Mutational analysis of the estrogen receptor ligand-binding domain: influence of ligand structure and stereochemistry on transactivation

Abstract

The mouse estrogen receptor (mER) exhibits ligand stereochemical specificity for indenestrol A (IA), a stilbestrol estrogen. IA has a chiral C3 methyl group, and the mER preferentially binds the S-enantiomer (IA-S), resulting in elevated biological activity when compared with the IA-R enantiomer. To elucidate the mechanisms for this stereochemical recognition, we have constructed a series of mERs with individual amino acid substitutions at Met521, His528, Met532, and Val537. The abilities of yeast-expressed wild-type and mutant mERs to transactivate an estrogen-responsive reporter gene construct were measured in the presence of diethylstilbestrol (DES) and IA enantiomers. The concentration of IA-S required to induce half-maximal transactivation by wild-type mER was 10-fold lower than IA-R, which is attributed to the 15-fold greater binding affinity for IA-S. Wild-type mER displayed similar dose-response curves for IA-R and demethyl IA, which lacks a C3 methyl group, demonstrating that the presence and correct orientation of the C3 methyl group on the IA compound is required for high-affinity ligand binding and transcriptional activity. Each mutant exhibited a reduced preference for IA-S enantiomer with respect to transactivation, suggesting that this region of the mER functions in ligand stereochemical recognition and activation. A mutation at Met532 diminished DES- and IA-S-induced transactivation by 7.5-fold and 40-fold respectively, with minimal change on their binding affinity. These data suggest that Met532 is required for transactivation induced by the potent agonist, IA-S, and the M532G mutation effectively uncouples IA-S ligand binding from transactivation. Use of these stereochemically different ligands in combination with mutagenesis of the mER demonstrates that ligand structure could influence transactivation by specifically altering the conformation of the mER AF-2 region.

Enantiomer‐specific biomagnification of α‐Hexachlorocyclohexane and selected chiral chlordane‐related compounds within an arctic marine food web

“…The stereochemical selectivity of enzymes is well documented [45], including CYP‐dependent monooxygenase activity [46] and CYP1A and epoxide hydrolase isozymes [47,48]. However, highly selective, non‐CYP mediated processes also influence the stereochemically specific bioactivity of chiral molecules [11,49,50]. As a result, the term “biotransformation” in this manuscript refers to all possible processes related to CYP‐mediated metabolism, receptor‐mediated disposition, and other unidentified mechanisms responsible for enantiomer‐specific accumulation of chiral OCs in biota.…”

Enantiomer-Specific Accumulation of PCB Atropisomers in the Bowhead Whale (Balaena mysticetus)

“…To our knowledge, the stereoselectivity of PCB metabolism has not been addressed. However, highly selective, non-CYP mediated processes also influence the stereochemically specific bioactivity of chiral molecules (4,47,48). As a result, the term "biotransformation" in this manuscript will refer to all possible processes related to CYP-mediated metabolism, receptor-mediated disposition, and other unidentified mech- The change in the EF values of chiral PCBs from Calanus to the bowhead was less than the change in EF values of chiral OCs observed between prey and predator in other studies (7,49,50).…”

Pyrazolo[1,5-a]pyrimidines: Estrogen Receptor Ligands Possessing Estrogen Receptor β Antagonist Activity

“…Thus, we have pursued an alternative approach, substituting the pyrazolo[1,5- a ]pyrimidine core with aromatic rings at the 2 and 3 positions. By making at least one of these aromatic rings a phenol, we would provide a functionality that is capable of hydrogen-bonding that can potentially act as an A-ring mimic of the natural ligand E2, in a “pendant phenol” orientation thought to be adopted by certain other fused heterocyclic systems − and illustrated in certain crystal structures, at least in ERβ. , In addition to orienting the phenolic hydroxyl group para to the heterocyclic core, we wanted to examine systems with two para hydroxyl groups, as well as systems having one para and one meta hydroxyl group. Changing the size of the substituents at the 5 and 7 positions would vary the hydrophobicity and the size of the system, which might lead to improved binding.…”