Megan K. Dennis

In vivo Effects of a GPR30 Antagonist

Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30/GPER, in addition to classical nuclear estrogen receptors (ERα/β), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized a selective agonist of GPR30, G-1 (1). To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of a G-1 analog, G15 (2) that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 reveals that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.

Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity

GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERα ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERα. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERα, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERα. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases.

Beneficial role of the GPR30 agonist G-1 in an animal model of multiple sclerosis.

The beneficial effects of estrogens in multiple sclerosis are thought to be mediated exclusively by the classical nuclear estrogen receptors ERalpha and ERbeta. However, recently many reports revealed that estrogens are able to mediate rapid signals through a G protein-coupled receptor (GPCR), known as GPR30. In the present study, we set out to explore whether effects mediated through this receptor were anti-inflammatory and could account for some of the beneficial effects of estrogen. We demonstrate that GPR30 is expressed in both human and mouse immune cells. Furthermore a GPR30-selective agonist, G-1, previously described by us, inhibits the production of lipopolysaccharide (LPS)-induced cytokines such as TNF-alpha and IL-6 in a dose-dependent manner in human primary macrophages and in a murine macrophage cell line. These effects are likely mediated solely through the estrogen-specific receptor GPR30 since the agonist G-1 displayed an IC(50) far greater than 10 microM on the classical nuclear estrogen receptors as well as a panel of 25 other GPCRs. Finally, we show that the agonist G-1 is able to reduce the severity of disease in both active and passive EAE models of multiple sclerosis in SJL mice and that this effect is concomitant with a G-1-mediated decrease in proinflammatory cytokines, including IFN-gamma and IL-17, in immune cells harvested from these mice. The effect of G-1 appears indirect, as the GPR30 agonist did not directly influence IFN-gamma or IL-17 production by purified T cells. These data indicate that G-1 may represent a novel therapeutic agent for the treatment of chronic autoimmune, inflammatory diseases.

G Protein-Coupled Estrogen Receptor-Selective Ligands Modulate Endometrial Tumor Growth

Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembrane spanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ERα and ERβ. High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediated signaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ERα-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK and PI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeutic agents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the “ERα-selective” agonist propylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth with G-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have important implications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of “ER-targeted” therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported in many clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivo prevents estrogen-mediated tumor growth.

Synthesis and Characterization of Iodinated Tetrahydroquinolines Targeting the G Protein-coupled Estrogen Receptor GPR30

A series of iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines was synthesized as potential targeted imaging agents for the G protein-coupled estrogen receptor GPR30. The affinity and specificity of binding to GPR30 versus the classical estrogen receptors ER alpha/beta and functional responses associated with ligand-binding were determined. Selected iodo-substituted tetrahydro-3H-cyclopenta[c]quinolines exhibited IC(50) values lower than 20 nM in competitive binding studies with GPR30-expressing human endometrial cancer cells. These compounds functioned as antagonists of GPR30 and blocked estrogen-induced PI3K activation and calcium mobilization. The tributylstannyl precursors of selected compounds were radiolabeled with (125)I using the iodogen method. In vivo biodistribution studies in female ovariectomized athymic (NCr) nu/nu mice bearing GPR30-expressing human endometrial tumors revealed GPR30-mediated uptake of the radiotracer ligands in tumor, adrenal, and reproductive organs. Biodistribution and quantitative SPECT/CT studies revealed structurally related differences in the pharmacokinetic profiles, target tissue uptake, and metabolism of the radiolabeled compounds as well as differences in susceptibility to deiodination. The high lipophilicity of the compounds adversely affects the in vivo biodistribution and clearance of these radioligands and suggests that further optimization of this parameter may lead to improved targeting characteristics.

Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.

OCA2 is used as a model melanosome cargo protein to define primary sequence elements required for acidic dileucine–motif binding to adaptors AP-1 and AP-3. OCA2 must bind to AP-3 for melanosome localization. BLOC-1 is also required and thus can cooperate with either adaptor for cargo delivery to lysosome-related organelles.

BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery

Quantitative analyses of melanosome cargo localization and trafficking and of endosomal membrane dynamics in immortalized melanocytes from mouse Hermansky–Pudlak syndrome models show that BLOC-2 functions to specify the delivery of recycling endosomal cargo transport intermediates to maturing melanosomes.

BLOC-1 and BLOC-3 regulate VAMP7 cycling to and from melanosomes via distinct tubular transport carriers

Dennis et al. analyze cycling of the v-SNARE VAMP7 during melanosome biogenesis in melanocytes. VAMP7 is targeted to and retrieved from maturing melanosomes in separate tubular carriers whose formation requires distinct BLOCs, each defective in variants of Hermansky–Pudlak syndrome.

Synthesis and Characterization of Tricarbonyl-Re/Tc(I) Chelate Probes Targeting the G Protein-Coupled Estrogen Receptor GPER/GPR30

The discovery of the G protein-coupled estrogen receptor GPER (also GPR30) and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPER-mediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer.

Influence of charge on cell permeability and tumor imaging of GPR30-targeted 111in-labeled nonsteroidal imaging agents.

Recent clinical studies implicate the role of G protein-coupled estrogen receptor, GPR30, in aggressive forms of breast, ovarian, and endometrial cancers. However, the functional role of GPR30 at cellular and molecular levels remains less clear and controversial, particularly its subcellular location. The primary objective of this study was to develop radiolabeled neutral and charged GPR30-targeted nonsteroidal analogues to understand the influence of ligand charge on cell binding, cellular permeability, and in vivo tumor imaging. Therefore, we developed a series of GPR30-targeted (111/113)In(III)-labeled analogues using macrocyclic and acyclic polyamino-polycarboxylate chelate designs that would render either a net negative or neutral charge. In vitro biological evaluations were performed to determine the role of negatively charged analogues on receptor binding and activation using calcium mobilization and phosphoinositide 3-kinase assays. In vivo evaluations were performed on GPR30-expressing human endometrial Hec50 tumor-bearing mice to characterize the biodistribution and potential application of GPR30-targeted imaging agents for translational research. In vitro functional assays revealed an effect of charge, such that only the neutral analogue activated GPR30-mediated rapid signaling pathways. These observations are consistent with expectations for initial rates of membrane permeability and suggest an intracellular rather than the cell surface location of functional receptor. In vivo studies revealed receptor-mediated uptake of the radiotracer in target organs and tumors; however, further structural modifications will be required for the development of future generations of GPR30-targeted imaging agents with enhanced metabolic properties and decreased nonspecific localization to the intestines.