A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

Abstract

We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of structurally altered estradiol analogs was used in which either an amino- or a nitro group had been added to the 2 or 4 position on the phenolic A-ring. The binding affinity of both amino analogs and the 4-nitro analog for the estrogen receptor was reduced relative to that of estradiol, as measured by competitive binding assay; the values were between 0.008% and 8% of estradiol's affinity. The slope of the displacement curve for the 4-nitro analog was also significantly different from that of estradiol (p < 0.05), indicating that the binding mechanism of these two ligands was different. The affinity of the 2-nitroestradiol ligand for the receptor was too low to be measured. The binding mechanism was then further investigated by measuring the Hill coefficient of [3H]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [3H]estradiol-estrogen receptor interaction; the Hill coefficient of [3H]estradiol binding in the presence of the analog was 0.99 compared with 1.7 for [3H]estradiol alone. Most interestingly, the presence of an amino group on either C2 or C4 brought about a switch from a positive to a negative cooperative binding interaction; the Hill coefficients of [3H]estradiol binding in the presence of the analogs were between 0.6 and 0.7. These results provide additional support for an induced-fit mechanism of ligand-estrogen receptor interactions.

Cooperativity and Dimerization of Recombinant Human Estrogen Receptor Hormone-binding Domain

“…Cooperativity of estradiol binding to the estrogen receptor has been most extensively studied using the protein from calf uterine cytosol (30,34), and the maximal Hill coefficient cited in these reports is ϳ1.6 at a concentration of ϳ5 nM receptor. Using recombinant full-length human estrogen receptor expressed in Sf9 insect cells, Obourn et al (31) also found a maximal Hill coefficient of ϳ1.6; their data suggest that at receptor concentrations below ϳ1 nM, the receptor does not exhibit cooperativity, while maximal cooperativity is observed at 10 -20 nM receptor concentration.…”

Changes in the Mutagenic and Estrogenic Activities of 17β-Estradiol after Treatment with Nitrite

“…There are several reports about the estrogenic activity of 2-nitro-17-estradiol. 24,25) These reports have demonstrated that the binding potency of 17-estradiol to the estrogen receptor was changed because the physicochemical properties of 17-estradiol were transformed by the nitrite treatment. The electronegative characteristic produces conformational changes in the alicyclic backbone of the estrogen, which could interfere with the binding of 2-nitro-17-estradiol to the estrogen receptor.…”

Synthesis, Photophysical and Electrochemical Properties, and Protein‐Binding Studies of Luminescent Cyclometalated Iridium(III) Bipyridine Estradiol Conjugates

“…Additionally, the emission lifetimes (< 1 ms) were noticeably [ 19a, 33b,c] On the basis of these observations, we argue that the emissive state of this complex possesses predominant 3 MLCT (dp(Ir)!p*(bpy-C6-est)) character. A shift of excited-state character from the expected 3 IL to 3 MLCT of this iridium-A C H T U N G T R E N N U N G (III)-pq bipyridine complex is probably a consequence of the electron-withdrawing amide substituent that stabilizes the p* orbitals of the diimine ligand.…”

“…[2] Owing to the important roles of both estradiol and its receptor in female physiology, the design of new biological probes for estradiol-binding proteins has attracted much attention. [3][4][5][6][7][8][9][10][11][12][13][14][15][16] Radioactive hormone derivatives, such as those containing tritium [3,4] and technetium, [5,6] are the most frequently used reagents. However, the potential health hazards of these reagents and the long experimental time required have prompted the search for nonisotopic reagents.…”

“…+ , [17b,c, 18a, 19d, 20, 22, 23, 25b, 26b, 30, 31, 33] the emission usually originates from a 3 MLCT state that may be mixed with some 3 LLCT [20b-d, 21c,d, 23b, 25b, 28b] or 3 SBLCT [18, 19d, 20, 25b, 27b] character in some cases. If more-conjugated cyclometalating ligands such as pq À[19a, 33b,c] and bsb À[33d] are used, the emissive state involves predominant 3 IL (p!p*) (pq À or bsb À ) character.…”