STEROIDAL AFFINITY LABELS OF THE ESTROGEN-RECEPTOR .2. 17-ALPHA-[(HALOACETAMIDO)ALKYL]ESTRADIOLS

In a previous study, Re described affinity labeling of the lamb uterine estrogen receptor by 17 alpha-[(bromoacetoxy)alkyl/alkynyl]estradiols. However, the intrinsic receptor-alkylating activities of these compounds were probably very hampered by their poor hydrolytic stability in estrogen receptor-containing tissue extracts. Therefore, (i) to develop affinity labels of the receptor not susceptible to hydrolysis and (ii) to specify the structural requirements for 17 alpha-electrophilic estradiol derivatives to be potent affinity labels of the receptor, we prepared four 17 alpha-[(haloacetamido)alkyl]estradiols. Three were bromoacetamides differing at the alkyl substituent (methyl, ethyl, or propyl), and the last was an [(iodoacetamido)propyl]estradiol prepared under both nonradioactive and H-3-labeled forms. Although their affinities for the estrogen receptor were very low (from 0.008% to 0.02% that of estradiol), they appeared to be efficient affinity labels of the receptor due to their irreversible inhibition of [H-3]estradiol specific binding in lamb uterine cytosol. The effect of the compounds was time-, pH-, and concentration-dependent, with >50% and >80% estrogen-binding sites inactivated at 0 degrees C and pH 8.5, for the less active and more active compounds, respectively; the corresponding IC50 values varied from similar to 20 nM to similar to 10 mu M. The order of efficiency was [(bromoacetamido)methyl]estradiol < [(bromoacetamido)ethyl]estradiol much less than [(bromoacetamido)propyl]estradiol < [(iodoacetamido)propyl]estradiol. Affinity labeling was directly demonstrated by ethanol-resistant binding of [H-3][(iodoacetamido)propyl]estradiol to the receptor. The irreversible inactivation of the hormone-binding site by the four haloacetamides was prevented by treatment of the cytosol with the thiol-specific reagent methyl methanethiosulfonate, suggesting that the target of these compounds was probably the -SH of cysteines. Negative results obtained with other 17 alpha-electrophilic estradiol derivatives suggested that affinity labeling of the receptor by such derivatives required a minimal distance, including at least four C-C or C-N bonds, between the steroid and the electrophilic carbon. We therefore concluded that target cysteines in the hormone-binding site were not in direct contact with the steroid but probably in the immediate neighborhood of the D ring of the bound steroid.