The Journal of Steroid Biochemistry and Molecular Biology
Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity
Introduction
Estrogens mediate a range of physiological processes, including roles in reproduction, the immune, nervous and cardiovascular systems [1]. Additionally, estrogen signaling plays a role in breast, ovarian and other types of cancer [2]. Estrogen signaling is mediated through at least three receptors, including the soluble nuclear receptors ERα and ERβ, and GPER, a seven transmembrane-spanning G protein-coupled receptor (GPCR) [3]. All three of these receptors can mediate gene transcription events, either directly, as in the case of ERα and ERβ [1], or indirectly, as in the case of GPER [4], [5]. ERα, ERβ and GPER can also mediate rapid signaling events through activation of MAPK, PI3K, Src kinase and related pathways [2], [3]. In vivo, these receptors vary in their tissue distribution and the estrogen responsiveness of a given tissue is determined both by receptor expression, co-regulator expression and by signaling interplay between receptors in response to estrogen [1], [3].
Estrogen receptors overlap in some physiological functions as well as in their ligand specificity. The triphenylethylene derivative tamoxifen is representative of the selective estrogen receptor modulator (SERM) anti-estrogen class of therapeutics that inhibit the binding of the endogenous ligand, 17β-estradiol (E2, 1), to ERα and ERβ, while fulvestrant (faslodex, ICI182,780) represents the “pure” antiestrogen class of therapeutics that initiates ERα and ERβ receptor down-regulation. Paradoxically, both of these compounds act as GPER agonists [6], [7], and the resulting agonist/antagonist properties of these compounds vary by receptor status and/or tissue [8]. Thus, compounds with selectivity towards a single receptor are of great use for probing receptor function in complex systems where multiple estrogen receptors are expressed. We have previously developed a GPER-selective agonist, G-1 2 [9], and a GPER-selective antagonist, G15 3 [10], which have been used to elucidate GPER function in a variety of systems, both in vitro as well as in vivo. For example, G-1 has been used to define a role for GPER in proliferation [4], [11], protein kinase C activation [12], PI3K activation [9] and calcium mobilization in a range of cells [9], [13], [14]. Additionally, G-1 has been used in in vivo systems to investigate the roles of GPER in uterine epithelial proliferation [10], thymic atrophy [15], immune regulation in experimental autoimmune encephalomyelitis (a murine model of multiple sclerosis) [15], [16] and vascular regulation [17]. The antagonist G15 has now been used by several groups to establish the role of GPER in estrogen-mediated events, including vasodilation [18], zebrafish oocyte maturation [19], neuroprotection [20] and inhibition of chondrogenesis [21]. These results indicate that selective ligands for GPER have a wide range of functional applications and can contribute to our understanding of the contributions of GPER signaling in complex systems expressing ERα and/or ERβ in addition to GPER.
Particularly in systems expressing multiple estrogen receptors, the utility of these compounds derives from and is limited by their selectivity for GPER vs. ERα and ERβ. These small molecules are based on a common tetrahydro-3H-cyclopenta[c]quinoline scaffold, with the key difference between agonist G-1 and antagonist G15 being the presence of an ethanone moiety on G-1 [9], [10]. Since G15 lacks this bulky substituent group and, as we demonstrate here, exhibits increased ERα/β activity compared to G-1, we synthesized G36, a G-1 analog that contains an isopropyl moiety substituted for the ethanone in G-1. We postulated that the increased bulk present in G-1 and G36 would increase steric clashes within the binding pocket of ERα and ERβ compared to G15, thus limiting the binding and downstream signaling activity observed when high doses of G15 are used.
In this report, we describe the identification and characterization of G36 4, a GPER-selective antagonist with enhanced selectivity for GPER and decreased activity towards ERα and ERβ compared to the previously described antagonist, G15. We show that high doses of G15 induce low levels of transcription via ERα and that G36 minimizes this off-target effect. Additionally, G36 maintains equal efficacy as an antagonist of GPER compared to G15 in a range of functional assays, both in vitro and in vivo.
Section snippets
Molecular docking
The crystal structure of the human estrogen receptor (ER) ligand-binding domain in complex with 17β-estradiol from the RCSB Protein Data Bank (PDB ID: 1ERE) was used [22]. The receptor was prepared for docking using the standard protocol implemented in fred_receptor, a wizard like graphical utility that prepares an active site for docking with FRED (version 2.2.5, OpenEye Scientific Software, Inc., Santa Fe, NM, USA, www.eyesopen.com, 2010). Three-dimensional conformations for the chosen
G-1/G15 ERE-mediated activity and design & synthesis of G36
In our original identification of the GPER antagonist G15 [10], we speculated that the ethanone moiety of G-1 might be critical for GPER activation through the formation of hydrogen bonds with the receptor and that loss of this bonding would result in a compound that could still bind but not activate GPER, thus acting as a competitive antagonist. In our initial characterization of G15, we detected weak binding of G15 to ERα and ERβ at high concentrations (≥10 μM) of G15; however, there were no
Conclusions
In summary, we have developed a second generation GPER antagonist G36 with improved performance and selectivity that exhibits significantly decreased off-target effects on ERα and ERβ compared to our previously described antagonist, G15. It is however important to note the context in which both G-1 and G15/G36 are likely to be used. G-1 has typically been used to determine the contribution of GPER activation to a cellular or physiological response in the absence of estrogen, either by its
Acknowledgments
This work was supported by NIH grants R01 CA127731 (ERP, JBA, TIO), CA118743 (ERP) and CA116662 (ERP), and MH084690 (LAS); the New Mexico Cowboys for Cancer Research Foundation (JBA, ERP); Oxnard Foundation (ERP); and the Stranahan Foundation (ERP). Data were generated in the Flow Cytometry and Fluorescence Microscopy (http://hsc.unm.edu/crtc/microscopy/Facility.html) Shared Resource Centers supported by the University of New Mexico Health Sciences Center and the University of New Mexico Cancer
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