Implications of the binding of tamoxifen to the coactivator recognition site of the estrogen receptor.

A number of studies have reported on the unusual pharmacological behavior of type I antiestrogens, such as tamoxifen. These agents display mixed agonist/antagonist activity in a dose-, cell-, and tissue-specific manner. Consequently, many efforts have been made to develop so-called 'pure' antiestrogens that lack mixed agonist/antagonist activity. The recent report of the structure of estrogen receptor (ER) beta with a second molecule of 4-hydroxytamoxifen (HT) bound in the coactivator-binding surface of the ligand-binding domain (LBD) represents the first direct example of a second ER ligand-binding site and provides insight into the possible origin of mixed agonist/antagonist activity of type I antiestrogens. In this review, we summarize the biological reports leading up to the structural conformation of a second ER ligand-binding site, compare the ERbeta LBD structure bound with two HT molecules to other ER structures, and discuss the potential for small molecular inhibitors designed to directly inhibit ER-coactivator and, more generally, nuclear receptor (NR)-coactivator interactions. The studies support a departure from the traditional paradigm of drug targeting to the ligand-binding site, to that of a rational approach targeting a functionally important surface, namely the NR coactivator-binding (activation function-2) surface. Furthermore, we provide evidence supporting a reevaluation of the strict interpretation of the agonist/antagonist state with respect to the position of helix 12 in the NR LBD.

The contribution of "alternative approaches" to understanding steroid hormone action.

In the 47 yr since the first evidence for a steroid hormone receptor was presented at an international congress to an audience of five persons, the concept of "alternative approach" has played an important role in providing new understanding. By asking not what does an estrogenic hormone do to cellular processes in responsive tissues but what do these cells do to the hormone, it was shown that rat uterus contains a characteristic protein with which the hormone associates to promote growth. In the following decade, it was established that this substance is a true receptor, involved in hormonal action. Furthermore, estradiol was found not to undergo a chemical change as it exerts its effect. Finally, estrogenic action was established as a two-step process in which association with the hormone converts the receptor from an inactive to an active form that can bind tightly in the nucleus to modify transcription. These findings, obtained by studying the fate of the hormone itself, disproved the then accepted concept that estrogens interact with enzyme systems, and opened a new field of research. Soon various laboratories identified intracellular receptors for all classes of steroid hormones, the action of which involves a similar two-step process. Several laboratories then attempted, without success, to obtain antibodies to these receptors by conventional techniques. The unconventional approach of gradient ultracentrifugation, using radioactive estradiol as a marker for the receptor, gave a means of recognizing the soluble immune complexes formed with estrogen receptor and provided the first antibodies to any steroid hormone receptor, permitting its cloning. Finally, the knowledge that estrogens act through a receptor suggested that measuring the receptor content of an excised tumor specimen could identify, in advance, two thirds of the human breast cancers that are not estrogen dependent. These patients will not benefit from endocrine ablation or antiestrogen treatment and should be placed directly on chemotherapy. This is now standard clinical practice.

The estrogen receptor: a model for molecular medicine.

The identification of the estrogen receptor (ER) in the laboratory provided a mechanism to describe the target site specificity of estrogen action in uterus, vagina, pituitary gland, and breast cancer. Most importantly, a test was established to predict the outcome of antihormonal therapy in breast cancer, and a target was identified to develop new drugs for the treatment and prevention of breast cancer. The development of tamoxifen for the treatment of all stages of ER-positive breast cancers has resulted in the improved survival of breast cancer patients. However, the recognition of selective ER modulation, i.e., estrogen-like action in bones and lowering circulating cholesterol but antiestrogenic actions in breast and uterus, has resulted in the development of multifunctional medicines with the goal of preventing not only breast and uterine cancer but also osteoporosis and coronary heart disease.

A two-site model for antiestrogen action.

Evidence is presented for a unified model for the reaction of antiestrogens with estrogen receptors that explains much of the unusual pharmacology of these clinically important agents. Agonist activity results from occupancy of the estradiol-binding (primary) site in the receptor and antagonism from the additional interaction with a secondary locus not recognized by hormone. In the case of type I antiestrogens, such as tamoxifen, this is weaker than primary site binding, so these substances are agonists at low concentrations and antagonists at higher levels. With type II antiestrogens, affinities for both sites are comparable, so one never has the agonist situation (only primary site occupied), and these agents are pure antagonists. Reproductive tissues of the mouse and guinea pig contain a small macromolecule that binds hydroxytamoxifen, but not estradiol, keeping the free antiestrogen concentration below that required for secondary binding. Thus, in these species, tamoxifen is a pure agonist.

Estrogen action: a historic perspective on the implications of considering alternative approaches.

In the 50 years since the initial reports of a cognate estrogen receptor (ER), much has been learned about the diverse effects and mechanisms of estrogens, such as 17beta-estradiol (E(2)). This expert narrative review briefly summarizes perspectives and/or recent work of the authors, who have been addressing different aspects of estrogen action, but take a common approach of using alternative considerations to gain insight into mechanisms with clinical relevance, and inform future studies, regarding estrogen action. Their "Top Ten" favorite alternatives that are discussed herein are as follows. 1 - E(2) has actions by binding to a receptor that do not require its enzymatic conversion. 2 - Using a different strategy for antibody binding could make the estrogen receptor (ER) more discernible. 3 - Blocking ERs, rather than E(2) production, may be a useful strategy for breast cancer therapy. 4 - Secretion of alpha-fetoprotein (AFP), rather than only levels of E(2) and/or progesterone, may influence breast cancer risk. 5 - A peptide derived from the active site of AFP can produce the same benefits of the entire endogenous protein in endocrine cancers. 6 - Differential distribution of ER subtypes in the body and brain may underlie specific effects of estrogens. 7 - ERbeta may be sufficient for the trophic effects of estrogen in the brain, and ERalpha may be the primary target of trophic effects in the body. 8 - ERbeta may play a role in the trophic effects of androgens, and may also be relevant in the periphery. 9 - Downstream of E(2)'s effects at ERbeta, there may be consequences for biosynthesis of progestogens and/or androgens. 10 - Changes in histones and/or other factors, which may be downstream of ERbeta, potentially underlie the divergent effects of E(2) in the brain and peripheral tissues.

A second binding site for hydroxytamoxifen within the coactivator-binding groove of estrogen receptor beta.

Evidence is presented that the estrogen antagonist 4-hydroxytamoxifen (HT) can occupy not only the core binding pocket within the ligand-binding domain of estrogen receptor (ER) beta but also a second site on its surface. The crystal structure of the ligand-binding domain (LBD) associated with HT was determined to 2.2 A and revealed two molecules of HT bound to the protein. One was located in the consensus ligand-binding pocket, whereas the other bound to a site that overlaps with the hydrophobic groove of the coactivator recognition surface. Relative to the ERalpha-tamoxifen structure, helix 12 has been displaced from the coactivator recognition surface and occupies a unique position. Although it has been demonstrated that association of the antagonist with the core ligand-binding pocket is sufficient to induce an antagonist ligand-binding domain conformation, this structure suggests that small molecules may directly antagonize receptor-coactivator interactions. These results provide a direct demonstration of two binding sites for HT in ERbeta, as has been previously suggested for ERalpha by using biochemical methods, and represent a crystal structure of a small nonpeptide molecule occupying the coactivator recognition site.