Enlarging the ligand binding pocket by reduction
Enlarging the ligand-binding pocket by reduction of the size of the residue F435 switched DES to an agonist indicating that F435 is involved in mediating the antagonistic effect of DES (Fig. 7E) . The conformation of the smaller side chain of L435 was either not altered by DES or was too small to displace H12 from its agonistic conformation. Diminishing the size of the ligand-binding pocket by enlarging the pocket-forming residue A272 reduced the effect of all the ligands (Fig. 7D). However, introduction of an additional large side chain to position 431 in order to decrease the size of the binding pocket even more did not show stronger effects than the single mutant (data not shown). This indicates that binding to the pocket might be completely blocked with the single mutant and that the residual increase of activity induced by equol and GSK4716 probably originates from effects not related to the pocket. Activation via residues outside the classical ligand-binding pocket has been described for mineralocorticoid receptor . However, this possibility remains to be addressed as a mutation (D328A) in the corresponding region of ERRγ did not abolish the agonistic effect of equol (data not shown). Reducing the size of the ligand-binding pocket by mutating V313 to a larger tryptophan did not influence equol responsiveness (Fig. 7G). Based on the structural models it seems plausible that this mutation creates unfavorable interactions with the phenolic group in 4-OHT and DES thus reducing the effects of these ligands. In contrast, equol could make stacking interactions with the indole of W313 and therefore maintains its agonistic effect. Y326 is highly conserved in several nuclear receptors and is located within the ligand-binding pocket of ERRγ. Y326 belongs to an area whose correspondent residues in mineralocorticoid receptor seem to be involved in MHC class II antigen (45-57) [Homo sapiens] binding and receptor activation . Diminishing the size of the ligand-binding pocket by mutating Y326 reduced the effects of GSK4716, 4-OHT and DES but had no effect on equol (Fig. 7H). In summary, mutations in the ERRγ ligand-binding domain modified the response to equol in a slightly different fashion than the responses to the other tested ligands. Our structural model and data of the mutagenesis experiments thus indicate that equol binds to the ERRγ ligand-binding pocket in a slightly different manner than the other examined agonist GSK4716 and the inverse agonists 4-OHT and DES. However, further structural studies are required to confirm these modeling studies. The concentration of equol required to stimulate the activity of ERRγ in our cell-based assays is fairly high. However, plasma concentrations of phytoestrogens in soya-consuming populations can reach levels as high as 1μM and even higher levels can be achieved by single-dose administration of the purified compounds , . Furthermore, equol and some other phytoestrogens have been shown to reach much higher concentrations in for example prostate fluid than in plasma , . Modulation of the transcriptional activity of nuclear receptors in transient transactivation assays or modulation of nuclear receptor functions in vitro often require ligand concentrations that exceed those required to modulate the receptor functions in vivo. Based on these observations, the concentrations of equol may well be sufficient to stimulate ERRγ in humans. Epidemiological investigations have indicated that phytoestrogens have many beneficial health effects such as a cancer-protective effect . Several studies have indicated that equol possesses higher biological activity than its precursor compound daidzein . According to our current study, equol enhances the transcriptional activity of ERRγ whereas daidzein has no effect. The higher bioactivity of equol could thus be at least partially explained by its ability to activate ERRγ in addition to ERs. Equol was recently shown to inhibit the growth of human breast and prostate cancer cells in vitro , . Only less than half of the human population is so called equol producers whose intestinal bacteria convert daidzein to equol . The ability to produce equol and high serum equol levels has been associated with a reduced prostate cancer risk indicating an important role for equol in prostate cancer prevention . ERRγ has been shown to suppress prostate cancer cell proliferation and tumor growth . In addition, ERRγ has been indicated as a marker of favorable prognosis in human prostate cancer . In this report, we have shown that the growth inhibitory effect of ERRγ on human prostate cancer cells is enhanced by equol (Fig. 8). The antiproliferative effects of equol on prostate and breast cancer cells may thus be at least partially mediated by ERRγ. In summary, our data demonstrate that equol acts as an ERRγ agonist and that at least some of the beneficial health effects of equol may be mediated by ERRγ.