Studies on the ligand specificity and potential identity of microsomal antiestrogen-binding sites.

Synthetic nonsteroidal antiestrogens are bound intracellularly by two high affinity saturable bindings sites, the estrogen receptor and the microsomal antiestrogen-binding site (AEBS). In order to further define the structural requirements for ligand binding to AEBS from rat liver and the MCF 7 human breast cancer cell line, the relative binding affinities of an extensive series of structurally related ligands were investigated using competitive binding assay techniques. The groups of compounds studied were: analogues of the triphenylethylene antiestrogens, Cl 628 and tamoxifen; analogues of cyclofenil; bibenzyl and stilbene derivatives; analogues of the cytochrome P-450 inhibitor SKF-525A; phenothiazine derivatives; and a series of structurally related compounds with a variety of pharmacological activities. High affinity binding to AEBS required the presence of both a hydrophilic basic aminoether side chain and a hydrophobic aromatic ring structure (di- or tricyclic for maximal affinity). Structural modifications to either influenced binding affinity. Aromatic substitution either raised (CF3) or lowered (OH, OCH3) affinity, apparently by electronic effects transmitted through the benzene nucleus. Side chain structure was the major determinant of binding affinity, but its influence was complex and dependent upon terminal amino group structure, side chain branching and substitution, and tissue source of AEBS. Optimal binding affinity was shown by side chains bearing basic heterocyclic amino terminal groups. Other cellular sites that are known to bind antiestrogens with relatively high affinity include calmodulin, cytochrome P-450, and histamine, dopamine, and muscarinic receptors. Binding studies using a variety of pharmacologically active and radiolabeled ligands selective for these sites, including those for dopamine D1 and D2 receptors ([3H]fluphenazine, [3H]flupenthixol, [3H]spiperone, and [3H]SCH 23390) and histamine H1 receptors ([3H]pyrilamine), demonstrated that several of these compounds interact with AEBS with high affinity. However, the ligand specificity and other binding properties of the AEBS as determined by competitive binding studies and Scatchard analysis show this site to be a molecular entity truly distinct from these other cellular binding sites.