Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (−/−) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

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The G‐protein estrogen receptor (GPER) has been implicated in a variety of disease states and conditions. Previously our group identified a novel GPER antagonist, CIMBA, that showed improved potency as compared to the G‐series antagonist, G‐36, and exhibited the ability to reduce the formation of gallstone in a murine model. Additional modifications were made to the CIMBA scaffold to improve potency and enhance solubility. In this study, particular emphasis was placed on examining the replacement of the cyclohexyl moiety in CIMBA. Groups explored included various cyclic aliphatic systems, aliphatic chains, conjugated ring systems, piperazine, piperidine, and morpholine. Additional modifications explored the tolerance of the additional groups at the methoxy of CIMBA as well as linker modifications. Compounds were initially screened for calcium mobilization at 10 mM. All compounds exhibiting either agonism or antagonism at 10 mM were screened further to determine the appropriate EC50 or IC50 values. In this series of compounds, it was determined that the absence of the amine in the linker was detrimental to activity. Additionally, the replacement of the cyclohexyl ring in CIMBA with a piperazine (referred to as PIMBA), showed improved aqueous solubility above 10 mM without effecting the potency drastically. Off‐target binding to the nuclear estrogen receptors, ERa and ERb, was determined with a fluorescence polarization. The improved solubility of PIMBA may increase the success of in vivo studies utilizing GPER‐specific ligands in understanding the pharmacology of the receptor in different disease states.

The G protein‐coupled estrogen receptor (GPER) has been implicated in the neuroprotective effects of 17β‐estradiol and selective GPER agonist, G‐1. Currently, G‐1 remains the standard for GPER agonists, necessitating the design and identification of novel candidates. Previously, our group synthesized and biologically evaluated a series of GPER antagonists. Modification of an aromatic moiety on the scaffold altered the pharmacological profile from antagonists to agonists. Using a fluorescence‐based binding assay for the classical estrogen receptors, ERa and ERb, we discovered off‐target binding. Due to the similarity in functionalization to estradiol, we hypothesized that the hydrogen bonding abilities of the initial candidate were responsible for the observed off‐target binding. To mitigate off‐target binding, modifications were made to reduce the hydrogen bonding ability. For each resulting candidate, the EC50 value was determined through in vitro biochemical screening. From these studies, we identified a lead compound that exhibited an EC50 value below that of our experimentally determined EC50 value of G‐1. Using E18 rat embryonic hippocampal neurons, the lead compound was shown to increase synaptogenesis. These results suggest that the identified lead compound for GPER may be a potential candidate for neurodegenerative diseases.Support or Funding InformationSaint Louis UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.