Binding affinity and synergy between agonists and peptides of the estrogen receptor‐beta (ERß) needed for arain‐selective SERB Activity

Abstract

The estrogen receptor (ER) exist predominantly in two isoforms (ERα and ERß). Both are primarily activated by the naturally occurring hormone 17ß-Estradiol (E2) which regulates a variety of physiological processes. One of ER's major role is acting as key neuroprotective modulator for hippocampal plasticity in the brain. The ERß isoform is predominantly found in the brain, which makes it a promising drug target for memory consolidation. Selectivity for ERß over ERα is crucial to the development of drug leads, since ERα–induced cell proliferation results in risk of cancer. We have developed a selective and potent ERß agonist with in vivo efficacy for improving cognitive function within mice. A prior study determined that selectivity for ER is related to the ligand's ability to induce the conformational change necessary for the activation of transcription. Selective estrogen receptor modulators (SERMs) like raloxifene & tamoxifen are used to treat breast cancer by selectively targeting ERα in mammary tissue. This is done through the synergistic binding of coactivator proteins to the ER in the presence of an agonist. Similarly, it is predicted that selective estrogen beta agonists (SERBAs) have the ability to be tissue specific in the presence of specific coactivators. Through the use of computational modeling, a splice variant of SRC1 (steroid receptor coactivator-1), SRC1-4, was identified to be the most favorable brain-relevant peptide for ERß. This study determines the binding affinity of agonist for ERß and ERα in the presence and absence of SRC1-4. In addition we dive into the extent to which binding synergy between agonist and coactivator exist, which could lead to selective activity in the brain. The synergy associated with the conformational change is measured by ΔΔGsynergy based on binding affinity for both agonist and coactivator. The greater the synergy between the agonist and coactivator, the more promising the lead compound will be as a SERBA. Preliminary studies have been done for the binding of SRC1-4 using TR-FRET assays where the concentration of coactivator was varied. Fluorescence polarization (FP) studies were also utilized to determine a Kd. In these studies, FL-SRC1-4 remained constant while ERß-LBD concentrations varied in the presence of 1 μM (saturating) E2. The two methods are compared and contrasted. The same FP assay was completed again in the absence of E2 (no agonoism). This provides a binding affinity for SRC1-4 to the receptor when an agonist is not present. Computational modeling studies were performed to compare ER's affinity for SRC1-4 peptides over other coactivator peptides. 3 unlabeled peptides (SRC1-1, SRC1-3 and PGC1a) were used in a competitive FP binding assay with FL-SRC1-4. The data was consistent with prior data, indicating SRC1-4 has the highest affinity for ERß. The agonistic-synergy effect will be calculated through the measurement of E2 affinity for ERß in the presence and absence of SRC1-4. The comparison of ΔΔG(synergy) for ERß vs. ERα will ultimately provide a measurement of selectivity and synergy that is biologically relevant for SERB activity. Hanson, A. M. et al. A-C estrogens as potent and selective estrogen receptor-beta agonists (SERBAs) to enhance memory consolidation under low-estrogen conditions. J. Med. Chem.61, 4720–4738 (2018).