Steroidal Affinity Labels of the Estrogen Receptor. 2. 17.alpha.-[(Haloacetamido)alkyl]estradiols

Abstract

In a previous study, we 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 3H-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 [3H]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 approximately 20 nM to approximately 10 microM. The order of efficiency was [(bromoacetamido)methyl]estradiol < [(bromoacetamido)ethyl]estradiol << [(bromoacetamido)propyl]estradiol < [(iodoacetamido)propyl]estradiol. Affinity labeling was directly demonstrated by ethanol-resistant binding of [3H][(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.

Identification of covalent attachment site of antiestrogenic estradiol 11β-derivatives on human estrogen receptor α ligand-binding domain

“…6. Ion identification was facilitated by comparison of the obtained spectrum with that of the homologous species involving the 14 (i) all detected ions involving an alkylated portion of CysLys included the Cys S and various portions of Cys or CysLys; (ii) no signal was found that would correspond to an alkylated portion of Lys; (iii) signals characterizing unsubstituted Lys (immonium, X and Y ions) were present, whereas no signal was found at m/z values which would correspond to unsubstituted Cys fragments.…”

“…14 C-labeled or non-radioactive electrophile (two-fold concentration relative to the amount of GST-LBD, ∼2-3 nmole) was added to glutathione-sepharose-bound GST-LBD in 0.5 mL of ice-cooled phosphate-buffered saline (pH 8.0), containing 5% dimethylformamide. The mixture was rotated for 30 min at 2 • C and then the matrix was drained, and washed with phosphate-buffered saline to eliminate unreacted electrophile.…”

“…Using peptide mass spectrometry (MS) analysis, we recently identified, within mouse [12] and human [13] LBDs (including whole and approximately half ER␣ C-terminal domain F, respectively), two cysteine residues (mouse Cys 421 and homologous human Cys 417 , designated Cys A, and mouse Cys 534 and corresponding human Cys 530 , designated Cys B) as exclusive and alternative covalent attachment sites of two estrogenic estradiol (E 2 ) derivatives, 17␣-bromoacetamidomethylE 2 (17BAME 2 ) and 17␣-bromoacetamidopropylE 2 (17BAPE 2 ) [14]. Although the two electrophiles differed only by an ethylene group in the 17␣-substituent and the human LBD and mouse LBD differed only at 7 out of 244 residues [15], in alkylated LBD the Cys A/Cys B balance considerably varied according to the LBD-electrophile pair used, with a balance close to one for mouse LBD alkylated by 17BAPE 2 [12] and close to 0.05 for human LBD alkylated by 17BAME 2 [13].…”

Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor α

“…This hypothesis is supported by recent reports [12,15,19,20], which indicated that it is difficult to account for the estrogenic/antiestrogenic activity of an ER ligand on the basis of the orientation of helix 12 in crystallized ER LBD-ligand complexes. To enhance the overall understanding of the molecular action of estrogens and antiestrogens, we sought to directly identify, within ER␣ LBD, the covalent attachment sites of estrogenic estradiol (E 2 ) 17␣-derivatives [21] and antiestrogenic E 2 11␤-derivatives [22] through mass spectrometry (MS) analysis and then to interpret the biochemical data by molecular modeling.…”

“…1), one previously tested ER␣ affinity label [21], directly derives from 17BAPE 2 by removal of the (CH 2 ) 2 module from the 17␣-(CH 2 ) 3 group. In 17BAME 2 the maximal distance between the steroidal C-17 and the electrophilic carbon (-carbon) is ∼35% lower than that of the homologous distance in 17BAPE 2 and the degree of freedom of the -carbon is two instead of four.…”