Binding affinity was measured by
Binding affinity was measured by a scintillation proximity binding assay using [H]4-OHT (ERRγ) or [H]estradiol (ERα/β) as radioligand. In all cases, the ERβ affinity was not significantly different from ERα. Compounds were compared to , which showed high affinity for both receptors and moderate (sixfold) selectivity for ERα. 4-Hydroxytoremifene () had an affinity profile nearly identical to that of . Surprisingly, 4-hydroxy--toremifene was nearly equipotent on the two receptors. An effect of chain length upon ERα/ERRγ affinity was also observed with CN and N. At =2, the nitrile substituent in decreased ERRγ affinity by 100-fold and further extension as in increased the affinity by almost 10-fold. In contrast, , an azide Microcystin-LR receptor with =2, showed increased ERRγ affinity, which was reduced by chain extension in . The SMe group in decreased the ERRγ affinity more than the affinity for ERα. Incorporation of acidic (COH), basic (NRR′), or highly polar (NHAc) groups led to a significant loss of affinity on both receptors, as no radioligand displacement was observed for – at the maximum concentration of the assay (3μM). The potency and selectivity of alcohol-containing tethers depended on chain length. Equivalent potency was observed with (=2), and the optimal compound in the series was where =3 in which a 25-fold improvement in selectivity for ERRγ over ERα was realized relative to , albeit at a lower overall affinity. Further extension of the chain bearing the hydroxyl (as in ) reversed the selectivity profile in favor of ERα (). In addition to the radioligand displacement assay, an ERRγ-RIP140 HTRF (FRET) assay was utilized to measure changes in coactivator interaction caused by compound treatment. A basal level of interaction between ERRγ and a peptide fragment from NR cofactor RIP140 was observed in the absence of added ligand and was inhibited by (IC=20nM). The interaction was decreased by a similar amount with , albeit at lower potency (IC=250nM), demonstrating that would function as an ERRγ inverse agonist. Given the improved selectivity in binding affinity of , we sought validation of its utility in a cellular context. HeLa cells were transfected with an expression plasmid encoding full-length ERRγ or ERα and a reporter plasmid 3xERE-TATA-LUC containing three copies of the vitellogenin ERE fused upstream of a TATA box sequence linked to luciferase (LUC). When normalized to transcriptional activity in the absence of compound treatment, shows dose-dependent repression of the activity of ERRγ and competitive antagonism of 100nM estradiol on ERα (). In contrast, there is no effect on ERα transcriptional activity by 1μM , but the ERRγ activity is repressed by 69% (). At 5μM , however, a partial repression of ERα activity is observed. These results suggest that there may be a workable concentration range in which the ERRγ and ERα effects of are separable. To confirm the molecular basis of the improved selectivity of , its complex with ERRγ LBD was crystallized. The X-ray structure was refined to a resolution of 2.30Å with a final factor of 18.7% and a free factor of 24.7%. (). The ligand binds in a mode similar to that observed in the 4-OHT/ERRγ structure where the phenol interacts with E275 and R316, and the basic side chain of the inhibitor interacts with D273. Distances from the alcohol functionality to Y326 and N346 indicate hydrogen bonding interactions with each residue: Y326 (O–O: 2.6Å) and N346 (O–O: 2.4Å). The X-ray crystal structure is consistent with our original design hypothesis. We have described the structure-based design of ERRγ selective inverse agonists. The 4-OHT scaffold has been modified to improve selectivity for ERRγ. A set of compounds was designed and prepared to utilize a polar interaction possible in ERRγ but not in ERα. When compared with the starting compound , a 25-fold improvement in binding selectivity for ERRγ over ERα has been achieved with , and the presence of the predicted hydrogen bonding interactions has been confirmed by the X-ray cocrystal structure of and ERRγ. The observed improvement in binding selectivity is reflected in the cellular activity of , which has an improved ERRγ selectivity profile relative to . As such, has potential for use as an inverse agonist chemical tool in the elucidation of ERRγ function.