Identification of Non-steroidal Aromatase Inhibitors via In silico and In vitro Studies

INTRODUCTION: Breast cancer is the most common cancer affecting women worldwide, including Pakistan. More than half of breast cancer patients have hormone-dependent breast cancer, which is developed due to the over-production of estrogen (the main hormone in breast cancer). METHOD: The biosynthesis of estrogen is catalyzed by the aromatase enzyme, which thus serves as a target for the treatment of breast cancer. During the current study, biochemical, computational, and STD-NMR methods were employed to identify new aromatase inhibitors. A series of phenyl-3- butene-2-one derivatives 1-9 were synthesized and evaluated for human placental aromatase inhibitory activity. Among them, four compounds 2, 3, 4, and 8 showed a moderate to weak inhibitory activity (IC50 = 22.6 - 47.9 µM), as compared to standard aromatase inhibitory drugs, letrozole (IC50 = 0.0147 ± 1.45 µM), anastrozole (IC50 = 0.0094 ± 0.91 µM), and exemestane (IC50 = 0.2 ± 0.032 µM). Kinetic studies on two moderate inhibitors, 4 and 8, revealed a competitive- and mixed-type of inhibition, respectively. RESULT: Docking studies on all active compounds indicated their binding adjacent to the heme group and interaction with Met374, a critical residue of aromatase. STD-NMR further highlighted the interactions of these ligands with the aromatase enzyme. CONCLUSION: STD-NMR-based epitope mapping indicated close proximity of the alkyl chain followed by an aromatic ring with the receptor (aromatase). These compounds were also found to be non-cytotoxic against human fibroblast cells (BJ cells). Thus, the current study has identified new aromatase inhibitors (compounds 4, and 8) for further pre-clinical and clinical research.

Switching from Aromatase Inhibitors to Dual Targeting Flavonoid-Based Compounds for Breast Cancer Treatment.

Despite the significant outcomes attained by scientific research, breast cancer (BC) still represents the second leading cause of death in women. Estrogen receptor-positive (ER+) BC accounts for the majority of diagnosed BCs, highlighting the disruption of estrogenic signalling as target for first-line treatment. This goal is presently pursued by inhibiting aromatase (AR) enzyme or by modulating Estrogen Receptor (ER) α. An appealing strategy for fighting BC and reducing side effects and resistance issues may lie in the design of multifunctional compounds able to simultaneously target AR and ER. In this paper, previously reported flavonoid-related potent AR inhibitors were suitably modified with the aim of also targeting ERα. As a result, homoisoflavone derivatives 3b and 4a emerged as well-balanced submicromolar dual acting compounds. An extensive computational study was then performed to gain insights into the interactions the best compounds established with the two targets. This study highlighted the feasibility of switching from single-target compounds to balanced dual-acting agents, confirming that a multi-target approach may represent a valid therapeutic option to counteract ER+ BC. The homoisoflavone core emerged as a valuable natural-inspired scaffold for the design of multifunctional compounds.

Dual aromatase-steroid sulfatase inhibitors (DASI's) for the treatment of breast cancer: a structure guided ligand based designing approach.

Dual aromatase-steroid sulfatase inhibitors (DASIs) lead to significant deprivation of estrogen levels as compared to a single target inhibition and thereby exhibited an additive or synergistic effect in the treatment of hormone-dependent breast cancer (HDBC). Triazole-bearing DASI's having structural features of clinically available aromatase inhibitors are identified as lead structures for optimization as DASI's. To identify the spatial fingerprints of target-specific triazole as DASI's, we have performed molecular docking assisted Gaussian field-based comparative 3D-QSAR studies on a dataset with dual aromatase-STS inhibitory activities. Separate contours were generated for both aromatase and steroid sulphates showing respective pharmacophoric structural requirements for optimal activity. These developed 3D-QSAR models also showed good statistical measures with the excellent predictive ability with PLS-generated validation constraints. Comparative steric, electrostatic, hydrophobic, HBA, and HBD features were elucidated using respective contour maps for selective target-specific favourable activity. Furthermore, the molecular docking was used for elucidating the mode of binding as DASI's along with the MD simulation of 100 ns revealed that all the protease-ligand docked complexes are overall stable as compared to reference ligand (inhibitor ASD or Irosustat) complex. Further, the MM-GBSA study revealed that compound 24 binds to aromatase as well as STS active site with relatively lower binding energy than reference complex, respectively. A comparative study of these developed multitargeted QSAR models along with molecular docking and dynamics study can be employed for the optimization of drug candidates as DASI's.Communicated by Ramaswamy H. Sarma.

Design, synthesis, molecular modeling, DFT, ADME and biological evaluation studies of some new 1,3,4-oxadiazole linked benzimidazoles as anticancer agents and aromatase inhibitors.

Breast cancer is the most frequent female cancer and second cause of cancer-related deaths among women around the world. Two thirds of breast cancer patients have hormone-dependent tumors, which is very likely be treated with hormonal therapy. Aromatase is involved in the biosynthesis of estrogen thus a critical target for breast cancer. In this study, in order to identify new aromatase enzyme inhibitors, a series of benzimidazole-1,3,4-oxadiazole derivatives were synthesized and characterized by 1H NMR, 13C NMR, and MS spectra analyses. In the in vitro anticancer assay, all the compounds tested anticancer activities using MTT-based assay against five cancer cell lines (MCF-7, A549, HeLa, C6, and HepG2). Among them, compound 5a exhibited the most potent activity with IC50 values of 5.165 ± 0.211 µM and 5.995 ± 0.264 µM against MCF-7 and HepG2 cell lines. Compound 5a was included in the BrdU test to determine the DNA synthesis inhibition effects for both cell types. Furthermore, compound 5c was also found to be more effective than doxorubicin on the HeLa cell line. The selectivity of anticancer activity was evaluated in NIH3T3 cell line. In vitro, enzymatic inhibition assays of aromatase enzyme were performed for compound 5a acting on the MCF-7 cell line. For compound 5a, in silico molecular docking and dynamics simulations against aromatase enzyme was performed to determine possible protein-ligand interactions and stability. DFT study was performed to evaluate the quantum mechanical and electronic properties of compound 5a. Finally, the theoretical ADME properties of the potential aromatase inhibitor compound 5a were analyzed by calculations.Communicated by Ramaswamy H. Sarma.

Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

Human aromatase is the cytochrome P450 catalyzing the conversion of androgens into estrogens in a three steps reaction essential to maintain steroid hormones balance. Here we report the capture and spectroscopic characterization of its compound I (Cpd I), the main reactive species in cytochromes P450. The typical spectroscopic transitions indicating the formation of Cpd I are detected within 0.8 s when mixing aromatase with meta-chloroperoxybenzoic acid. The estrogen product is obtained from the same reaction mixture, demonstrating the involvement of Cpd I in aromatization reaction. Site-directed mutagenesis is applied to the acid-alcohol pair D309 and T310 and to R192, predicted to be part of the proton relay network. Mutants D309N and R192Q do not lead to Cpd I with an associated loss of activity, confirming that these residues are involved in proton delivery for Cpd I generation. Cpd I is captured for T310A mutant and shows 2.9- and 4.4-fold faster rates of formation and decay, respectively, compared to wild-type (WT). However, its activity is lower than the WT and a larger amount of H2 O2 is produced during catalysis, indicating that T310 has an essential role in proton gating for generation of Cpd 0 and Cpd I and for their stabilization. The data provide new evidences on the role of threonine belonging to the conserved "acid-alcohol" pair and known to be crucial for oxygen activation in cytochromes P450.

Novel 1,2,3-Triazole Derivatives as Mimics of Steroidal System-Synthesis, Crystal Structures Determination, Hirshfeld Surfaces Analysis and Molecular Docking.

Herein, we present the synthesis and crystal structures determination of five 4-(1-phenyl-1H-1,2,3-triazol-4-yl)phenol derivatives containing halogen atoms, 6a-e, which may be used as an excellent mimic of steroids in the drug development process. Good quality crystals obtained for all of the synthesized compounds allowed the analysis of their molecular structures. Subsequently, the determined crystal structures were used to calculate the Hirshfeld surfaces for each of the synthesized compounds. Furthermore, results of our docking studies indicated that synthesized derivatives are able to bind effectively to the active sites of selected enzymes and receptors involved in the hormone biosynthesis and signaling pathways, analogously to the native steroids.

A review on diverse heterocyclic compounds as the privileged scaffolds in non-steroidal aromatase inhibitors.

Breast cancer, emerging malignancy is common among women due to overexpression of estrogen. Estrogens are biosynthesized from androgens by aromatase, a cytochrome P450 enzyme complex, and play a pivotal role in stimulating cell proliferation. Therefore, deprivation of estrogen by blocking aromatase is considered as the effective way for the inhibition and treatment of breast cancer. In recent years, various non-steroidal heterocyclic functionalities have been extensively developed and studied for their aromatase inhibition activity. This review provides information about the structural-activity relationship of heterocycles (Type II) towards aromatase. This aids the medicinal chemist around the significance of different heterocyclic moieties and helps to design potent aromatase inhibitors.

Base mediated synthesis of functionalized 2-(alkynyl)arylnitriles and their molecular docking study with aromatase receptor.

A simple, efficient, and transition metal-free approach to synthesize functionalized 2-(alkynyl)benzonitriles has been developed using suitably functionalized 2H-pyran-2-ones and 4-phenyl/trimethylsilanyl-but-3-yn-2-ones as precursors. The reaction proceeds in the presence of a base at room temperature to yield internal as well as terminal alkynes. The structure of the synthesized compound was confirmed by single-crystal X-ray analysis. The molecular docking study was performed to evaluate the binding mode of action of newly synthesized alkyne derivatives with known human breast cancer target receptor aromatase (PDB ID: 3EQM).

Molecular and Structural Evolution of Cytochrome P450 Aromatase.

Aromatase is the cytochrome P450 enzyme converting androgens into estrogen in the last phase of steroidogenesis. As estrogens are crucial in reproductive biology, aromatase is found in vertebrates and the invertebrates of the genus Branchiostoma, where it carries out the aromatization reaction of the A-ring of androgens that produces estrogens. Here, we investigate the molecular evolution of this unique and highly substrate-selective enzyme by means of structural, sequence alignment, and homology modeling, shedding light on its key role in species conservation. The alignments led to the identification of a core structure that, together with key and unique amino acids located in the active site and the substrate recognition sites, has been well conserved during evolution. Structural analysis shows what their roles are and the reason why they have been preserved. Moreover, the residues involved in the interaction with the redox partner and some phosphorylation sites appeared late during evolution. These data reveal how highly substrate-selective cytochrome P450 has evolved, indicating that the driving forces for evolution have been the optimization of the interaction with the redox partner and the introduction of phosphorylation sites that give the possibility of modulating its activity in a rapid way.

Melatonin as an Oncostatic Molecule Based on Its Anti-Aromatase Role in Breast Cancer.

Breast cancer is the most common type of cancer. In the developmental stages of breast cancer, estrogens are strongly involved. As estrogen synthesis is regulated by the enzyme aromatase, targeting the activity of this enzyme represents a therapeutic option. The pineal hormone melatonin may exert a suppressive role on aromatase activity, leading to reduced estrogen biosynthesis. A melatonin-mediated decrease in the expression of aromatase promoters and associated genes would provide suitable evidence of this molecule's efficacy as an aromatase inhibitor. Furthermore, melatonin intensifies radiation-induced anti-aromatase effects and counteracts the unwanted disadvantages of chemotherapeutic agents. In this manner, this review summarizes the inhibitory role of melatonin in aromatase action, suggesting its role as a possible oncostatic molecule in breast cancer.

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase.

Aromatase (CYP19A1) catalyzes the synthesis of estrogens from androgens and is an invaluable target of pharmacotherapy for estrogen-dependent cancers. CYP19A1 is also one of the most primordial human CYPs and, to the extent that its fundamental dynamics are conserved, is highly relevant to understanding those of the more recently evolved and promiscuous enzymes. A complementary approach employing molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to interrogate the changes in CYP19A1 dynamics coupled to binding androstenedione (ASD). Gaussian-accelerated molecular dynamics and HDX-MS agree that ASD globally suppresses CYP19A1 dynamics. Bimodal HDX patterns of the B'-C loop potentially arising from at least two conformations are present in free 19A1 only, supporting the possibility that conformational selection is operative. Random-acceleration molecular dynamics and adaptive biasing force simulations illuminate ASD's binding pathway, predicting ASD capture in the lipid headgroups and a pathway to the active site shielded from solvent. Intriguingly, the predicted access channel in 19A1 aligns well with the steroid binding sites of other human sterol-oxidizing CYPs.

Synthesis, investigation of biological effects and in silico studies of new benzimidazole derivatives as aromatase inhibitors.

Inhibition of aromatase enzymes is very important in the prevention of estrogen-related diseases and the regulation of estrogen levels. Aromatase enzyme is involved in the final stage of the biosynthesis of estrogen, in the conversion of androgens to estrogen. The development of new compounds for the inhibition of aromatase enzymes is an important area for medicinal chemists in this respect. In the present study, new benzimidazole derivatives have been designed and synthesized which have reported anticancer activity in the literature. Their anticancer activity was evaluated against human A549 and MCF-7 cell lines by MTT assay. In the series, concerning MCF-7 cell line, the most potent compounds were the 4-benzylpiperidine derivatives 2c, 2g, and 2k with IC50 values of 0.032 ± 0.001, 0.024 ± 0.001, and 0.035 ± 0.001 µM, respectively, compared to the reference drug cisplatin (IC50 = 0.021 ± 0.001 µM). Then, these compounds were subject to further in silico aromatase enzyme inhibition assays to determine the possible binding modes and interactions underlying their activity. Thanks to molecular docking studies, the effectiveness of these compounds against aromatase enzyme could be simulated. Consequently, it has been found that these compounds can be settled very properly to the active site of the aromatase enzyme.

Effector role of cytochrome P450 reductase for androstenedione binding to human aromatase.

Cytochromes P450 constitute a large superfamily of monooxygenases involved in many metabolic pathways. Most of them are not self-sufficient and need a reductase protein to provide the electrons necessary for catalysis. It was shown that the redox partner plays a role in the modulation of the structure and function of some bacterial P450 enzymes. Here, the effect of NADPH-cytochrome reductase (CPR) on human aromatase (Aro) is studied for what concerns its role in substrate binding. Pre-steady-state kinetic experiments indicate that both the substrate binding rates and the percentage of spin shift detected for aromatase are increased when CPR is present. Moreover, aromatase binds the substrate through a conformational selection mechanism, suggesting a possible effector role of CPR. The thermodynamic parameters for the formation of the CPR-Aro complex were studied by isothermal titration calorimetry. The dissociation constant of the complex formation is 4.5 folds lower for substrate-free compared to the substrate-bound enzyme. The enthalpy change observed when the CPR-Aro complex forms in the absence of the substrate are higher than in its presence, indicating that more interactions are formed/broken in the former case. Taken together, our data confirm that CPR has a role in promoting aromatase conformation optimal for substrate binding.

A requirement for an active proton delivery network supports a compound I-mediated C-C bond cleavage in CYP51 catalysis.

CYP51 enzymes (sterol 14α-demethylases) are cytochromes P450 that catalyze multistep reactions. The CYP51 reaction occurs in all biological kingdoms and is essential in sterol biosynthesis. It removes the 14α-methyl group from cyclized sterol precursors by first forming an alcohol, then an aldehyde, and finally eliminating formic acid with the introduction of a Δ14-15 double bond in the sterol core. The first two steps are typical hydroxylations, mediated by an electrophilic compound I mechanism. The third step, C-C bond cleavage, has been proposed to involve either compound I (i.e. FeO3+) or, alternatively, a proton transfer-independent nucleophilic ferric peroxo anion (compound 0, i.e. Fe3+O2-). Here, using comparative crystallographic and biochemical analyses of WT human CYP51 (CYP51A1) and its D231A/H314A mutant, whose proton delivery network is destroyed (as evidenced in a 1.98-Å X-ray structure in complex with lanosterol), we demonstrate that deformylation of the 14α-carboxaldehyde intermediate requires an active proton relay network to drive the catalysis. These results indicate a unified, compound I-based mechanism for all three steps of the CYP51 reaction, as previously established for CYP11A1 and CYP19A1. We anticipate that our approach can be applied to mechanistic studies of other P450s that catalyze multistep reactions, such as C-C bond cleavage.

Conformational selection is present in ligand binding to cytochrome P450 19A1 lipoprotein nanodiscs.

Cytochromes P450 (CYPs) display remarkable plasticity in their ability to bind substrates and catalyze a broad array of chemical reactions. Herein we evaluate binding of androstenedione, testosterone, and 7-hydroxyflavone to CYP19A1, also known as aromatase, in phospholipid nanodiscs by stopped-flow UV-vis spectroscopy. Exponential fitting of the kinetic traces supports the possibility of a multi-step binding mechanism. Subsequent global fitting of the data to the solutions of the coupled differential equations describing the fundamental mechanisms of induced fit and conformational selection, consistently support presence of the latter. To our knowledge, this is the first discrimination of conformational selection from induced fit for a mono-disperse CYP in a native-like membrane environment. In addition, 7-hydroxyflavone binds to CYP19A1 nanodiscs with comparable affinity to the substrates and induces an unusual spectral response likely attributable to hydrogen bonding to, rather than displacement of the heme-coordinated water molecule.

Long Noncoding RNA HUPCOS Promotes Follicular Fluid Androgen Excess in PCOS Patients via Aromatase Inhibition.

CONTEXT: Androgen excess is a key feature of polycystic ovary syndrome (PCOS), but the underlying molecular mechanism remains unclear. OBJECTIVE: To determine the role and mechanism of novel long noncoding RNA (lncRNA) highly up-regulated in PCOS (HUPCOS) in the androgen excess of PCOS patients. DESIGN: The lncRNA expression profile in granulosa cells derived from PCOS and non-PCOS women were analyzed by using microarray assay. Human granulosa cell line KGN was used for mechanism investigation. SETTING: This was a university-based study. PATIENTS: Thirty-eight PCOS and 38 control patients were recruited: 8 PCOS and 8 control samples used for microarray discovery, the remaining 30 PCOS cases and 30 controls for quantitative RT-PCR validation. MAIN OUTCOME MEASURES: The aberrant expression lncRNA profile of PCOS patients was measured using microarray. The relationship of HUPCOS and follicular fluid testosterone was measured. Aromatase expression were analyzed after HUPCOS downregulation. HUPCOS interaction protein was confirmed by RNA pull-down. RESULTS: The significantly elevated lncRNA in PCOS granulosa cells was named HUPCOS, which was positively correlated with follicular fluid testosterone of PCOS patients. HUPCOS downregulation increased aromatase expression and promoted conversion of androgen to estrogen. RNA-binding protein with multiple splicing (RBPMS) was the most likely protein that combined with HUPCOS. CONCLUSIONS: Our findings suggested that HUPCOS mediated androgen excess in follicular fluid of PCOS patients by suppressing aromatase expression via interaction with RBPMS.

Towards an Understanding of the Mode of Action of Human Aromatase Activity for Azoles through Quantum Chemical Descriptors-Based Regression and Structure Activity Relationship Modeling Analysis.

Aromatase is an enzyme member of the cytochrome P450 superfamily coded by the CYP19A1 gene. Its main action is the conversion of androgens into estrogens, transforming androstenedione into estrone and testosterone into estradiol. This enzyme is present in several tissues and it has a key role in the maintenance of the balance of androgens and estrogens, and therefore in the regulation of the endocrine system. With regard to chemical safety and human health, azoles, which are used as agrochemicals and pharmaceuticals, are potential endocrine disruptors due to their agonist or antagonist interactions with the human aromatase enzyme. This theoretical study investigated the active agonist and antagonist properties of "chemical classes of azoles" to determine the relationships of azole interaction with CYP19A1, using stereochemical and electronic properties of the molecules through classification and multilinear regression (MLR) modeling. The antagonist activities for the same substituent on diazoles and triazoles vary with its chemical composition and its position and both heterocyclic systems require aromatic substituents. The triazoles require the spherical shape and diazoles have to be in proper proportion of the branching index and the number of ring systems for the inhibition. Considering the electronic aspects, triazole antagonist activity depends on the electrophilicity index that originates from interelectronic exchange interaction (ωHF) and the LUMO energy ( E LUMO PM 7 ), and the diazole antagonist activity originates from the penultimate orbital ( E HOMONL PM 7 ) of diazoles. The regression models for agonist activity show that it is opposed by the static charges but favored by the delocalized charges on the diazoles and thiazoles. This study proposes that the electron penetration of azoles toward heme group decides the binding behavior and stereochemistry requirement for antagonist activity against CYP19A1 enzyme.

Mechanistic Insights into the Regio- and Stereoselectivities of Testosterone and Dihydrotestosterone Hydroxylation Catalyzed by CYP3A4 and CYP19A1.

The hydroxylation of nonreactive C-H bonds can be easily catalyzed by a variety of metalloenzymes, especially cytochrome P450s (P450s). The mechanism of P450 mediated hydroxylation has been intensively studied, both experimentally and theoretically. However, understanding the regio- and stereoselectivities of substrates hydroxylated by P450s remains a great challenge. Herein, we use a multi-scale modeling approach to investigate the selectivity of testosterone (TES) and dihydrotestosterone (DHT) hydroxylation catalyzed by two important P450s, CYP3A4 and CYP19A1. For CYP3A4, two distinct binding modes for TES/DHT were predicted by dockings and molecular dynamics simulations, in which the experimentally identified sites of metabolism of TES/DHT can access to the catalytic center. The regio- and stereoselectivities of TES/DHT hydroxylation were further evaluated by quantum mechanical and ONIOM calculations. For CYP19A1, we found that sites 1ß, 2ß and 19 can access the catalytic center, with the intrinsic reactivity 2ß>1ß>19. However, our ONIOM calculations indicate that the hydroxylation is favored at site 19 for both TES and DHT, which is consistent with the experiments and reflects the importance of the catalytic environment in determining the selectivity. Our study unravels the mechanism underlying the selectivity of TES/DHT hydroxylation mediated by CYP3A4 and CYP19A1 and is helpful for understanding the selectivity of other substrates that are hydroxylated by P450s.

In silico molecular docking and in vitro antioxidant activity studies of novel α-aminophosphonates bearing 6-amino-1,3-dimethyl uracil.

In the present study, a new series of α-Aminophosphonates bearing 6-amino-1,3-dimethyluracil was synthesized in good to excellent yields (78-95%) by one-pot, three-component reaction of 6-amino-1,3-dimethyluracil, aromatic aldehydes and diethylphosphite via Kabachnik-Fields reaction by using an eco-friendly Eaton's reagent. All the compounds were screened for in vitro antioxidant studies by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H2O2) methods. Among the synthesized bioactive molecules, 4a, 4d, 4g, and 4h exhibited promising antioxidant activity compared with the standard drug Ascorbic acid. Furthermore, in order to support the biological results of the compounds, molecular docking studies were performed against Aromatase enzyme for four compounds which revealed that the compounds 4a, 4d, 4g, and 4h have significant binding modes, with docking scores of -8.6, -8.4, -8.1 and -8.1 respectively and the compound 4b specifically has equal dock score of -8.0 when compared with the standard drug Exemestane.

All-Atom Simulations Disclose How Cytochrome Reductase Reshapes the Substrate Access/Egress Routes of Its Partner CYP450s.

Cytochromes P450 enzymes (CYP450s) promote the oxidative metabolism of a variety of substrates via the electrons supplied by the cytochrome P450 reductase (CPR) and upon formation of a CPR/CYP450 adduct. In spite of the pivotal regulatory importance of this process, the impact of CPR binding on the functional properties of its partner CYP450 remains elusive. By performing multiple microsecond-long all-atom molecular dynamics simulations of a 520 000-atom model of a CPR/CYP450 adduct embedded in a membrane mimic, we disclose the molecular terms for their interactions, considering the aromatase (HA) enzyme as a proxy of the CYP450 family. Our study strikingly unveils that CPR binding alters HA's functional motions, bolstering a change in the shape and type of the channels traveled by substrates/products during their access/egress to/from the enzyme's active site. Our outcomes unprecedentedly contribute to extricate the many entangled facets of the CYP450 metabolon, redrafting its intricate panorama from an atomic-level perspective.