Chinnasamy Ramesh

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.

G Protein-coupled Estrogen Receptor Protects from Atherosclerosis

Coronary atherosclerosis and myocardial infarction in postmenopausal women have been linked to inflammation and reduced nitric oxide (NO) formation. Natural estrogen exerts protective effects on both processes, yet also displays uterotrophic activity. Here, we used genetic and pharmacologic approaches to investigate the role of the G protein-coupled estrogen receptor (GPER) in atherosclerosis. In ovary-intact mice, deletion of gper increased atherosclerosis progression, total and LDL cholesterol levels and inflammation while reducing vascular NO bioactivity, effects that were in some cases aggravated by surgical menopause. In human endothelial cells, GPER was expressed on intracellular membranes and mediated eNOS activation and NO formation, partially accounting for estrogen-mediated effects. Chronic treatment with G-1, a synthetic, highly selective small molecule agonist of GPER, reduced postmenopausal atherosclerosis and inflammation without uterotrophic effects. In summary, this study reveals an atheroprotective function of GPER and introduces selective GPER activation as a novel therapeutic approach to inhibit postmenopausal atherosclerosis and inflammation in the absence of uterotrophic activity.

Preclinical Development of a Neutral, Estrogen Receptor–Targeted, Tridentate 99mTc(I)-Estradiol-Pyridin-2-yl Hydrazine Derivative for Imaging of Breast and Endometrial Cancers

Breast and endometrial cancers are the most common invasive malignancies in women, with more than 217,000 new diagnoses per year in the United States. These cancers are often classified into 2 subtypes based on the expression of the classical estrogen receptor. In this study, we describe a new structural class of neutral tridentate 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivatives for potential use in breast and endometrial cancer imaging. Methods: The 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative was synthesized via the Sonogashira cross-coupling reaction and radiolabeled via the tricarbonyl approach. Radiochemical purity was assessed by high-performance liquid chromatography. Cell-binding studies were performed with human breast adenocarcinoma MCF-7 cells. The in vivo biodistribution of the 99mTc(I) derivative was evaluated in virgin female C57BL/6 mice in defined phases of the estrous cycle. Biodistribution and SPECT/CT studies were performed with mice bearing MCF-7 and primary human endometrial tumors. Results: Radiochemical analysis demonstrated that the postpurification purity of the 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative was ≥95%, with a specific activity of 99mTc of 47.5 TBq/mmol. Cell-binding studies yielded a dissociation constant (mean ± SEM) of 11 ± 1.5 nM. In vivo studies revealed that receptor-mediated uptake was present in all phases of the estrous cycle in reproductive organs and mammary glands but was highest during the diestrous phase of the estrous cycle. Despite high nonspecific uptake in the liver, significant receptor-mediated uptake was observed in target tissues and estrogen receptor–expressing tumors (0.67% for MCF-7 tumors and 0.77% for endometrial tumors). Tumor uptake was reduced by approximately 50% on coinjection with 17β-estradiol. Conclusion: We have characterized a novel neutral tridentate 99mTc(I)-estradiol-pyridin-2-yl hydrazine derivative for potential use in breast and endometrial cancer imaging. This study represents the first step on a path toward the design of estrogen-based Tc-labeled tracers with improved targeting and SPECT imaging characteristics.

Design and Synthesis of a New Class of Membrane Permeable Triazaborolopyridinium Fluorescent Probes

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are tunable through variation of pyridyl or hydrazone substituents, and they offer favorable photophysical characteristics featuring large Stokes shifts and general insensitivity to solvent or pH. The stability, neutral charge, cell membrane permeability, and favorable relative influences on the water solubility of HPY conjugates are complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes. This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target. Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.

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.

Highly efficient synthesis and characterization of the GPR30-selective agonist G-1 and related tetrahydroquinoline analogs

The GPR30 agonist probe G-1 and structural analogs were efficiently synthesized using multicomponent or stepwise Sc(III)-catalyzed aza-Diels-Alder cyclization. Optimization of solvent and reaction temperature provided enhanced endo-diastereoselectivity.

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.

Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity.

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.