Takatoshi Tokunaga

Direct Evidence Revealing Structural Elements Essential for the High Binding Ability of Bisphenol A to Human Estrogen-Related Receptor-γ

Background Various lines of evidence have shown that bisphenol A [BPA; HO-C6H4-C(CH3)2-C6H4-OH] acts as an endocrine disruptor when present in very low doses. We have recently demonstrated that BPA binds strongly to human estrogen-related receptor-γ (ERR-γ ) in a binding assay using [3H]4-hydroxytamoxifen ([3H]4-OHT). We also demonstrated that BPA inhibits the deactivation activity of 4-OHT. Objectives In the present study, we intended to obtain direct evidence that BPA interacts with ERR-γ as a strong binder, and also to clarify the structural requirements of BPA for its binding to ERR-γ. Methods We examined [3H]BPA in the saturation binding assay using the ligand binding domain of ERR-γ and analyzed the result using Scatchard plot analysis. A number of BPA derivatives were tested in the competitive binding assay using [3H]BPA as a tracer and in the luciferase reporter gene assay. Results [3H]BPA showed a KD of 5.50 nM at a Bmax of 14.4 nmol/mg. When we examined BPA derivatives to evaluate the structural essentials required for the binding of BPA to ERR-γ , we found that only one of the two phenol-hydroxyl groups was essential for the full binding. The maximal activity was attained when one of the methyl groups was removed. All of the potent BPA derivatives retained a high constitutive basal activity of ERR-γ in the luciferase reporter gene assay and exhibited a distinct inhibitory activity against 4-OHT. Conclusion These results indicate that the phenol derivatives are potent candidates for the endocrine disruptor that binds to ERR-γ.

ERRγ tethers strongly bisphenol A and 4-α-cumylphenol in an induced-fit manner

A receptor-binding assay and X-ray crystal structure analysis demonstrated that the endocrine disruptor bisphenol A (BPA) strongly binds to human estrogen-related receptor gamma (ERRgamma). BPA is well anchored to the ligand-binding pocket, forming hydrogen bonds with its two phenol-hydroxyl groups. In this study, we found that 4-alpha-cumylphenol lacking one of its phenol-hydroxyl groups also binds to ERRgamma very strongly. The 2.0 A crystal structure of the 4-alpha-cumylphenol/ERRgamma complex clearly revealed that ERRgammas Leu345-beta-isopropyl plays a role in the tight binding of 4-alpha-cumylphenol and BPA, rotating in a back-and-forth induced-fit manner.

Receptor binding characteristics of the endocrine disruptor bisphenol A for the human nuclear estrogen‐related receptor γ

Bisphenol A, 2,2‐bis(4‐hydroxyphenyl)propane, is an estrogenic endocrine disruptor that influences various physiological functions at very low doses, even though bisphenol A itself is ineffectual as a ligand for the estrogen receptor. We recently demonstrated that bisphenol A binds strongly to human estrogen‐related receptor γ, one of 48 human nuclear receptors. Bisphenol A functions as an inverse antagonist of estrogen‐related receptor γ to sustain the high basal constitutive activity of the latter and to reverse the deactivating inverse agonist activity of 4‐hydroxytamoxifen. However, the intrinsic binding mode of bisphenol A remains to be clarified. In the present study, we report the binding potentials between the phenol‐hydroxyl group of bisphenol A and estrogen‐related receptor γ residues Glu275 and Arg316 in the ligand‐binding domain. By inducing mutations in other amino acids, we evaluated the change in receptor binding capability of bisphenol A. Wild‐type estrogen‐related receptor γ‐ligand‐binding domain showed a strong binding ability (KD = 5.70 nm) for tritium‐labeled [3H]bisphenol A. Simultaneous mutation to Ala at positions 275 and 316 resulted in an absolute inability to capture bisphenol A. However, individual substitutions revealed different degrees in activity reduction, indicating the chief importance of phenol‐hydroxyl⇆Arg316 hydrogen bonding and the corroborative role of phenol‐hydroxyl⇆Glu275 hydrogen bonding. The data obtained with other characteristic mutations suggested that these hydrogen bonds are conducive to the recruitment of phenol compounds by estrogen‐related receptor γ. These results clearly indicate that estrogen‐related receptor γ forms an appropriate structure presumably to adopt an unidentified endogenous ligand.

Exploration of endocrine-disrupting chemicals on estrogen receptor α by the agonist/antagonist differential-docking screening (AADS) method: 4-(1-Adamantyl)phenol as a potent endocrine disruptor candidate

We established a novel screening method to survey endocrine-disrupting chemicals by means of in silico docking calculations. Endocrine disruptors target the human nuclear receptor, which bind a chemical in a pocket presenting in the ligand-binding domain (LBD). The LBD alters its conformation, depending upon the binding of either agonist or antagonist. We discovered that the chemicals can be differentiated into either agonist or antagonist by the docking calculations of the chemical for the LBD. We used the crystal structures of both agonist-bound LBDs and antagonist-bond LBDs as templates in the docking calculations, and estimated binding energies to discriminate between agonist and antagonist bindings. This agonist/antagonist differential-docking screening (AADS) method predicted, for example, 4-(1-adamantyl)phenol as an agonist of the human estrogen receptor alpha (hERalpha). Indeed, this compound, one of the essential raw materials for nanoporous organosilicate thin films, was confirmed to exhibit strong agonist activity in the reporter-gene assay for hERalpha with a high binding affinity. The AADS method is an approach that appears to foresee both the binding ability and the agonist/antagonist function of chemicals for the target nuclear receptors.

Direct measure of fluorescence intensity for efficient receptor-binding assay: Conjugates of ethinylcarboxyestradiol and 5(and 6)-carboxyfluorescein via α,ω-diaminoalkanes as a tracer for estrogen receptor

Steroidal nuclear receptors (NRs) have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of NRs. We designed and synthesized a series of conjugates of 17alpha-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with alpha,omega-diaminoalkanes. The resulting fluorescein-linked estradiol derivatives E2(n)cF (n=2, 4, 6, 8, 10 and 12) were evaluated for their fluorescence and receptor-binding characteristics. E2(4)cF and E2(8)cF exhibited the sufficient binding affinity to the recombinant estrogen receptor (ER) in the radiolabel binding assay using [(3)H]17beta-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. They exhibited sufficiently large specific binding characteristics with adequate K(d)- and B(max)-values. When these fluorescent ligands were used as a tracer for the binding assay against the ER, assay data of various compounds were shown to be compatible with those obtained from the ordinary binding assay using [(3)H]17beta-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.