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

Abstract

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.

Figure 1

Chemical structures of BPA and its related chemicals. BPA and 20 chemicals exhibiting binding ability stronger than or comparable to BPA are shown, in with BPC having the highest affinity to ERα. 9,9-Bis(4-hydroxyphenyl)fluorine exerted comparable binding ability as that observed for BPA. Four chemicals (bottom) represent tandem tri-ring bisphenols showing ERα–antagonistic activity.

Figure 2

Agonist activities of E2 and BPA-related compounds according to luciferase-reporter assay. White bars indicate the transcriptional activity in the absence of the compounds. Transcriptional activities were evaluated by calculating fold induction from the luminescence versus that observed in the absence of each compound. The dose-dependent transcriptional activities associated with each compound are indicated by bars in light grey (0.1 μM), grey (1 μM), and black (10 μM), respectively. Compound numbers are indicated in Table 1 and Fig. 1.

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.

Figure 4

Molecular superposition of tandem tri-ring bisphenols onto the BPA-bound ERα structure. Superposition of tandem tri-ring bisphenols and BPA via the in silico Molecular Superpose function show that the tandem tri-ring bisphenols clash with the side chains of the human ERα structure (PDB ID: 3UUA) in its active conformation. BPM (magenta), α,α,α′-tris(4-hydroxyphenyl)-1-ethyl-4-isopropylbenzene (yellow), BPP (blue), and α,α′-bis(4-hydroxy-3,5-dimethylphenyl)-1,4-diisopropylbenzene (orange) are superposed onto the HO-C₆H₆-C-C₆H₆ moiety of BPA. The molecular surface of the H524 residue located in close proximity to the N-terminal of Helix 11 is illustrated in transparent green. (a) BPM clashes with L349, A350 (in Helix 3), L387, M388, L391(in Helix 5), M421, I424, and F425 (in Helix 7), (b) α,α,α′-Tris(4-hydroxyphenyl)-1-ethyl-4-isopropylbenzene clashes with L346, A350 (in Helix 3), L387, M388, L391(in Helix 5), M421, I424, F425, L428 (in Helix 7), G521(in Helix 10), H524 and M528 (in Helix 11), (c) BPP clashes with L346, T347 (in Helix 3), M421, I424 (in Helix 7), and H524 (in Helix 11), (d) α,α′-Bis(4-hydroxy-3,5-dimethylphenyl)-1,4-diisopropylbenzene clashes with A350 (in Helix 3), L387, M388, L391 (in Helix 5), M421, I424 (in Helix 7), and H524 (in Helix 11).

Figure 5

Clipped coordinates and evaluation using three compensating methods or ab initio calculation. Terminal atoms were generated by compensating with (a) typically utilized protonation methods involving the in-filling of hydrogen atoms (Case 1), (b) reconstructing each clipped amino acid residue as an individual amino acid (Case 2), and c expanding the selected region to include the nitrogen atom at the i − 1 position and protonate conventionally (Case 3), respectively. Clipped atoms from the deposited coordinates of the human ERα crystal structure (PDB ID: 3UUC) were from ranges of (d) 4 Å, (e) 5 Å, (f) 6 Å, (g) 7 Å, (h) 8 Å, and (i) 9 Å.

Figure 6

The calculated bond overlap population of chlorine and carbon atoms. The calculated values of bond overlap populations are shown for (a) Case 1, (b) Case 2, and (c) in Case 3 (HIVE chip method). The values of the bond overlap populations converged in Case 3 as upon expansion of the calculated coordinates form the chlorine atoms.

Figure 7

The HOMO and LUMO calculated using the coordinates clipped 7 Å from the chlorine atoms via the HIVE clip method. The calculated molecular orbitals are illustrated as electron clouds. The electron levels of each orbital are indicated under each figure. The LUMO was restricted in close proximity to the ligand in the ERα structure.