconazole exposure increased serum testosterone level but lowered estradiol level at a dose of 100 mg kg−1, without affecting serum luteinizing hormone and follicle-stimulating hormone concentrations. Tebuconazole up-regulated the expression of testicular Cyp11a1, Hsd11b1, and Fshr genes as well as their proteins at a dose of 100 mg kg−1. However, tebuconazole did not stimulate the proliferation of Leydig cells. Tebuconazole in vitro inhibits aromatase activity in primary rat Leydig cells with IC50 value of 40 μmol/L. In conclusion, tebuconazole exposure stimulates pubertal Leydig cell differentiation via inhibiting aromatase activity.

Acute exposure to 4-OH-A, not PCB1254, alters brain aromatase activity but does not adversely affect growth in zebrafish

Acute developmental exposure to pharmaceuticals or environmental contaminants can have deleterious, long lasting effects. Many of these compounds are endocrine disruptors (EDCs) that target estrogen signaling, with effects on reproductive and non-reproductive tissues. We recently reported that zebrafish larvae transiently exposed to the pharmaceutical EDC 4-OH-A display visual deficits as adults. Here, we examine whether these long-term effects are due to compound-induced morphological and/or cellular changes. Zebrafish aged 24 h, 48 h, 72 h, or 7 days post-fertilization (larvae) or 3-4mos (adults) were exposed to either 4-OH-A or PCB1254 for 24 h. After that time, notochord length, eye diameter, inter-eye distance, and heart rate were measured from larvae; and aromatase (estrogen synthase) activity was measured in homogenates of adult brain tissue. In general, indices of larval growth and development were not altered by 24 h exposure to either compound. 4-OH-A potently inhibited aromatase activity, while PCB1254 did not, with inhibition continuing even after removal from treatment. These results support differential function of EDCs and indicate that developmental exposure to 4-OH-A causes sustained inhibition of aromatase, which could be associated with altered adult behaviors.

The Multiple Biological Targets of Hops and Bioactive Compounds.

Botanical dietary supplements for women's health are increasingly popular. Older women tend to take botanical supplements such as hops as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Especially extracts from spent hops, the plant material remaining after beer brewing, are enriched in bioactive prenylated flavonoids that correlate with the health benefits of the plant. The chalcone xanthohumol (XH) is the major prenylated flavonoid in spent hops. Other less abundant but important bioactive prenylated flavonoids are isoxanthohumol (IX), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN). Pharmacokinetic studies revealed that these flavonoids are conjugated rapidly with glucuronic acid. XH also undergoes phase I metabolism in vivo to form IX, 8-PN, and 6-PN. Several hop constituents are responsible for distinct effects linked to multiple biological targets, including hormonal, metabolic, inflammatory, and epigenetic pathways. 8-PN is one of the most potent phytoestrogens and is responsible for hops' estrogenic activities. Hops also inhibit aromatase activity, which is linked to 8-PN. The weak electrophile, XH, can activate the Keap1-Nrf2 pathway and turn on the synthesis of detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 and glutathione S-transferase. XH also alkylates IKK and NF-κB, resulting in anti-inflammatory activity. Antiobesity activities have been described for XH and XH-rich hop extracts likely through activation of AMP-activated protein kinase signaling pathways. Hop extracts modulate the estrogen chemical carcinogenesis pathway by enhancing P450 1A1 detoxification. The mechanism appears to involve activation of the aryl hydrocarbon receptor (AhR) by the AhR agonist, 6-PN, leading to degradation of the estrogen receptor. Finally, prenylated phenols from hops are known inhibitors of P450 1A1/2; P450 1B1; and P450 2C8, 2C9, and 2C19. Understanding the biological targets of hop dietary supplements and their phytoconstituents will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.

Differential Toxicity of Alkylphenols in JEG-3 Human Placental Cells: Alteration of P450 Aromatase and Cell Lipid Composition.

Alkylphenols (APs) are a diverse class of chemicals that can cross the placental barrier and interfere with embryonic and fetal development. This work investigates the comparative toxicity, ability to inhibit aromatase activity, and to alter the lipid composition of 10 alkylphenols in the human placenta choriocarcinoma cell line JEG-3. Among the selected APs, 4-dodecylphenol (DP), 4-heptylphenol (HP), and 4-cumylphenol (CP) showed the highest cytotoxicity (EC50: 18-65 µM). Aromatase inhibition was closely related to the hydrophobicity of APs. HP significantly induced the generation of reactive oxygen species (ROS) (43-fold), inhibited placental aromatase activity (IC50: 41 µM), and induced a general dose-dependent depletion of polyunsaturated lipids (10-20 µM), which were attributed to high levels of oxidative stress. In contrast, 2,4,6-tri-tert-butylphenol (TTBP) significantly induced the intracellular accumulation of triacylglycerides (TGs), whereas DP increased the synthesis of phosphatidylcholines (PCs) and TGs at the expense of diacylglycerides (DGs). Overall, this study evidences the different modes of action of alkylphenols in human placental JEG-3 cells, describes differential lipidomic fingerprints, and highlights DP, HP, CP, and TTBP as the ones that caused the most harmful effects.

Aromatase inhibition by 2-methyl indole hydrazone derivatives evaluated via molecular docking and in vitro activity studies

A causal association is reported between prolonged exposures to elevated levels of estrogen and breast cancer. Therefore inhibiting aromatase (CYP19A), which catalyses the conversion of androgens to estrogens, is an important approach in prevention and treatment of estrogen receptor positive (ER+) breast cancer.Melatonin, a natural indolic hormone, is reported to prevent free radical induced carcinogenesis and block local estrogen synthesis in breast tissue via aromatase inhibition. However several features of melatonin limit its therapeutic use.In the present study aromatase inhibiting potential of 2-methyl indole hydrazones are investigated, and compared with melatonin, by two in vitro models; a cell-free assay using a fluorescence substrate and a cell-based assay where cell proliferation was determined in ER + human breast cancer cells (MCF-7 BUS) in the absence of estrogen and the presence of testosterone. Aromatase inhibitory effect is also explored by molecular modelling studies.In biological activity assays monochloro substituted indole hydrazones were found to have stronger aromatase inhibitory activity among all tested derivatives and were more active than melatonin. This finding is further confirmed by molecular modelling.These results may be useful in the design and synthesis of novel melatonin analogues with higher inhibitory potency against aromatase.

DRUG LIKENESS SCORING AND STRUCTURE ACTIVITY/PROPERTY RELATIONSHIPS OF 1,2,3-TRIAZOLE DERIVATEVES AS AROMATASE INHIBITOR

Molecular geometry, electronic structure and effect of the substitution for 1,2,3-triazole derivatives, have been studied by molecular mechanics, ab initio/HF and DFT /B3LYP. The molecular electrostatic potential surface (MESP) which displays the activity centers of a molecule and the substitution effects on its systems have been performed by HSAB (Hard Soft Acid and Base) principle application. Also, the structure-activity/property relationship studies were applied on twenty-two molecules of 1, 2, 3-triazole derivatives, all have the aromatase inhibitory activity. Results such as net charges, bond length, dipole moment, QSAR properties, Lipinski’s parameters, and Lipophilic Efficiency (LipE), are reported in this paper.

Integrated in silico-in vitro strategy for screening of some traditional Egyptian plants for human aromatase inhibitors

Ethnopharmacological relevanceAromatase enzyme (CYP19) is widely known as a critical target protein for treating hormone-dependent breast cancer. Natural products from traditional medicinal plants continue to be an active source of aromatase inhibitors. Meanwhile, high cost of experimental work and low hit rate associated with HTS have stimulated the implementation of in-silico virtual screening to resolve these pitfalls, where coupling of both classical wet lab procedure and VS may offer a more deepened access to bioactive compounds with less work and time waste. Aim of the studyIn this work, a sequential structure-based and ligand-based virtual screening strategy was utilized for investigating an in-house database of 1720 phytochemical constituents of 29 medicinal plants and natural products used in traditional Egyptian medicine to search for compounds with the potential to be used as inhibitors of the human aromatase enzyme. Materials and methodsThe suggested strategy included using Glide docking with its feature ‘extra precision (XP)’ for carrying out structure-based virtual screening (SBVS) where the resulting hits were further promoted to ligand-based virtual screening (LBVS) through the development of two pharmacophore and QSAR models; one for steroidal and the other for non-steroidal aromatase inhibitors. ResultsThe combined results revealed that Artemisia annua, Zingiber officinale, Cicer arietinum, Annona muricata and Vitex agnus castus were the top scoring plants in terms of in-silico activity scores, respectively. The hydro-alcoholic extracts and different solvent fractions of the top scoring plants were subsequently tested experimentally for their aromatase inhibitory activity, by the aid of in-vitro fluorometric assay. The rank ordering of the activities for the plants agreed with the ordering predicted on the basis of SBVS and LBVS workflow implemented. ConclusionThe suggested strategy provides a reliable means of prospecting in-silico screening of natural products databases in the search for new dug leads as aromatase inhibitors. The hits so obtained can then be subjected to further phytochemical studies, to isolate and identify suitable compounds for further in-vitro testing.

Synthesis and molecular docking of N,N′-disubstituted thiourea derivatives as novel aromatase inhibitors

A three series of thioureas, monothiourea type I (4a–g), 1,4-bisthiourea type II (5a–h) and 1,3-bisthiourea type III (6a–h) were synthesized. Their aromatase inhibitory activities have been evaluated. Interestingly, eight thiourea derivatives (4e, 5f–h, 6d, 6f–h) exhibited the aromatase inhibitory activities with IC50 range of 0.6–10.2 μM. The meta-bisthiourea bearing 4-NO2 group (6f) and 3,5-diCF3 groups (6h) were shown to be the most potent compounds with sub-micromolar IC50 values of 0.8 and 0.6 μM, respectively. Molecular docking also revealed that one of the thiourea moieties of these two compounds could mimic steroidal backbone of the natural androstenedione (ASD) via hydrophobic interactions with enzyme residues (Val370, Leu477, Thr310, and Phe221 for 6f, Val370, Leu477, Ser478, and Ile133 for 6h). This is the first time that the bisthioureas have been reported for their potential to be developed as aromatase inhibitors, in which the 4-NO2 and 3,5-diCF3 analogs have been highlighted as promising candidates.

Pyridine-substituted thiazolylphenol derivatives: Synthesis, modeling studies, aromatase inhibition, and antiproliferative activity evaluation.

Drugs used in breast cancer treatments target the suppression of estrogen biosynthesis. During this suppression, the main goal is to inhibit the aromatase enzyme that is responsible for the cyclization and structuring of estrogens either with steroid or non-steroidal-type inhibitors. Non-steroidal derivatives generally have a planar aromatic structure attached to the triazole ring system in their structures, which inhibits hydroxylation reactions during aromatization by coordinating the heme group. Bioisosteric replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase the selectivity for aromatase enzyme inhibition. In this study, pyridine-substituted thiazolylphenol derivatives, which are non-steroidal triazole bioisosteres, were synthesized using the Hantzsch method, and physical analysis and structural determination studies were performed. The IC50 values of the compounds were determined by a fluorescence-based aromatase inhibition assay. Then, their antiproliferative activities on the MCF7 and HEK 293 cell lines were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the crystal structure of human placental aromatase was subjected to a series of docking experiments to identify the possible interactions between the most active structure and the active site. Lastly, an in silico technique was performed to analyze and predict the drug-likeness, molecular and ADME properties of the synthesized molecules.

Discrete Fourier Transform-Based Multivariate Image Analysis: Application to Modeling of Aromatase Inhibitory Activity.

We recently generalized the formerly alignment-dependent multivariate image analysis applied to quantitative structure-activity relationships (MIA-QSAR) method through the application of the discrete Fourier transform (DFT), allowing for its application to noncongruent and structurally diverse chemical compound data sets. Here we report the first practical application of this method in the screening of molecular entities of therapeutic interest, with human aromatase inhibitory activity as the case study. We developed an ensemble classification model based on the two-dimensional (2D) DFT MIA-QSAR descriptors, with which we screened the NCI Diversity Set V (1593 compounds) and obtained 34 chemical compounds with possible aromatase inhibitory activity. These compounds were docked into the aromatase active site, and the 10 most promising compounds were selected for in vitro experimental validation. Of these compounds, 7419 (nonsteroidal) and 89 201 (steroidal) demonstrated satisfactory antiproliferative and aromatase inhibitory activities. The obtained results suggest that the 2D-DFT MIA-QSAR method may be useful in ligand-based virtual screening of new molecular entities of therapeutic utility.

3-(Azolylmethyl)-1H-indoles as Selective P450 Aromatase Inhibitors

The synthesis of 1-(halobenzyl) and 1-tosyl-3-(1H-imidazol-1-ylmethyl)-1H-indoles, 1-(halobenzyl) and 1-tosyl-3-(1H-1, 2, 4-triazol-1-ylmethyl)-1H-indoles and 1-(halobenzyl)-3-(4H-1, 2, 4-triazol-4-ylmethyl)-1H-indoles is described. 3-(Azolylmethyl)-1H-indoles were obtained in three steps from 1H-indole-3-carbaldehyde (1) by benzylation or tosylation, reduction and azole moiety fixation. In an alternative method, the bromo intermediates issued from the corresponding alcohols were condensed with the azolium sodium salts. Inhibitory activity against P450arom and P45017α, and influence on retinoic acid metabolism were evaluated. Three imidazole compounds exhibited potent and selective aromatase inhibitory activity.

3D-QSAR study of steroidal and azaheterocyclic human aromatase inhibitors using quantitative profile of protein\u2013ligand interactions

Aromatase is a member of the cytochrome P450 superfamily responsible for a key step in the biosynthesis of estrogens. As estrogens are involved in the control of important reproduction-related processes, including sexual differentiation and maturation, aromatase is a potential target for endocrine disrupting chemicals as well as breast cancer therapy. In this work, 3D-QSAR combined with quantitative profile of protein–ligand interactions was employed in the identification and characterization of critical steric and electronic features of aromatase-inhibitor complexes and the estimation of their quantitative contribution to inhibition potency. Bioactivity data on pIC50 values of 175 steroidal and 124 azaheterocyclic human aromatase inhibitors (AIs) were used for the 3D-QSAR analysis. For the quantitative description of the effects of the hydrophobic contact and nitrogen–heme–iron coordination on aromatase inhibition, the hydrophobicity density field model and the smallest dual descriptor Δf(r)S were introduced, respectively. The model revealed that hydrophobic contact and nitrogen–heme–iron coordination primarily determines inhibition potency of steroidal and azaheterocyclic AIs, respectively. Moreover, hydrogen bonds with key amino acid residues, in particular Asp309 and Met375, and interaction with the heme–iron are required for potent inhibition. Phe221 and Thr310 appear to be quite flexible and adopt different conformations according to a substituent at 4- or 6-position of steroids. Flexible docking results indicate that proper representation of the residues’ flexibility is critical for reasonable description of binding of the structurally diverse inhibitors. Our results provide a quantitative and mechanistic understanding of inhibitory activity of steroidal and azaheterocyclic AIs of relevance to adverse outcome pathway development and rational drug design.

Integrating regression and classification-based QSARs with molecular docking analyses to explore the structure-antiaromatase activity relationships of letrozole-based analogs

Aromatase is a multienzyme complex responsible for the biosynthesis of estrogen and its level has been found higher in breast cancer. Although the designing strategy of aromatase inhibitors (AIs) has continued for more than four decades, it may still be in demand to design highly effective and less toxic AIs. In this study, several chemometric approaches have been used to explore the important structural features of a series of letrozole-based analogs for their promising aromatase inhibitory activity. All techniques are statistically validated individually and in turn, validated with each other along with the structure–activity relationship (SAR) observations. The imidazole ring has been found to interact with the heme iron, whereas the triazole ring system has not shown any interaction. Moreover, imidazole function is better than 1,2,3-triazole, whereas 1,2,3-triazole is better than the 1,2,5-triazole ring system. Additionally, a bulky aryl substitution in the azole ring along with the orientation of the azole nitrogens and the cyanophenyl function has an essential role in the inhibition of aromatase. Furthermore, a cyano group substituted at the phenyl moiety interacts with Arg115, Met374, and Ser478 at the enzyme active site to form hydrogen bonding interactions. These observations are useful for designing potential AIs in the future.

AROMATASE INHIBITORY AND CYTOTOXIC ACTIVITIES OF CHEMICAL CONSTITUENTS FROM THE VIETNAMESE MEDICINAL PLANT BAN-CHI-LIEN (SCUTELLARIA BARBATA D. DON)

Aromatase inhibitory and cytotoxic activities were determined for apigenin, luteolin and the new diterpene named scutebarbalactone VN, which were obtained by bioassay-guided fractionation and isolation from the methanol extract of the Vietnamese medicinal plant Banchi-lien (Scutellaria barbata D. Don). In the aromatase inhibition assay, an IC50 value of 3.36 mM was found for scutebarbalactone VN, while IC50 values of 7.2 mM and 7.95 mM were found for the positive controls aminoglutethimide and b-estradiol, respectively. In the cytotoxicity assays using a panel of human cancer cell lines, scutebarbalactone VN showed promising anticancer activity with IC50 ranging from 2.15 to 8.3 mM compared with those of the positive control ellipticine ranging from 1.0 to 2.1 mM. Apigenin and luteolin were found to be inactive in both assays.

Ergostane-Type Sterols from King Trumpet Mushroom (Pleurotus eryngii) and Their Inhibitory Effects on Aromatase.

Two new ergostane-type sterols; (22E)-5α,6α-epoxyergosta-8,14,22-triene-3β,7β-diol (1) and 5α,6α-epoxyergost-8(14)-ene-3β,7α-diol (2) were isolated from the fruiting bodies of king trumpet mushroom (Pleurotus eryngii), along with eight known compounds (3–10). All isolated compounds were evaluated for their inhibitory effects on aromatase. Among them, 4 and 6 exhibited comparable aromatase inhibitory activities to aminoglutethimide.