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The bulky side chains of antiestrogens hinder folding of the ligand binding domain (LBD) of estrogen receptors (ERs) into a transcriptionally active conformation. The presence of a tertiary amine in the side chain of raloxifene, which interacts with a negatively charged residue in helix H3 of the ER LBD [Asp351 in human (h)ER␣], is important for antiestrogenicity in animal and cellular models. To better understand the molecular basis of the differential activity of tamoxifen and raloxifene, we have examined the influence of tertiary amine substituents and of mutations at position 351 in hER␣ on the activity profiles of tamoxifen derivatives. Results obtained in several cellular model systems suggest that the degree of antagonist activity of tamoxifen derivatives does not strictly correlate with the basicity of the side chain but depends on an optimal spatial relationship between the tertiary amine of these antiestrogens and the negative charge at position 351. Although altering the position of the negative charge at residue 351 (mutation D351E) had little effect on transcriptional activity in the presence of tamoxifen, it drastically increased the partial agonist activity of a tamoxifen derivative with improved antagonist activity as well as that of raloxifene. Our results suggest that contrary to raloxifene, tamoxifen and most of its derivatives do not interact with Asp351 in an optimal manner, although this can be improved by modifying tertiary amine substituents.

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