Eur. J. Biochem. 262, 240±245 (1999) q FEBS 1999
Structural requirements of para-alkylphenols to bind to estrogen receptor Yukiko Tabira1, Makoto Nakai2, Daisuke Asai1, Yoshikuni Yakabe2, Yoshiko Tahara1, Teruo Shinmyozu3, Masato Noguchi4, Mineo Takatsuki2 and Yasuyuki Shimohigashi1 1
Laboratory of Structure±Function Biochemistry, Department of Molecular Chemistry, Graduate School of Science, Kyushu University, Fukuoka, Japan; 2Chemicals Assessment Center, Chemicals Inspection and Testing Institute, Kurume, Japan; 3Institute for Fundamental Research of Organic Chemistry, Fukuoka, Japan; 4Department of Medical Biochemistry, Kurume University School of Medicine, Japan
Octyl- and nonylphenols in the environment have been proposed to function as estrogens. To gain insight into their structural essentials in binding to the estrogen receptor, a series of phenols with saturated alkyl groups at the para position, HO-C6H4-CnH2n+1 (n = 0±12), were examined for their ability to displace [3H]17b-estradiol in the recombinant human estrogen receptor, which was expressed in Sf9 cells using the vaculovirus expression system. All tested para-alkylphenols were found to bind fully to the estrogen receptors in a dose-dependent manner. The interaction of alkylphenols with the receptor became stronger with increase in the number of the alkyl carbons and the activity was maximized with n = 9 of nonylphenol. Phenol (n = 0) exhibited weak but full binding to the receptor, whereas anisole with a protected phenolic hydroxyl group was completely inactive. Also, alkanes such as n-octane, 2,2,4-trimethylpentane corresponding to tert-octane, and n-nonane exhibited no binding. The results indicate that the binding of para-alkylphenols to the estrogen receptor is due to the effect of covalent bonding of two constituents of the phenol and alkyl groups, which correspond to the A-ring and hydrophobic moiety of the steroid structure, respectively. When alkylphenols were examined for their receptor binding conformation by 1H-NMR measurements and ab initio molecular orbital calculations, it was suggested that nonbranched alkyl groups are in an extended conformation, while branched alkyl groups are in a folded conformation. These results suggest that branched and nonbranched alkyl moieties of alkylphenols interact differently with the lipophilic ligand binding cavity of the estrogen receptor when compared to the binding of 17b-estradiol. Keywords: estrogen receptor; xenoestrogens; para-alkylphenols; 17b-estradiol; structure±activity relationships; receptor-binding conformation.
The endocrine-disrupting effects of environmental chemicals are interpreted as interference with the steroidal regulation of the normal development and function of the male and female reproductive tracts [1±3]. In recent years, a large number of chemicals that bind to the estrogen receptor have been identified [4±6]. However, these chemicals, so-called endocrine disrupters, usually bear little structural resemblance to natural estrogens. Structure-activity studies have been developed primarily for polychlorinated chemicals such as dioxins and PCBs [7,8], and Waller et al. [9] have extended their data set with plant, physiological, and pharmacological estrogens. Clarification of the relevant structure±activity relationships between endocrine hormone receptors and their disrupters, especially of xenoestrogens, appears to be essential to better understand the chemical and biochemical basis of the receptor binding properties. This is particularly important for elucidation of the structural elements essential for receptor binding and activation of steroid hormone receptors, and also for exploring probable xenobiotics which possess a latent structure to bind to the estrogen receptor. Correspondence to Y. Shimohigashi, Laboratory of Structure-Function Biochemistry, Department of Molecular Chemistry, Graduate School of Science, Kyushu University, Fukuoka 812-8581, Japan. Tel.: and Fax: +81 92 642 2584, E-mail:
[email protected] Abbreviations: RP-HPLC, reversed-phase high-performance liquid chromatography. (Received 14 January 1999, accepted 22 March 1999)
The initial step in the molecular mechanism of hormonal action of estrogens is a binding of the steroid to its receptor [10], which is a member of specific transcription factors present in only a restricted range of cell types. Xenoestrogens are one of the most critical environmental chemicals, and alkylphenols have been identified as potential xenoestrogens [11,12]. Any xenoestrogens should compete with the natural estrogen in binding to the receptor, and this requires the xenoestrogens to hold or capture a certain structure essential for receptor binding. At this moment, no systemic structural evaluation of xenoestrogens has been performed in relation to the structure of receptor binding sites, although the three-dimensional crystal structures of the ligand binding site of several steroid hormone receptors, including the estrogen receptor, have been reported. In the present study, we examined a series of phenols with branched and nonbranched alkyl groups at the para position together with their parent constituents (phenol and various alkanes), and evaluated their structural requirements for binding to the estrogen receptor (Fig. 1). In particular, the structure and activity of octylphenols, namely, branched tertoctylphenol and nonbranched n-octylphenol, were compared with those of 17b-estradiol. Our results clearly indicate that alkylphenols interact with estrogen receptors only when phenol is conjugated with hydrophobic alkyl groups and the hydrophobic interfaces of alkylphenols are insufficient to sustain a conformation of estradiol for full receptor binding (Fig. 1).
q FEBS 1999
Estrogen receptor binding of alkylphenols (Eur. J. Biochem. 262) 241
Estrogen receptor binding assay Inhibition of radiolabeled estrogen to its receptor was evaluated as follows. A solution (10 mL) of recombinant human estrogen receptor, which was expressed in Sf9 cells using the vaculovirus expression system (a kind gift from Sumitomo Chemical Co., Ltd), at a concentration of
