A-RING NITRO-SUBSTITUTED AND AMINO-SUBSTITUTED ESTRADIOL ANALOGS PRODUCE A NEGATIVE COOPERATIVE OR NONCOOPERATIVE [H-3] ESTRADIOL ESTROGEN-RECEPTOR BINDING MECHANISM

We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of str ucturally 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 phenoli c A-ring. The binding affinity of both amino analogs and the 4-nitro a nalog for the estrogen receptor was reduced relative to that of estrad iol, 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 tha t of estradiol (p < 0.05), indicating that the binding mechanism of th ese two ligands was different. The affinity of the 2-nitroestradiol li gand for the receptor was too low to be measured. The binding mechanis m was then further investigated by measuring the Hill coefficient of [ H-3]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [H-3]e stradiol-estrogen receptor interaction; the Hill coefficient of [H-3]e stradiol binding in the presence of the analog was 0.99 compared with 1.7 for [H-3]estradiol alone. Most interestingly, the presence of an a mino group on either C2 or C4 brought about a switch from a positive t o a negative cooperative binding interaction; the Hill coefficients of [H-3]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.