A yeast sensor of ligand binding.

We describe a biosensor that reports the binding of small-molecule ligands to proteins as changes in growth of temperature-sensitive yeast. The yeast strains lack dihydrofolate reductase (DHFR) and are complemented by mouse DHFR containing a ligand-binding domain inserted in a flexible loop. Yeast strains expressing two ligand-binding domain fusions, FKBP12-DHFR and estrogen receptor-alpha (ERalpha)-DHFR, show increased growth in the presence of their corresponding ligands. We used this sensor to identify mutations in residues of ERalpha important for ligand binding, as well as mutations generally affecting protein activity or expression. We also tested the sensor against a chemical array to identify ligands that bind to FKBP12 or ERalpha. The ERalpha sensor was able to discriminate among estrogen analogs, showing different degrees of growth for the analogs that correlated with their relative binding affinities (RBAs). This growth assay provides a simple and inexpensive method to select novel ligands and ligand-binding domains.     

Estrogen receptor ligands. Part 4: The SAR of the syn-dihydrobenzoxathiin SERAMs

A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERalpha selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.     

m-Carborane bisphenol structure as a pharmacophore for selective estrogen receptor modulators

A series of m-carborane derivatives was prepared based upon the structures of antiestrogenic drugs and their activities were evaluated by estrogen receptor alpha (ERalpha) binding assay and transactivation assay using human breast cancer cell line, MCF-7 cells. The m-carborane bisphenol 5 exhibited about a thousand times more potent ER agonistic activity than the o-carborane bisphenol 11. The m-carborane bisphenol structure appears to be a favorable hydrophobic pharmacophore for the development of novel selective estrogen receptor modulators (SERMs).     

CoMSIA and docking study of rhenium based estrogen receptor ligand analogs

OPLS all atom force field parameters were developed in order to model a diverse set of novel rhenium based estrogen receptor ligands whose relative binding affinities (RBA) to the estrogen receptor alpha isoform (ERalpha) with respect to 17beta-estradiol were available. The binding properties of these novel rhenium based organometallic complexes were studied with a combination of Comparative Molecular Similarity Indices Analysis (CoMSIA) and docking. A total of 29 estrogen receptor ligands consisting of 11 rhenium complexes and 18 organic ligands were docked inside the ligand-binding domain (LBD) of ERalpha utilizing the program Gold. The top ranked pose was used to construct CoMSIA models from a training set of 22 of the estrogen receptor ligands which were selected at random. In addition scoring functions from the docking runs and the polar volume (PV) were also studied to investigate their ability to predict RBA ERalpha. A partial least-squares analysis consisting of the CoMSIA steric, electrostatic and hydrophobic indices together with the polar volume proved sufficiently predictive having a correlation coefficient, r(2), of 0.94 and a cross-validated correlation coefficient, q(2), utilizing the leave-one-out method of 0.68. Analysis of the scoring functions from Gold showed particularly poor correlation to RBA ERalpha which did not improve when the rhenium complexes were extracted to leave the organic ligands. The combined CoMSIA and polar volume model ranked correctly the ligands in order of increasing RBA ERalpha, illustrating the utility of this method as a prescreening tool in the development of novel rhenium based estrogen receptor ligands.     

Synthesis and receptor-binding examinations of the normal and 13-epi-D-homoestrones and their 3-methyl ethers

An effective epimerization of the normal estrone 3-methyl and 3-benzyl ethers by using o-phenylenediamine and AcOH made the possibility for facile entry into the 13alpha-estrone series. Combination of this synthetic methodology with an isolation step carried out by means of the Girard-P reagent, the corresponding ethers of 13-epi-estrone were obtained in excellent yields. The 3-hydroxy and 3-methoxy D-homoestrone derivatives in both the normal and the 13alpha-estrone series were then synthesized and tested in vitro in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities (RBAs) are lower than that of the reference compound 3,17beta-estradiol. The progesterone receptor-binding affinities of the four D-homo derivatives were also tested showing low values for 13alpha-D-homoestrone and its 3-methyl ether. Pharmacologically, these 13alpha-D-homoestrone derivatives are estrogen receptor-selective molecules.     

In vitro fluorescence anisotropy analysis of the interaction of full-length SRC1a with estrogen receptors alpha and beta supports an active displacement model for coregulator utilization

Binding of full-length P160 coactivators to hormone response element-steroid receptor complexes has been difficult to investigate in vitro. Here, we report a new application of our recently described fluorescence anisotropy microplate assay to investigate binding and dissociation of full-length steroid receptor coactivator-1a (SRC1a) from full-length estrogen receptor alpha (ERalpha) or estrogen receptor beta (ERbeta) bound to a fluorescein-labeled (fl) estrogen response element (ERE). SRC1a exhibited slightly higher affinity binding to flERE.ERbeta than to flERE.ERalpha. Binding of SRC1a to flERE.ERalpha and to flERE.ERbeta was 17beta-estradiol (E2)-dependent and was nearly absent when ICI 182,780, raloxifene, or 4-hydroxytamoxifen were bound to the ERs. SRC1a binds to flERE.E2-ERalpha and flERE.E2-ERbeta complexes with a t1/2 of 15-20 s. Short LXXLL-containing nuclear receptor (NR) box peptides from P160 coactivators competed much better for SRC1a binding to flERE.E2-ER than an NR box peptide from TRAP220. However, approximately 40-250-fold molar excess of the P160 NR box peptides was required to inhibit SRC1a binding by 50%. This suggests that whereas the NR box region is a primary site of interaction between SRC1a and ERE.E2-ER, additional contacts between the coactivator and the ligand-receptor-DNA complex make substantial contributions to overall affinity. Increasing amounts of NR box peptides greatly enhanced the rate of dissociation of SRC1a from preformed flERE.E2-ER complexes. The data support a model in which coactivator exchange is facilitated by active displacement and is not simply the result of passive dissociation and replacement. It also shows that an isolated coactivator exhibits an inherent capacity for rapid exchange.