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A series of indole-thiazole derivatives act as GPER agonists and inhibit breast cancer cell growth Austin O'Dea, Chelsea Sondergard, Patrick Sweeney, and Christopher Kent Arnatt ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00212 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018
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ACS Medicinal Chemistry Letters
A series of indole-thiazole derivatives act as GPER agonists and inhibit breast cancer cell growth Austin O’Dea,†,‡ Chelsea Sondergard,†,‡ Patrick Sweeny,† Christopher Kent Arnatt†,* †Department of Chemistry, College of Arts and Sciences, Saint Louis University, St. Louis, MO, USA GPER, GPR30, estrogen receptor negative breast cancer, cAMP, GPCR, SERM ABSTRACT: The G Protein-Coupled Estrogen Receptor (GPER, GPR30) represents a promising target for the treatment of estrogen receptor α and β negative breast cancers. Previously reported agonists of GPER have shown that activation of GPER inhibits breast cancer cell proliferation. We report herein a new GPER agonist scaffold based upon in silico pharmacophore screening. Three of these compounds were found to increase cAMP at similar levels as the known GPER-selective agonist G-1. Compound 5 was found to be selective for GPER (over estrogen receptor α and β) and inhibit breast cancer cell proliferation at levels consistent with G-1. Docking studies go on to suggest that both 5 and G-1 bind within the same binding pocket in GPER and point to possible key residues that are important in GPER activation.
In 1996, the cDNA encoding for a 375- amino-acid orphan G protein-coupled receptor (GPCR) was isolated and identified as GPR30 (now G protein-coupled estrogen receptor, GPER).1 The endogenous agonist of GPER was discovered when mitogen-activated protein kinase (ERK 1/2) activation was present with treatment of 17-β estradiol (E2, Figure 1) and in the absence of estrogen receptor α or estrogen receptor β (ERα and ERβ respectively) expression.2 Later, in a separate study, Revankar et al. showed the first binding curve for E2 and E2-induced calcium mobilization mediated by GPER in a GPER-overexpressed COS-7 cell line.3 While the exact physiological functions of GPER are still being elucidated, there have been several influential discoveries of its involvement in disease states.4-6 Due to the lack of traditional therapeutic targets (e.g. ERα and ERβ) for estrogen receptor negative breast cancers, the expression and function of GPER in those cancers presents a possible treatment option.2,7-10 Clinically, GPER has been shown to be expressed in over 50% of breast carcinomas and its expression has been correlated to increased tumor size, metastasis, and poor clinical outcomes.11-15 A study using two estrogen receptor negative cell lines, SkBr3 (human breast cancer, ERα and ERβ negative) and MDA-MB-231 (human breast cancer, ERα negative, and ERβ positive), found that GPER activation by G-1 could inhibit breast cancer cell proliferation in vitro and in vivo.16 Using robust standard methods and complementary experiments, Wei et al. found that multiple signaling pathways (upregulation of p21 and p53, activation of ERK1/2) were involved in G-1-induced inhibition of breast cancer proliferation.16 While prior groups have shown a GPER-dependent effect on breast cancer, this was the first study to methodically approach the signaling pathway needed for this effect in vitro and in vivo.17,18 Several groups have discovered a variety of GPER agonists and antagonists via virtual screening.17,19-25 Bologa et al. were
the first to discover a selective GPER agonist by using virtual screening for E2-like compounds.19 The selective agonist produced from this study, G-1 (Figure 1), had nanomolar affinity for GPER and did not bind to ERα or ERβ at concentrations up to 1 µM.19 Subsequently, two selective GPER antagonists, G-15 and G-36 (Figure 1), were found by a similar screening method looking for G-1-like compounds.20,21 While these studies were successful, they all relied on using the binding pocket of ERα, rather than GPER, to predict ligand binding and function. Therefore, no knowledge of any receptor interactions could be gained. Additionally, with only three compounds in the series, no structure-activityrelationship (SAR) studies can reliably be performed. However, these GPER selective chemical probes have played a crucial role in every major study to determine GPER’s functions in vitro and in vivo.6
Figure 1. Structures of GPER agonists and antagonists.
The success of G-1, G-15, and G-36 has led to other compounds being found through computational modeling and screening.17,22-25 Our initial GPER homology models (active and inactive state), based upon the agonist and antagonistbound β2 adrenergic receptor (PDB codes: 2RH1 and
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3SN6)26,27 crystal structures, have allowed us to explore differences in the GPER-agonist and antagonist binding site.28,29 This homology model approach revealed several potentially key residues needed for the GPER-agonist interaction. In this study, we have developed a pharmacophore to screen for GPER agonists to explore those key GPERagonist interactions and discovered a new class of GPER agonists which inhibit breast cancer proliferation in vitro. To focus the design and synthesis of new GPER selective compounds, G-1, G-15, and G-36 were docked into the active and inactive GPER homology model, and subsequently superimposed within the binding pocket. As reported before, several favorable interactions were observed between GPER and the compounds.29 A 3D pharmacophore model for GPER binding (both agonist and antagonist) was generated based upon observed interactions within the binding pocket and screened against the ZINC database to search for molecules that shared similar features (Figure S1).30 After virtual screening and further inspection, the top hits were chosen and docked into the GPER active and inactivestate homology models using GLIDE. Based upon the calculated GlideScores, the top scoring molecule in the ZINC database for binding to GPER was ZINC65156419 (Figure 2, 1). Like G-1, G-15, and G-36, 1 consists of an aminecontaining heterocycle connected to a hydrophobic/aromatic group. A subsequent search of the ZINC database revealed four other compounds (Figure 2, 2-5) which shared key characteristics with 1: an indole moiety, a thiazole ring, and an amide linker between them.
Due to the ability of estrogenic compounds to bind to ERα, ERβ, and GPER, the selectivity of 1 and 5 for GPER over either of the classical estrogen receptors was assessed through fluorescence polarization binding studies and cAMP antagonism studies. At both ERα and ERβ, binding of E2 was dose-dependent (Figure 3b and 3c). For G-1, our binding study results confirmed that no appreciable binding exists at either ERα and ERβ. Additionally, no appreciable binding was observed for 5. However, at the highest concentration, 1 µM, 1 showed a moderate level of binding at both ERα and ERβ. Since binding to ERα and ERβ is a possibility, antagonism studies with 1 and 5 were performed to confirm that observed signaling occurs through GPER and not via an off-target interaction. During the antagonism studies, the determined IC80 concentration of G-15, was utilized to antagonize various concentrations of G-1, 1 and 5. There was a statistically significant increase in cAMP response to 10 µM of G-1, 1, and 5 (p
