Discovery of novel oestrogen receptor α agonists and antagonists by screening a revisited privileged structure moiety for nuclear receptors

Bisphenol A (BPA) is used as an industrial raw material for polycarbonate plastics and epoxy resins; however, various concerns have been reported regarding its status as an endocrine-disrupting chemical. BPA interacts not only with oestrogen receptors (ERs) but constitutive androstane receptor, pregnane X receptor, and oestrogen-related receptor γ (ERRγ); therefore, the bisphenol structure represents a privileged structure for the nuclear-receptor superfamily. Here, we screen 127 BPA-related compounds by competitive-binding assay using [3H]oestradiol and find that 20 compounds bind to ERα with high affinity. We confirm most of these as ERα agonists; however, four compounds, including bisphenol M and bisphenol P act as novel antagonists. These structures harbour three benzene rings in tandem with terminal hydroxy groups at para-positions, with this tandem tri-ring bisphenol structure representing a novel privileged structure for an ERα antagonist. Additionally, we perform an ab initio calculation and develop a new clipping method for halogen bonding or non-covalent interaction using DV-Xα evaluation for biomolecules.


Results eRα preferentially binds bisphenol structures.
We performed the competitive binding assays using [ 3 H]E2 against ERα to evaluate 127 commercially available bisphenol or benzylphenol derivatives, some of which are used as industrial raw materials for polycarbonate plastics. The CAS registry numbers, their common names, and the IUPAC names of all tested chemicals are provided in Supplementary Table 1. Those chemicals include 56 bisphenol-structure-containing chemicals, 10 benzylphenol-structure-containing chemicals, and 61 bisphenol or benzylphenol derivatives with hydroxyl groups substituted primarily with ester groups. Eighteen of the chemicals possess more than three phenyl rings. We found that 70 compounds (>55% of the compounds tested) bound to the ligand-binding domain (LBD) of ERα responsible for ligand-dependent activation function (i.e., the activation function-2 motif). Bisphenol C (BPC) bound to ERα with the highest affinity of all tested compounds, exhibiting a 50% inhibitory concentration (IC 50 ) of 2.81 nM, followed by 4,4′-(1,3-dimethylbuthylidene)bisphenol exhibiting an IC 50 of 5.75 nM. Notably, 20 compounds exhibited stronger or almost the same affinity than BPA as a weak ERα agonist, with their chemical structures shown in Fig. 1 and their respective binding affinity were determined by independently performed competitive binding assays using [ 3 H]E2 and summarized in Table 1. Their representative curves and the R 2 values are shown in Supplementary Fig. 1. The chemical names of the compounds that exhibited lower affinity than BPA are listed in Supplementary Table 2. Sixteen of the 21 compounds (76%; the 20 compounds plus BPA) harboured bisphenol moieties, with a total of 43 of 56 (77%) bisphenol-structure-containing compounds binding to ERα.
Bisphenol derivatives containing three tandem benzene rings exhibit eRα-antagonist activity.
Clipping of nitrogen atoms at the i-1 position is essential for ab initio calculations of LBD conformation. We used the crystal structure of the BPC-bound ERα LBD (PDB ID: 3UUC) to perform ab initio calculations to elucidate the mechanisms associated with the high binding affinity of BPC for the ERα-LBD,   Table 1 and www.nature.com/scientificreports www.nature.com/scientificreports/ which contains two halogen atoms, i.e., two chlorines. We selected amino acid residues near the chlorine atoms from the deposited structure for ab initio calculation. To increase the precision of the results, we used three compensating methods for calculations involving the terminal regions. Case 1 involved in-filling hydrogen atoms, Case 2 reconstructed each clipped amino acid residue as individual amino acids, and Case 3 expanded the analysed region to include the nitrogen atom at the i-1 position along with conventional protonation (Fig. 5a-c).
We performed ab initio calculations using the coordinate data of from 4 Å to 9 Å for the chlorine atoms of BPC ( Fig. 5d-i) in the ERα-BPC complex under the three conditions. The calculated bond overlap population of covalent bonds between chlorines and the carbon atom in the complex are summarized in Fig. 6 and Supplementary Table 3. The average values and their standard deviations of bond overlap population from 7 Å to 8 Å were 0.7072 ± 0.0104, 0.7268 ± 0.0098, and 0.7258 ± 0.0070 in Cases 1, 2, and 3, respectively. The bond overlap population is used to estimate the contribution of covalency in target bonds. The results showed that the compensating method used in Case 3 was useful for ab initio calculations of the LBD, because the calculated bond overlap populations were relatively stable and converged upon expansion of the target regions used for calculation. We defined Case 3 as the "halogen or non-covalent interaction by DV-Xα evaluation" clipping method (HIVE clip), with this representing a novel method for ab initio calculations of regions of large protein structures.
Determination of ligand-related space constraints for ab initio calculations. We evaluated the appropriate distance from the target ligand (BPC) necessary for ab initio calculations using the DV-Xα molecular-orbital calculation method. The crystal structure of the ERα-BPC complex was protonated and clipped based on the HIVE method in 0.5-Å increments from 4 Å to 9 Å from the chlorine atoms of BPC. The calculated energy level of the molecular orbitals and the energy diagram density of the chlorine atoms in the clipped region are illustrated in Supplementary Fig. 2. The bond overlap population of the chlorine and carbon atoms converged as we expanded the calculated region. The region from 7 Å to 9 Å, the bond overlap population of C-Cl(1) returned a value of 0.7295, with differences between each bond overlap population and the average value, [|ΔC-Cl(1)| = |C-Cl(1) n − C-Cl(1) average (7-9 Å) |]of 0.0020 (at 9 Å) and 0.0164 (at 4.5 Å). All calculated bond overlap populations are summarized in Supplementary Table 4. The results indicated that clipping the coordinates of the residues with protonated nitrogen atoms at the i-1 position within 7 Å of the chlorine atoms was appropriate for DV-Xα ab initio molecular-orbital calculation. Under this condition, the net charges of the chlorine atoms in the ERα-bound structure were −0.18934 and −0.20076, whereas those in the unbound structure were −0.16108 and −0.17064, respectively, suggesting that a net-charge shift occurred upon ERα binding to BPC. the lowest unoccupied molecular orbital (LUMo) is located close to bound BpC. We analysed the highest occupied molecular orbital (HOMO) and LUMO from the calculation and illustrated using VESTA 33 (Fig. 7), revealing a LUMO-HOMO energy gap of 1.0328 eV. Moreover, the LUMO was restricted to a position in close proximity to the BPC ligand in the ERα LBD.

Discussion
We screened 127 BPA derivatives and their related compounds, revealing that bisphenol derivatives containing three tandem benzene rings (i.e., tandem tri-ring bisphenols) representing novel privileged structures for ERα antagonists. Competitive-binding assays using [ 3 H]E2 showed that BPC bound to ER with the highest affinity, followed by 4,4′-(1,3-dimethylbuthylidene)bisphenol, and that BPM, α,α,α′-tris(4-hydroxyphenyl)-1-ethyl -4-isopropylbenzene, BPP, and α,α′-bis(4-hydroxy-3,5-dimethylphenyl)-1,4-diisopropylbenzene also bound to ERα. These tandem tri-ring bisphenols elicited antagonistic effects according to reporter-gene assays in HeLa cells and mainly due to structural conflicts between the C-ring in the tandem tri-ring bisphenols and Helix 11 of ERα. Additionally, bulky groups located on the sp 3 -carbon connecting the B-ring and the C-ring displace Helix 12 (i.e., the activation helix) of ERα. HeLa cells are generally used in the first screening according to the Organisation for Economic Co-operation and Development test guidelines No.455 (OECD TG455) 34 ; however, oestrogen receptor antagonists frequently show opposing activities in different tissue types, and future studies would provide clues for this unknown mechanism. Moreover, we confirmed that halogen-containing bisphenol derivatives (e.g. BPC) Figure 3. Antagonist activity of 4-OHT and BPA-related compounds according to luciferase-reporter assay. Antagonistic activities by the compounds are indicated as transcriptional activity relative to the luciferase activity induced by 10 nM E2 in the absence of a compound (indicated as control). One-way analysis of variance was performed to analyse significant inhibition of E2-induced activity relative to the activity observed in the absence of the compounds (control). *p < 0.001; **p < 0.0001. www.nature.com/scientificreports www.nature.com/scientificreports/ bound to ERα with high affinity. Common methods used for ab initio analysis of large biomolecules include fragmentation methods 35,36 , although improved systematic fragmentation methods are required. Our results of the ab initio calculation of the ERα-BPC complex indicated that the novel HIVE clip method described in this study was beneficial for evaluating the ligand-bound conformation of the large biomolecules.
Previous studies reported the binding affinities of bisphenol derivatives using competitive binding assay [37][38][39][40] , and screening experiments for ERα−related transcriptional activity of ERα were commonly performed using reporter-gene assays. Two studies analysed the transcriptional activities of structurally related BPA derivatives as ERα agonists, with 19 and 55 compounds systematically examined, respectively, although these included neither  www.nature.com/scientificreports www.nature.com/scientificreports/ BPC nor 4,4′-(1,3-dimethylbuthylidene)bisphenol have not been analyzed in both papers 41,42 . The 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are essential for ERα-related transcriptional activity, with the bridging alkyl groups changing their respectively influences 42 . Bisphenol moieties connected by oxime esters show agonistic activity for ERα with suppress cell proliferation in cancer cells 43,44 . The estrogenic activities of contaminants in BPA used for industrial purposes were previously analysed by yeast two-hybrid assays 45 , identifying 4,4′-(1,3-dimethylbuthylidene)bisphenol as exerting 8-fold higher estrogenic activity than laboratory grade BPA. In the present study, our binding assays revealed that 4,4′-(1,3-dimethylbuthylidene) bisphenol bound the to ERα LBD with an IC 50 value of 5.75 nM, which was sufficient to induce ERα-related activity. A previous study reported high-affinity BPC-ERα binding in ERα-expressing cells, with BPC displaying both agonistic and partially antagonistic activities 46 . The crystal structure of the ERα LBD-BPC complex shows the antagonistic form of BPC binding to ERα 46 . In the present study, we confirmed the high affinity of BPC for the ERα LBD and its role as an ERα agonist to a similar degree as that reported for BPA 46 ; however, we did not observe any antagonistic activity by BPC on ERα. A possible explanation might be the use of different cell types, as the previous study used and an HELN cell line stably expressing an ERα-reporter gene 46 , whereas we analysed ERα-related transcriptional activity using transiently transfected HeLa cells. In some cases, high copy numbers introduced by transfected plasmids result in high levels of protein expression, and elevated ERα concentrations www.nature.com/scientificreports www.nature.com/scientificreports/ in cells might affect their associated transcriptional activity; it is also well known that some oestrogen-receptor ligands act as selective oestrogen receptor modulators (SERMs); therefore further study is needed to clarify this point. We demonstrated for the first time that 9,9-bis(4-hydroxyphenyl)fluorine directly binds to the ERα LBD according to a competitive binding assay using [ 3 H]E2. This result agrees with the results of a recent report warning that a BPA substitute, 9,9-bis(4-hydroxyphenyl)fluorine, shows apparent anti-estrogenic activity both in vitro and in vivo 47 . Furthermore, we found that 9,9-bis(4-hydroxy3-methylphenyl)fluorine bound to the ERα LBD with www.nature.com/scientificreports www.nature.com/scientificreports/ higher affinity than 9,9-bis(4-hydroxyphenyl)fluorine, suggesting that increased focus should be given to this compound in order to assess to its safety when it is utilized as a BPA alternative.
BPC and BPA share a similar structure; however, BPC binds to the ERα LBD with higher affinity than BPA. Because BPC contains two chlorine atoms, halogen bonds derived from chlorine atoms are possible causes for these strong interactions 31,48,49 . To elucidate the binding mechanism, we performed the ab initio molecular orbital calculations using the coordinates of the BPC-bound ERα LBD crystal structure and the HIVE clip method to mimic the peptide bonds. The results showed the calculated LUMO located near the bound ligand moiety, representing the first demonstration of this in a ligand-receptor complex. It is reasonable that LUMO would be restricted near the bound ligand according to use of the HIVE clip method based on its application of a specific clipping strategy using coordinates of protein structures. Further studies including full-electron ab initio calculations are needed to reveal the detailed significance of these results.
In summary, we identified a tandem tri-ring bisphenol moiety, such as that found in BPM and BPP, as a novel privileged structure for ERα antagonists. Our results illustrated the LUMO of the BPC-ERα complex crystal structure by ab initio calculation according to coordinates selected using our novel HIVE clip method. These results demonstrated the efficacy of this method to accelerate protein-structure analysis and/or simulation for the purpose of screening of ligand-receptor interactions. Radioligand binding assays. We performed the radioligand binding assays for ERα according to previously reported by method 50  for 2 h at 20 °C in order to estimate total binding. Specific binding was calculated by subtracting the nonspecific binding determined by addition of 10 μM non-radiolabeled E2 to another set of working solutions from the observed total binding. After incubation, free radioligand was removed by addition and incubation of 100 μL 0.4% dextran-coated charcoal (Sigma-Aldrich) in phosphate-buffered saline (PBS; pH 7.4) on ice for 10 min. After centrifugation for 10 min at 15,000 rpm, the radioactivity of each supernatant was measured using a liquid scintillation counter (LS6500; Beckman Coulter, Fullerton, CA, USA). The data of the calculated specific binding of [ 3 H]E2 were assessed by Scatchard plot analysis 51 .

Methods
The binding affinities of the test compounds were evaluated by competitive binding assays. Each chemical was dissolved in DMSO to prepare a 1.0-mM stock solution, followed by subsequent incubation of serial dilutions (1 pM to 10 μM) with GST-ERα-LBD (20 ng) and [ 3 H]E2 (5 nM, final) for 2 hrs at 20 °C to assess their ability to hinder the binding of [ 3 H]E2. Free radioligand was absorbed by 0.4% dextran-coated charcoal for the saturation binding assay and removed by the vacuum filtration system using a 96-well filtration plate (MultiScreenHTS HV; 0.45-mm pore size; Merck KGaA, Darmstadt, Germany). The radioactivities of the eluents were measured using a TopCount NXT system (PerkinElmer) for 3 min/well. To determine the binding affinity of each test compound, IC 50 values were calculated from the dose-response curves generated by nonlinear regression analysis using the software package Prism (GraphPad Software Inc., La Jolla, CA, USA).
Luciferase-reporter assay for evaluating agonist activity. HeLa cells were maintained in Eagle's minimum essential medium (MEM; Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) supplemented with 10% (v/v) foetal bovine serum treated with dextran-coated charcoal at 37 °C under 5% CO 2 . To analyse the agonist activity, HeLa cells were incubated at a density of 5 × 10 5 cells per 60-mm dish for 24 h, followed by transfection of 3 μg of the reporter plasmid (3 × ERE/pGL4.23) and 1 μg of the ERα expression plasmid (ERα/pcDNA3.1) using Lipofectamine LTX reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. After 24 h, cells were harvested, suspended in Eagle's MEM, and seeded into 96-well plates at 5 × 10 4 cells/well, followed by treatment with a series of the test compounds diluted with 1% bovine serum albumin (BSA)/PBS (v/v) to prepare different concentrations (0-10 μM). After a 24-h incubation, luciferase activity was measured using a Luciferase Assay system (Promega, Madison, WI, USA) according to the manufacturer's instructions. Briefly, cells were lysed using reporter lysis buffer (Promega), and luminescence was measured using a Wallace 1420 ARVOsx multilabel counter (PerkinElmer). Cells treated only with 1% BSA/PBS were used as a vehicle control. Each assay was performed in triplicate and repeated at least three times. (2019) 9:9954 | https://doi.org/10.1038/s41598-019-46272-y www.nature.com/scientificreports www.nature.com/scientificreports/ To evaluate antagonistic activity, we examined serial concentrations of test compounds (0-10 μM, final) in the presence of 1 nM or 10 nM concentrations of E2, which normally induces basal levels of ERα-related transcriptional activity 1 .
First principal molecular orbital calculation. We performed the first principal molecular orbital calculation (i.e., ab initio calculation) in order to illuminate the strong affinity of halogen-containing bisphenol derivatives. We used the DV-Xα cluster method 30 , to calculate the electron states of a solo BPC compound and the BPC-ERα structure using Slater's exchange potential and numerical basis functions. We calculated bond overlap population and net charge using Mulliken population analysis. The bond overlap population obtained using this method represents a measure of the covalent bonding between target atoms. The 3D coordinates of the BPC-ERα complex were obtained from the PDB (PDB ID: 3UUC).