STEROIDAL AFFINITY LABELS OF THE ESTROGEN-RECEPTOR .1. 17-ALPHA-(BROMOACETOXY)ALKYL ALKYNYLESTRADIOLS

To develop steroidal affinity labels for the estrogen receptor, we prepared five electrophilic estradiol derivatives bearing the 17alpha-propyl, 17alpha-(1'-butynyl), or 17alpha-(1'-octynyl) chain, with either a terminal epoxy function (for the 17alpha-propyl substituent) or a terminal bromoacetoxy function (for all three 17alpha-substituent types). These compounds displayed low affinity for the lamb uterine estrogen receptor; with apparent relative affinity constants ranging from 0.02 % to 0.24 % that of estradiol. They were also rapidly transformed in cytosol, probably to the corresponding vicinal diols (epoxy compounds) or primary alcohols (bromoacetoxy compounds). Nevertheless, bromoacetates induced irreversible inactivation of the hormone-binding site but only with ligand-free binding sites. The effect of bromoacetates was prevented by treatment of the cytosol with the thiol-specific reagent methyl methanethiosulfonate. Inactivation of the receptor at 0-degrees-C was rapid (<1 h) and strongly dependent on both compound concentration and pH, with significant effects obtained at either > 150 nM(at pH 9) or pH > 7.5(at 5 muM). Regardless of the conditions used, the order of efficiency for bromoacetates was always: 17alpha-propyl derivative < 17alpha-butynyl derivative < 17alpha-octynyl derivative, with maximal inactivation of approximately 30 % and approximately 70 % of the hormone-binding sites obtained for the less active and more active compounds, respectively. Characteristics of the receptor inactivation suggest that (i) prepared bromoacetates are highly reactive affinity labels for the estrogen receptor, (ii) they react with similar (or even a single) nucleophilic amino acid residues located within or near the hormone-binding site of the receptor; these residues are probably the -SH of cysteines, and (iii) position 17alpha of steroidal ligands is suitable for introducing electrophilic substituents to develop efficient affinity labels for the receptor.