Steroidal Affinity Labels of the Estrogen Receptor α. 4. Electrophilic 11β-Aryl Derivatives of Estradiol

Abstract

Ten electrophilic estradiol 11beta-aryl derivatives were synthesized, with three different types of 11beta-substituent: (i) pOO(CH(2))(2)X (compounds: 6, X = OSO(2)CH(3); 7, X = I; 13, X = NHCOCH(2)Cl; 15, X = N(CH(3))COCH(2)Br; and 16, X = N(CH(3))COCH(2)Cl); (ii) pOO(CH(2))(5)X (compounds: 17, X = I; 20, X = NHCOCH(2)Br; and 22, X = N(CH(3))COCH(2)Br); and (iii) pOC(triple bond)CCH(2)X (compounds: 27, X = NHCOCH(2)Cl; and 29, X = N(CH(3))COCH(2)Cl). The range of their apparent affinity constants for binding the lamb uterine estrogen receptor alpha (ERalpha) was 3-40% that of estradiol. Six electrophiles, chloroacetamides 13, 16, 27, and 29, iodide 17, and bromoacetamide 20 (whose arm linking the electrophilic carbon to the 11beta-phenyl group includes at least six bonds), were able to irreversibly inhibit the binding of [(3)H]estradiol to ER (25-60% decrease in binding sites), with the following compound effectiveness order: 17 < 13 < 16 approximately 20 approximately 27 approximately 29. Mesylate 6, iodide 7 (whose linking arm includes only three bonds), and bromoacetamides 15 and 22 (which differ from 16 by the Cl to Br change and from 20 by the NH to NCH(3) change, respectively) were much less effective (<10% decrease in binding sites, if any). The fact that the inactivation of estradiol-binding sites by the six electrophiles was totally prevented by estradiol indicated that they were ER affinity labeling agents. When ER was modified by methyl methanethiosulfonate, an SH-specific reagent, the different compounds led to very contrasting results in ER affinity labeling. With modified ER, iodide 17 and chloroacetamides 27 and 29 were practically inactive, chloroacetamides 13 and 16 and bromoacetamide 20 were still active but less effective than on the native ER, whereas tertiary bromoacetamides 15 and 22, found to be practically inactive on native ER, became the most effective electrophiles ( approximately 45% and approximately 65% binding sites inactivated, respectively). The results indicate that in the steroid-filled hormone-binding pocket: (i) nucleophilic residues are localized on the beta-side but relatively remote from the steroid nucleus (distance from C-11 > "seven bonds"); (ii) relatively discrete changes in the electrophilic functionality, such as Cl to Br or NH to NCH(3) of haloacetamido compounds, can markedly modify the positioning of the electrophilic center which could no longer react with the nucleophilic residues; and (iii) cysteine residues (probably homologues of human ERalpha cysteine 381 and/or cysteine 530) are, at least partly, the covalent attachment sites of the electrophiles. Moreover, modification of cysteine residues by methyl methanethiosulfonate changes the structure of the hormone-binding pocket, whose labeling by the various electrophiles is profoundly altered.

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

“…In this study, the Cys 530 S of human ER␣ LBD was identified as the quasi-exclusive (11BAEOPE 2 ) or main (11BAPOPE 2 ) covalent attachment site of 11BAEOPE 2 and 11BAPOPE 2 , two potent and high affinity antiestrogens [16]. Cys 530 was previously found to be the covalent attachment site of low affinity non-steroidal electrophiles, ketononestrol aziridine, a diphenylethane estrogen and tamoxifen aziridine, a triphenylethylene antiestrogen [22], whereas by means of an indirect approach based on the use of various Cys → Ala ER␣ mutants [17], Cys 530 appeared to be involved in the covalent attachment of E 2 11␤-aziridinylalkoxyphenyl derivatives (two compounds, related to 11BAEOPE 2 and 11BAPOPE 2 ) [18].…”

“…Trypsin (sequencing grade) was from Promega, and ␣-cyano-4-hydroxycinnamic acid was from Sigma-Aldrich. Unlabeled 11BAEOPE 2 and 11BAPOPE 2 had been previously synthesized [16]. Solvents and compounds used for HPLC and electrospray ionization (ESI) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS analyses were HPLC grade (for HPLC, Millipore Q water was passed through a C18 preparative column and then sterilized).…”

“…A few years ago, a study involving ten electrophilic E 2 11␤-aryl derivatives showed that most of these compounds specifically inactivated the ligand-binding site of the lamb ER␣ [16]. This indicated that these antiestrogenic and high affinity compounds were efficient ER affinity labeling ligands.…”

Stereochemical effects during [M-H]− dissociations of epimeric 11-OH-17β-estradiols and distant electronic effects of substituents at C(11) position on gas phase acidity

“…Moreover, an electrostatic interaction involving the methyl group at C (13) and the oxygen atom at C (11) could take place when the hydroxyl group is in the axial orientation ([III ␤ -H] Ϫ ). This interaction is reinforced by the steric repulsion occur- ⌬E ϭ E considered structure Ϫ E of the most stable structure .…”

“…The E 2 /ER␣ complex is stabilized by hydrogen contacts occurring between, on the one hand, the hydroxyl group of phenolic A ring (E 2 ) and Glu-353 as well as Arg-394 of the hormone binding pocket (HBP), and on the other hand, between the 17␤-hydroxyl of D-ring and His-524 (Scheme 1a) [5][6][7]. Moreover, position around the functionalized site at the C (11) position (C ring) seems to contribute to the anchorage of the hormone within this pocket of the receptor [7][8][9][10][11][12][13], suggesting that steric effects at this position are important for binding affinity. Accordingly, we may consider that the proton donor character efficiency could be reinforced through electrostatic effects mediated by the nature and the stereochemistry of substituent in position 11 [14].…”