Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.
Benzothiopyran (2) was prepared and the bioactive (+)-2 was found to exhibit a reduced affinity toward the estrogen receptors (ERα/β) when compared to the corresponding dihydrobenzoxathiin (+)-1.
In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water solubility of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water solubility and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (−)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (Ki 53 ± 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp8 binding pocket. We correlated the SARs at both the C4 and the Trp8 binding pockets with calculations of the electrostatic potentials of the diverse C4 aromatic substituents using Spartan 3-21G(*) MO analysis. These calculations provide an approximate analysis of a molecule's ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp8 binding pocket of sst4. The Spartan 3-21G(*) MO analysis reveals significant negative electrostatic potential in the region of the π-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic aromatic rings typified by pyridine and pyrazine not only enhances water solubility and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the π-cloud of the aromatic substituent to interact with side chains of an aromatic binding pocket such as that for Trp8 of SRIF-14. Conversely, these calculations accommodate the experimental findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of (+)-2. This result is consistent with the high electron density in the plane of the heterocycle depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analogue (+)-14 more closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14 represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor. We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the free hydroxyl at C4 optimizes affinity.
