Antagonists for Constitutively Active Mutant Estrogen Receptors: Insights into the Roles of Antiestrogen-Core and Side-Chain

Abstract

A major risk for patients having estrogen receptor α (ERα)-positive breast cancer is the recurrence of drug-resistant metastases after initial successful treatment with endocrine therapies. Recent studies have implicated a number of activating mutations in the ligand-binding domain of ERα that stabilize the agonist conformation as a prominent mechanism for this acquired resistance. There are several critical gaps in our knowledge regarding the specific pharmacophore requirements of an antagonist that could effectively inhibit all or most of the different mutant ERs. To address this, we screened various chemotypes for blocking mutant ER-mediated transcriptional signaling and identified RU58668 as a model compound that contains structural elements that support potent ligand-induced inhibition of mutant ERs. We designed and synthesized a focused library of novel antagonists and probed how small and large perturbations in different ligand structural regions influenced inhibitory activity on individual mutant ERs in breast cancer cells. Effective inhibition derives from both nonpolar and moderately polar motifs in a multifunctional side chain of the antagonists, with the nature of the ligand core making important contributions by increasing the potency of ligands possessing similar types of side chains. Some of our new antagonists potently blocked the transcriptional activity of the three most common mutant ERs (L536R, Y537S, D538G) and inhibited mutant ER-mediated cell proliferation. Supported by our molecular modeling, these studies provide new insights into the role of specific components, involving both the ligand core and multifunctional side chain, in suppressing wild-type and mutant ER-mediated transcription and breast cancer cell proliferation.

Figure 1.. Chemical Structures.

(A) Structure of initial lead compound, RU58668. (B) Segmented elements for the design of a focused library of novel antagonists based on RU58668: Ligand core, Linker, Central Polar group, and Terminal Component.

Figure 2.

Downregulation of ERα levels in MCF-7 cells. Cells were treated with the indicated concentration of compounds for 24 hours, and ERα levels were determined by cell Western blot analysis.

Figure 3.

Inhibition of MCF-7 cell proliferation driven by WT or mutant ERα. MCF-7 cells that were expressing doxycycline-inducible WT or mutant ER were treated with various concentrations of the indicated compound in regular medium and proliferation was measured after 5 days using the MTT assay. Graphs were plotted with the mean ±SD of 6 biological replicates.

Figure 4.

i) Superimposed Protein Data Bank (PDB) entries of the Y537S mutant in the agonist conformation (with and without protein ribbon representations in panels A and B, respectively) exhibit a channel, which is defined by H11, H12, and the H11–H12 loop and which is stabilized by a hydrogen bond between S537 and D351. ii) Binding models for compounds 5, 3, and 1 (depicted in panels A, B, and C, respectively) were developed in a wild-type hERα protein structure (PDB:1ERR). Compounds 5 and 3 benefit from a water-mediated hydrogen bond involving Y526 and the sulfoxide oxygen atom; compound 1 cannot achieve this interaction without the loss of important interactions with the protein’s hydrophobic surface. iii) The binding models, which also are presented in the previous figure section, for compounds 5, 3, and 1 in wild-type hERα (PDB:1ERR), as depicted in panels A, B, and C, respectively, are superimposed on the rERβ (PDB:1HJ1) X-ray complex (yellow carbon atoms) with its ligand, ICI 164,384. Hydrogen bonds are indicated by dashed, yellow lines. iv) A water-mediated hydrogen bond, which involves Y526 and the sulfoxide oxygen atom, is observed in the wild-type hERα (PDB:1ERR) binding models for compounds 3, 5, and 11 (panels A and B). For compound 11, the linker adopts a lower-energy linear conformation (panel B), and the ligand’s core is further stabilized by a hydrogen bond between His524 and the oxygen atom of the rotatable phenol group of the ligand. The origin of the linker conformational difference is the rotation of the phenoxy group at the base of the linker (in compound 11 vs. compounds 3 and 5), as shown in the superimposition (panel C). v) In the wild-type hERα (PDB:1ERR) binding models for compound 11 (panel A) and fulvestrant (panel B), the perfluorinated end of the flexible chain binds more deeply in a surface pocket when fulvestrant is compared to compound 11. The protein model in each case is represented by a color-coded surface, with green indicating a mainly hydrophobic region. The water of the hydrogen bond involving Y526 is also shown. Hydrogen bonds are indicated by dashed, magenta lines.

Scheme 1.

General scheme for synthesis of novel antagonists (1–11). See SI for further details of the synthetic procedures for all the antagonists mentioned in Table 1.