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eLife. 2017; 6: e26857.
PMCID: PMC5548489

Estrogen receptor coregulator binding modulators (ERXs) effectively target estrogen receptor positive human breast cancers

Jeffrey Settleman, Reviewing editor
Jeffrey Settleman, Calico Life Sciences, United States;

Abstract

The majority of human breast cancer is estrogen receptor alpha (ER) positive. While anti-estrogens/aromatase inhibitors are initially effective, resistance to these drugs commonly develops. Therapy-resistant tumors often retain ER signaling, via interaction with critical oncogenic coregulator proteins. To address these mechanisms of resistance, we have developed a novel ER coregulator binding modulator, ERX-11. ERX-11 interacts directly with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. ERX-11 is orally bioavailable, with no overt signs of toxicity and potent activity in both murine xenograft and patient-derived breast tumor explant models. This first-in-class agent, with its novel mechanism of action of disrupting critical protein-protein interactions, overcomes the limitations of current therapies and may be clinically translatable for patients with therapy-sensitive and therapy-resistant breast cancers.

DOI: http://dx.doi.org/10.7554/eLife.26857.001

Research Organism: E. coli, Mouse, Other

eLife digest

Around 70% of breast cancers in women need one or both of the female hormones (estrogen and progesterone) to grow. To treat these 'hormone-dependent' cancers, patients receive drugs that either block the production of estrogen or directly target a receptor protein that senses estrogen in the cancer cells. Unfortunately, many breast cancers develop resistance to these drugs. This resistance is often caused by genetic mutations that alter the estrogen receptor; for example, the receptor may develop the ability to interact with other proteins in the cell known as coregulators to promote tumor growth.

Developing new drugs that prevent estrogen receptors from interacting with coregulators may provide more options for treating hormone-dependent breast cancers. Here, Raj et al. developed a new small molecule named ERX-11 that is able to inhibit the growth of human breast cancer cells that are sensitive to existing drugs as well as cells that have become drug-resistant.

For the experiments, hormone-dependent breast cancer cells from humans were transplanted into mice. This procedure usually causes the mice to develop tumors, but giving the mice ERX-11 by mouth stopped estrogen receptors from interacting with coregulators and blocked the growth of tumors. Furthermore, ERX-11 does not appear to have any toxic effects on the mice, indicating that it may also be safe for humans.

The findings of Raj et al. suggest that ERX-11 is a promising new drug candidate for treating some breast cancers. The next steps are to examine the effects of ERX-11 on mice and other animals in more detail before deciding whether this molecule is suitable for clinical trials. In the longer term, molecules similar to ERX-11 could also be developed into drugs to treat other types of cancer that are also caused by abnormal interactions of coregulator proteins.

DOI: http://dx.doi.org/10.7554/eLife.26857.002

Introduction

Endocrine therapies for estrogen receptor alpha (ER)-positive breast cancer involve modulation of ER signaling using either antiestrogens (AE) or aromatase inhibitors (AI). However, most patients develop resistance to these drugs, and disease progression is common, with progression to metastases (Musgrove and Sutherland, 2009; Ma et al., 2015). ER signaling is complex and involves coregulators (McDonnell and Norris, 2002; O'Malley and Kumar, 2009). Therapy-resistant tumors often retain ER-expression and ER-signaling. While multiple mechanisms maintain ER signaling in therapy-resistant tumors, ER signaling is mediated by the interactions between activated ER and critical coregulator proteins (Dasgupta and O'Malley, 2014; Lonard et al., 2007).

Alterations in the concentration or activity of selective coregulators enable ER-signaling from AE-ER complexes, effectively converting the antagonist to an agonist (O'Hara et al., 2012; Kurebayashi, 2003). Over a third (38%) of ER coregulators identified in breast cancer are over-expressed (Lonard et al., 2007; Lonard and O'Malley, 2012; Cortez et al., 2014), such as SRC3 (AIB1) (List et al., 2001; Azorsa et al., 2001), SRC2 (Kurebayashi et al., 2000), and PELP1 (Habashy et al., 2010). These deregulated coregulators contribute to mammary tumorigenesis (Cortez et al., 2014), therapy resistance and metastases (Kumar et al., 2009; Shou et al., 2004; Burandt et al., 2013; Girard et al., 2014).

Recent studies revealed that breast tumors acquire mutations in the ER ligand binding domains (L536N, Y537S, Y537N and D538G) that facilitate constitutive activity of these mutant ER (MT-ER) in the absence of ligand (Toy et al., 2013; Robinson et al., 2013; Jeselsohn et al., 2014; Merenbakh-Lamin et al., 2013). Tumors with MT-ER interact with oncogenic coregulators to drive ER-dependent transcriptional programs and proliferation and are poorly responsive to AEs and AIs (Toy et al., 2013; Robinson et al., 2013; Jeselsohn et al., 2014; Merenbakh-Lamin et al., 2013; Toy et al., 2017).

Thus, there is a strategic need for drugs that disrupt interactions between ER and critical coregulators to block ER signaling. In this study, we have synthesized a series of small organic molecules to emulate the nuclear receptor (NR) box motif, important for ER coregulator interactions. We have identified a small molecule named as ER coregulator binding modulator-11 (ERX-11), with potent anti-proliferative activity against ER-driven breast tumors. ERX-11 interacts with ER and blocks the interaction between ER and coregulators. In ER-expressing breast cancer cells, ERX-11 blocks the proliferation and induces apoptosis. ERX-11 has no activity against ER-negative breast cancer cells.

Results

Screening and identification of ER coregulator binding modulator 11 (ERX-11)

The peptidomimetic D2 blocks the interaction between the androgen receptor (AR) and NR-box containing coregulators, such as PELP1, with an IC50 of 40 nM (Ravindranathan et al., 2013). String analyses of the PELP1 interactome suggested an equally robust interaction between PELP1 and ER as that between PELP1 and AR (Figure 1—figure supplement 1A). However, D2 was unable to block the interaction between PELP1 and ER (data not shown) and required much higher concentrations (µM range) to block the proliferation of ER-driven MCF-7 breast cancer cells (Figure 1—figure supplement 1B).

Since sequences flanking the NR-box may influence the affinity and selectivity of coregulator interactions (McInerney et al., 1998), we hypothesized that a longer oligo-benzamide scaffold may more effectively target the interaction between ER and coregulators. This strategy generated a series of tris-benzamides (see Appendix 1—chemical structures 122) (Figure 1A) that added a functional group (R) to the D2 bis-benzamide, corresponding to the amino acid side chain groups found at the i-3/4 or i + 7 position surrounding the NR-box sequences (Figure 1A, Figure 1—figure supplement 2G). Importantly, each of these small molecules were named as ER coregulator binding modulators (ERXs, where X refers to their multiple potential and unknown targets) had differential activities in ER-positive breast cancer cells (Figure 1A, Figure 1—figure supplement 1C), confirming our hypothesis that the sequences flanking the core LXXLL motif could determine specificity and activity. The ERXs maintained the structural requirement for mimicking helices (confirmed by molecular modeling (MacroModel, Schrodinger, NY) (Figure 1—figure supplement 2A, shown for ERX-11).

Figure 1.
Derivation and characterization of ERX-11.

Within this series of tris-benzamides, one compound (ERX-11) with a hydroxyethyl functional group mimicking a serine residue (Figure 1A) was consistently able to block 17-β-estradiol (E2)-induced proliferation in 8/8 ER-positive breast cancer cell lines (Figure 1B, Figure 1—figure supplements 1C, D and and2B),2B), with an IC50, ranging from 250 to 500 nM. In contrast, no effect of ERX-11 was noted in 11/12 ER-negative cells, with modest activity in the MDA-MB-157 cell line (Figure 1C). ERX-11 was as effective as tamoxifen or ICI in reducing the growth of ZR-75 and MCF-7 cells (Figure 1—figure supplements 1E and 2C,D,E), and the combination of ERX-11 and tamoxifen was not additive (Figure 1—figure supplement 2F). While several compounds, including selective estrogen receptor modulators, have been shown to have similar activity against ER-positive breast cancers, the novel chemical structure, potential for a unique mechanism of action led to the designation of ERX-11 as a lead compound. We then established a protocol for large-scale batch synthesis (Figure 1—figure supplement 3A–C).

ERX-11 interacts with ER

Since ERX-11 mimics the NR-box, we expected its direct binding to ER. Modeling studies using Autodock (The Scripps Research Institute, La Jolla, CA) indicated that ERX-11 could bind to the AF-2 domain of ER (Figure 1D). Using biotinylated ERX-11 (synthesis described in Figure 1—figure supplement 3D), we showed that ERX-11 interacts in vitro with purified ER protein (Figure 1E). Addition of ‘cold’ ERX-11 efficiently competed for the binding of purified ER protein to biotinylated ERX-11 (Figure 1F). Further, short 15mer peptides, corresponding to NR box sequences within the SRC1, SRC2, AIB1 and PELP1 proteins, efficiently disrupted the interaction between biotinylated ERX-11 and purified ER (Figure 1G). However, not all LXXLL peptides interfere ERX-11 interaction with ER, as only peptide surrounding the third PELP1 LXXLL motif, but not the first PELP1 LXXLL motif blocked ERX-11 interaction suggesting ERX-11 can only block some LXXLL interactions (Figure 1G, last two panels). Further, pre-incubation of the purified ER protein with selective estrogen receptor degraders (SERDs) GDC-0810 or AZD-9496, or fulvestrant (ICI) was unable to block the interaction between ER and ERX-11 (Figure 1H). In contrast, tamoxifen was able to block the interaction between purified ER and ERX-11 (Figure 1H), suggesting similarities in the ER-binding pockets of ERX-11 and tamoxifen.

We then demonstrated that biotinylated ERX-11 could pull down endogenous ER in ZR-75 nuclear extracts (Figure 2A). These data indicate that ERX-11 directly interact with ER, both as purified protein and within a cellular context. Unbiased evaluation, using immunoprecipitation mass spectrometric (IPMS) analyses of the biotinylated ERX-11 pulldown in MCF-7 cells, identified ER as one of the top ERX-11 interactors (Figure 2B, Table 1). Pathways analysis revealed that ERX-11-binding proteins were involved in the activation of transcriptional regulation (Figure 2—figure supplement 1). Importantly, ERX-11 pulldown included a number of proteins other than ER, including a weak affinity for the progesterone receptor (PGR) and several ER-associated proteins (Table 1). Immunoprecipitation analyses in MCF-7 cells validated the strong affinity of ERX-11 for ER, and weak affinity for the PR-A isoform but not GR, AR or PR-B isoforms (Figure 2C).

Figure 2.
ERX-11 interacts with ER and blocks its interactome.
Table 1.
Top proteins pulled down by biotinylated ERX-11 in MCF-7 cells, as identified by IP-MS. The column marked E2 represents spectral counts for the protein bound to biotinylated control eluted from avidin column, under conditions of E2 stimulation. The column ...

The interaction between ER and ERX-11 within the cells was partially disrupted by high doses of tamoxifen (Figure 2D). Further, in the tamoxifen-resistant cell line, MCF-7-TamR, even high doses of tamoxifen could not disrupt the interaction between ERX-11 and ER (Figure 1—figure supplement 3E). The differences between these results and the in vitro results may be attributed to the context in which ER is presented within the cell.

Using GST-fused ER domain constructs, we validated that ERX-11 interact with the GST-AF2 domain of ER but not with the GST-AF1 or GST-DNA-binding domain of ER (Figure 2E). Further, ER-AF2 interaction with ERX-11 was disrupted by tamoxifen but not ICI (Figure 2F). These data clearly establish the interaction between ER and ERX-11 through the AF-2 domain.

ERX-11 blocks ER interactions with coregulators

Using an unbiased approach with IPMS, we showed that ERX-11 significantly disrupted the interactions of 91 nuclear ER-binding proteins with ER in MCF-7 cells (Figure 2—figure supplement 2A), including well-characterized ER coregulators, such as SRC1, SRC3, and PELP1. Global analyses revealed that these proteins may be involved in a number of critical cellular pathways including transcription, cell cycle and regulation of cell death (Table 2). These findings were validated by IPMS studies in ZR-75 cells, which showed a significant overlap with MCF-7 cells in the coregulators disrupted by ERX-11 (Figure 2—figure supplement 2B). Of the top 10 coregulators, whose interactions with ER were negatively influenced by ERX-11, five contained LXXLL motifs with serine at i-3/4 and i+7/8 flanking position of the LXXLL motifs Table 3. Interestingly in the MDA-MB-231 TNBC model cells, we found that biotinylated ERX-11 was able to stringently interact only with a small number of proteins (n = 8) (Figure 2—figure supplement 2C).

Table 2.
Top biological processes of coregulators, whose interactions with ER are disrupted by ERX-11 in MCF-7 cells.
Table 3.
Selected pathways modulated by ERX-11 treatment. Differentially expressed genes were subjected to pathway analysis using IPA software and the selected top canonical pathways modulated by ERX-11 are shown. This data is related to Figure 3.

In MCF-7 cells, a majority of ER-binding proteins disrupted by ERX-11 were also blocked by tamoxifen (55/88 proteins or 62.5%) (Figure 2G and Figure 2—figure supplement 3). Importantly, a significant number of ER-binding proteins were disrupted by ERX-11 but not tamoxifen (33/88 or 37.5%) (Figure 2G and Figure 2—figure supplement 3). The combination of tamoxifen and ERX-11 had significant overlap with ERX-11 in its ability to block ER-binding proteins (Figure 2—figure supplement 3).

Co-immunoprecipitation studies validated that endogenous complexes containing ER and coregulator PELP1 do form in MCF-7 cells and that ERX-11 disrupts the formation of these complexes (Figure 2H). Proximity ligation assays confirmed the disruption of the endogenous interaction between ER and several coregulators including PELP1, SRC1 and SRC3 (Figure 2I, quantitation in Figure 2—figure supplement 4A). In contrast, ERX-11 has no effect on ER interaction with ARID1B (Figure 2I, quantitation in Figure 2—figure supplement 4B). Using an in vivo structural complementation NanoBiT assay, we found that the direct interaction between ER and PELP1 was enhanced by E2 treatment and that ERX-11 significantly reduced the interaction between ER and PELP1 (Figure 2J).

To confirm the specificity of ERX-11 for the ER AF-2 domain, we demonstrated using reporter-based assays, that ERX-11 failed to reduce the ERE-Luc reporter activity driven by a ERα-VP16 chimera that does not require AF-2 (Figure 2—figure supplement 4C). As expected, tamoxifen, did not affect the ERα-VP16 chimera-induced reporter activity, while ICI reduced the ERE-Luc reporter activity. Evaluation with an endometrial cancer cell line Ishikawa, which exhibits agonist activity via AF1, revealed that ERX-11 lacks AF1 agonist activity (Figure 2—figure supplement 4D). Collectively, these results confirm that the ERX-11 block signaling driven by functional AF2 domain but not by AF1 domain.

We then specifically evaluated whether interactions through the ER LXXLL motif was responsible for ERX-11 activity. Biotinylated ERX-11 was able to pull down both the wild-type ER and the L540Q point mutant ER (which retains E2 binding and does not interact with SRC1) and these interactions were not affected by tamoxifen (Figure 2K). Using ER L540Q point mutant, we showed that the mutant ER still interacts with biotinylated ERX-11 (Figure 2K). Interestingly, the ERX-11 binds strongly to ER▲12 mutant (helix 12 deleted ER) and this binding is blocked efficiently by tamoxifen (Figure 2K). These data suggest that the presence of helix 12 may regulate the conformation of the binding pocket and account for differences in the binding of ERX-11 and tamoxifen to ER. Our data would suggest that removal of helix 12 enables access of ERX-11 to the same binding pocket as tamoxifen and may reflect the in vitro data, where tamoxifen and ERX-11 compete efficiently for ER binding. In contrast, neither GDC-0810 nor AZD-9496 were able to block ERX-11 binding to ER or its mutants, suggesting that their binding to ER occurs through distinct pockets (Figure 2—figure supplement 5A and B).

In competition assays, tamoxifen fails to dislodge ERX-11 from ER (Figure 2—figure supplement 5C, Figure 1—figure supplement 3E). Increasing concentrations of tamoxifen is only able to dislodge ERX-11 from ER▲12 mutant at higher concentrations, suggesting that ERX-11 interaction with ER is through a second binding site (Figure 2—figure supplement 5D).

Further, using an ER restoration model, MTT cell viability assays revealed that introduction of either ER, ER▲12 or ER-L540Q into MDA-MB-231 cells, restored ERX-11 growth inhibitory activity in non-responsive MDA-MB-231 cells. These results further underscore the importance of ER in ERX-11 mode of action (Figure 2—figure supplement 5E).

Docking simulations model ERX-11 binding to ER

We have modeled ERX-11 interaction with ER using docking simulations of ERX-11 using known crystal structures (Figure 2—figure supplement 6). In the agonist-bound conformation (3ERD.pdb), helix 12 is relocated and forms a hydrophobic cleft (i.e. AF2-binding site) that is surrounded by helices 3, 4, 5 and 12: ERX-11 can be modeled to make hydrophobic contact with the AF2 site with its two isobutyl side chain groups (Figure 2—figure supplement 6A(A)). In addition, the hydroxyl group of ERX-11 may interact with a residue near AF2 domain. These data suggest that ERX-11 interacts with ER LBD differently than the agonist.

In the antagonist-bound conformation (3ERT.pdb), 4-hydroxytamoxifen induces conformational change and makes helix 12 occupy the AF2-binding site, blocking both coactivator and corepressor binding: here, ERX-11 may interact with an alternate pocket formed by helices 5, 11 and 12, as shown in the Figure 2—figure supplement 6A(B). These data could explain why the interaction between ERX-11 and purified ER could be blocked by tamoxifen. However, in a cellular context, tamoxifen cannot block ERX-11 binding to ER suggesting that the secondary binding site of ERX-11 on ER may be stabilized by coregulators. Docking simulation of ERX-11 on human ERα with affinity-tagged corepressor peptide (Figure 2—figure supplement 6A(C))(2JFA.pdb) or rat ERβ crystal structure with ICI bound (Figure 2—figure supplement 6A(D))(1HJ1.pdb) showed that ERX-11 can still bind to the AF2 domain, in a similar manner as it does when the ligand is bound. These data further support our biochemical findings that ICI does not block ERX-11 interaction with ER.

Detailed evaluation showed that (1L2I.pdb) two isobutyl groups of ERX-11 may dock into the AF2 binding site (black dashed box) (Figure 2—figure supplement 6B). The hydroxyl group of ERX-11 may interact with Gln375 of the helix five through a hydrogen bond. The carboxamide group of ERX-11 was docked into a pocket formed with Gln542, Asp545 and Ala546 on the helix 12. Additional docking experiment on ER crystal structures without helix 12 (3ERT.pdb and 5ACC.pdb) showed that ERX-11 was found to bind to the tamoxifen-binding site in the ER▲12 (Figure 2—figure supplement 6C(A)). The superimposition of tamoxifen (red) and ERX-11 (green) clearly shows the overlap of their binding sites (3ERT.pdb) (Figure 2—figure supplement 6C(B)). This may explain our experimental results showing the competition of tamoxifen with ERX-11 on ER▲12. In the presence of SERDs, ERX-11 can bind to the AF2 domain (Figure 2—figure supplement 6C(C), which do not overlap with the binding site of SERDs (Figure 2—figure supplement 6C(D)) (5ACC.pdb). A model to explain the potential interactions between ER and ERX-11 or between ER▲12 mutant and ERX-11 in the absence or presence of agonist, SERDs or tamoxifen is included (Figure 2—figure supplement 7).

ERX-11 blocks ER-driven breast cancer signaling pathways

RNA-seq analyses revealed that ERX-11 altered the expression of 880 E2-regulated genes (p<0.01) in ZR-75 cells compared to vehicle control (Figure 3A). Using stringent cutoffs (p<0.01 and RPKM FC > 1.5), ERX-11 down-regulated more genes (669) than upregulated (211) as evidenced by the volcano plot (Figure 3—figure supplement 1A and B) (complete list at GEO database, accession number GSE75664). RT-qPCR analyses validated the expression of the top down-regulated (Figure 3B and Figure 3—figure supplement 1C) and up-regulated genes (Figure 3H). Gene set enrichment analysis (GSEA) confirmed the correlation between ERX-11–regulated genes and both the tamoxifen-responsive and E2-responsive genes set (Figure 3C and D). In addition, ERX-11-regulated genes correlated well with signatures of early and late response E2 targets as well as genes driven by consensus ER motifs (Figure 3E and F).

Figure 3.
ERX-11 globally disrupts ER-mediated transcriptome.

Ingenuity pathway analysis (IPA) revealed that ERX-11 significantly down-regulated genes involved in ER-signaling, breast cancer, cell cycle, and MAPK signaling (Table 3). ERX-11 upregulated gene set positively correlated with apoptotic genes, on GSEA analyses (Figure 3G). Importantly, ERX-11 but not tamoxifen or ICI-treatment-induced apoptosis in ZR-75 (Figure 3I) and in T-47D cells (Figure 3—figure supplement 1D) as shown by induction of caspase 3/7 activity. Apoptosis can be seen as early as 24 hr, however, the effect is more pronounced at 72 hr (Figure 3—figure supplement 1E). These results suggest that ERX-11 both reduces the expression of genes involved in proliferation and enhances expression of genes that promote apoptosis.

ERX-11 inhibits ER-mediated transcription

Using ERE-Luc reporter based transcription assays, we found that ERX-11 significantly reduced the E2-induced ERE-Luc reporter gene activity in ZR-75 cells in a similar fashion as tamoxifen (Figure 4A). In HEK-293T cells, expression of AIB1 and SRC1 enhanced ER-driven ERE-Luc reporter activity and was blocked by both tamoxifen and ERX-11 in both a ligand-dependent (Figure 4B) and ligand-independent manner (Figure 4C). Further, ZR-75 cells stably overexpressing either PELP1 or AIB-1 were responsive to ERX-11 (Figure 4D). These results indicate that ERX-11 interferes with both ligand-dependent and ligand-independent transcriptional function of ER.

Figure 4.
ERX-11 affects ER ligand-dependent and independent transcriptional activity.

Using chromatin immunoprecipitation studies, we found that ERX-11 treatment significantly blocked ER recruitment to canonical ER target gene promoters following E2 treatment (Figure 4E). In contrast, ERX-11 did not affect AR recruitment to AR target genes (Figure 4—figure supplement 1A). Since ERX-11 binds to ER, we hypothesized that the effect of ERX-11 on ER DNA binding may be mediated via disruption of ER dimerization. Using the NanoBiT assay, we demonstrated that ERX-11 efficiently blocks the dimerization of ER (Figure 4F). In contrast, ERX-11 did not affect AR dimerization (Figure 4—figure supplement 1B).

Using E2 dendrimer conjugates (EDC), that are potent in activating ER non-genomic signaling but not ER genomic signaling (Chakravarty et al., 2010), we showed that ERX-11 was unable to influence the EDC-mediated non-genomic activation of the Src, AKT and MAPK pathways (Figure 4—figure supplement 1C). A detailed time course evaluation showed that ERX-11 treatment only modestly altered the stability of ER within 24 hr in ZR-75 and T-47D cells (Figure 4G, Figure 4—figure supplement 1D). However, similar to its inhibitory activity on ER transcription, after several days of ERX-11 treatment, decreased ER levels were detected (Figure 4H). Accordingly, RTqPCR results showed that ERX-11 reduce the ER transcript levels under conditions of long-term treatment (7 days). These results reflect the inhibition of ER signaling indirectly affected autoregulation of ER transcript by E2-ER signaling (Figure 4—figure supplement 1E).

ERX-11 suppresses ER-driven breast tumor growth in vivo

Our prior studies indicated that our peptidomimetics are orally bioavailable (Ravindranathan et al., 2013). We detected no overt signs of toxicity after 14 days of treatment of C57BL/6 mice (n = 3) with 10, 50 or 100 mg/kg/day of ERX-11 via oral gavage. ERX-11 treatment neither caused weight loss nor have uterotrophic effects or any observable hematologic, liver and kidney abnormalities (data not shown). We designated our highest dose (100 mg/kg) as the maximum tolerated dose and used 10% of this dose (10 mg/kg/day) for testing as therapeutic dose, so that we would have at least a 10:1 therapeutic to toxicity ratio.

Established ZR-75 xenografts (n = 8 tumors/group) in the mammary fat pad of nude mice were randomized to feed via oral gavage 5 days/week with either 10 mg/kg ERX-11 or vehicle (30% Captisol). ERX-11 treatment resulted in significantly smaller tumors (63% reduction compared to control) (Figure 5A). ERX-11–treated tumors exhibited less proliferation (Ki67 staining), and more apoptosis (TUNEL and caspase-3 staining) than controls (Figure 5A,B). Further, ERX-11 treatment group had lower ER but similar PELP1 protein expression levels within the tumor, compared to control (Figure 5B). The mice body weights in the control and ERX-11 treated groups were similar (Figure 5—figure supplement 1A). Mice treated with ERX-11 exhibited no uterotrophic effects, no changes in ovary, liver and kidney gross morphology on H and E staining, or acute phase injury to liver and kidney (Figure 5—figure supplement 2A–D). These data indicate that ERX-11 is a potent inhibitor of the growth of ER-positive breast tumors in vivo with no overt signs of toxicity in mice.

Figure 5.
ERX-11 inhibits the growth of ER-positive, syngeneic and coregulator-driven breast tumors in vivo.

To address the potential immunogenicity of ERX-11, D2A1 ER-positive breast cancer xenografts were established in a syngeneic BALB/c model system with an intact immune system. In D2A1 model cells, cellular gene int-5/aromatase in BALB/c mammary alveolar hyperplastic nodule (D2 HAN/D2 tumor cells) is activated as a result of mouse mammary tumor virus integration within the 3' untranslated region of the aromatase gene. Thus, these models also have ability to synthesize local estrogen via aromatase induction. Further, this model express ER, and represent a model of intra-tumoral estrogen-driven mammary cancer (Figure 5—figure supplement 1B(A,C). D2A1 cells are responsive to antiestrogen treatment (Figure 5—figure supplement 1B(B). Oral administration of ERX-11 dramatically limited the proliferation of these rapidly progressing tumors (Figure 5C). The proliferative indices of ERX-11-treated tumors were significantly lower than controls (Figure 5C, Figure 5—figure supplement 1C), while the apoptotic indices were higher than control (Figure 5—figure supplement 1C). Again, no overt signs of toxicity was noted in these mice; specifically, no enlargement of the spleen or evidence of immune complex deposition within the kidneys was detected (data not shown). These data further support the potential clinical translatability of ERX-11.

ERX-11 reduces growth of therapy-resistant breast cancer cells

To evaluate the effect of ERX-11 on coregulator-driven proliferation, we used ZR-75 cells stably overexpressing AIB-1 and PELP1. While these modified ZR-75 cells are highly proliferative (Figure 4D), ERX-11 was potent in blocking their proliferation (Figure 5D). ERX-11 was potent (73% reduction in tumor volume compared to control) against the growth of MCF-7-PELP1 xenografts, which overexpress PELP1 (3-fold higher than parental MCF-7) (Figure 5E). IHC analysis of ERX-11 treated tumors showed decreased Ki-67 staining (Figure 5E, Figure 5—figure supplement 1D).

Importantly, ERX-11 had activity against ER-driven breast cancer cell lines that were either resistant to tamoxifen (MCF-7-TamR, Figure 6A, or MCF-7-HER2, Figure 6B) or to letrozole (MCF-7-LTLT, Figure 6C). In these cell lines, ERX-11 was still able to interact with the ER, both in the absence and presence of tamoxifen (Figure 6D). In contrast to SERD (ICI), which had limited activity on the tamoxifen resistant cell lines, ERX-11 had potent activity (Figure 6A) (Figure 6—figure supplement 1A). ERX-11 was potent against the growth of MCF-7-LTLT xenografts (Figure 6E). IHC analysis of ERX-11-treated tumors showed decreased Ki-67 staining (Figure 6—figure supplement 1B).

Figure 6.
ERX-11 reduces the growth of ER positive and ER-MT endocrine-therapy-resistant tumors.

We then evaluated the effect of ERX-11 against two prevalent ER mutants (MT-ESR1-Y537S, MT-ESR1-D538G) (Toy et al., 2013; Robinson et al., 2013; Jeselsohn et al., 2014; Merenbakh-Lamin et al., 2013). Using biotinylated ERX-11, we showed that ERX-11 interacts directly with ESR1-MT (ESR1-MT-D538G, ESR1-MT-Y537S), with high affinity comparable to the affinity to WT-ER (Figure 6F). Using CRISPR/Cas9, we knocked down ER in ZR-75 cells and then stably transfected with WT-ESR1 or MT-ESR1 (Y537S, and D538G). While ESR1-MT expressing cells showed higher rates of proliferation than WT-ESR1-expressing cells (Figure 6—figure supplement 1C), they were still inhibited by ERX-11 (Figure 6G). Further the ability of these ESR1-MT to drive ligand-independent transcription from an ERE-Luc reporter was also efficiently blocked by ERX-11 (Figure 6H). Further, these ESR1-MT expressing cells were resistant to tamoxifen, however, were sensitive to ERX-11-mediated growth inhibition (Figure 6I). Further, oral ERX-11 administration had significant activity against the growth of ZR-75-ESR1MT-Y537S xenografts in vivo (Figure 6J), with significant reduction in proliferative indices (Figure 6K, Figure 6—figure supplement 1D). These data support the efficacy of ERX-11 against breast tumors driven by mutant ESR1.

ERX-11 has activity against primary patient derived breast tumor explants

We recently developed an ex vivo culture model of primary breast and prostate tumors, which allows for the evaluation of drugs on breast tumors while maintaining their native tissue architecture (Dean et al., 2012; Schiewer et al., 2012). In brief, surgically extirpated de-identified breast tissues are sliced into small pieces and grown ex vivo for a short term on a gelatin sponge in the absence or presence of ERX-11 (Figure 7A). Incubation of ERX-11 with ER-positive breast tumor samples (patient characteristics detailed in Table 4) dramatically decreased their proliferation in 11/12 patients (Ki67 staining) compared to untreated controls (Figure 7B). Further, ERX-11 treatment significantly reduced the ER staining (Figure 7C), but not PELP1 staining (Figure 7—figure supplement 1) in 12/12 ER-positive tumors. Importantly, ERX-11 treatment had no effect on the proliferation on 6/6 triple negative breast cancer (TNBC) tumors (Figure 7D). These results suggest that ERX-11 has the potential to selectively influence the growth of human breast tumors expressing ER.

Figure 7.
ERX-11 decreases the growth of patient-derived explants (PDEx): Schematic representation of ex vivo culture model is shown.
Table 4.
Clinicopathologic characteristics of the 12 patients, whose ER+, PR + status in breast tumors were analyzed by Ki67 and ER staining. This data is related to Figure 7B,C.

Discussion

The majority of breast cancer is ER positive. Therapeutic agents that suppress oncogenic ER activation by depletion of hormone-driven growth signaling or by blocking synthesis of hormone have become the mainstay of systemic treatment for breast cancer. However, both de novo and acquired therapy resistance are major clinical challenges. Importantly, ER signaling is intact in these therapy-resistant tumors. ER interaction with critical coregulator proteins appears to mediate ER signaling in these therapy-resistant and ER-positive metastatic tumors. While AIs and AEs may disrupt some of these ER-coregulator interactions, their ability to target these interactions is limited in therapy-resistant cells. In this study, we described the development of a novel inhibitor that targets ER interactions with coregulators. Using in vitro and in vivo assays, we demonstrated that (Musgrove and Sutherland, 2009) ERX-11 blocks both ligand-dependent and ligand-independent ER signaling, (Ma et al., 2015) ERX-11 effectively blocks ER signaling in both therapy-sensitive and therapy-resistant cells and (McDonnell and Norris, 2002) disruption of ER signaling by ERX-11 has biological activity against both therapy-sensitive and therapy-resistant cells, with minimal overt signs of toxicity in vitro and in vivo.

Until recently, protein–protein interactions have been viewed as undruggable. However, the recent advent of a transformative class of compounds (peptidomimetics) allowed us to rationally design and synthesize small organic molecules that structurally emulate target protein sequences in defined conformations. We have developed a novel oligo-benzamide scaffold for mimicking helical protein segments (Ravindranathan et al., 2013; Ahn and Han, 2007). The rigid framework of the oligo-benzamide scaffold can present functional groups mimicking amino acid residues in a helical conformation (e.g. ones in i, i-3/4, and i + 7 positions). Furthermore, we have established efficient synthetic routes to make bis- and tris-benzamides as alpha-helix mimetics (Lee and Ahn, 2011; Marimganti et al., 2009). Based on the helix mimicking tris-benzamide scaffold, we designed ERX-11 to block the interactions between ER and a subset of its coregulator proteins containing the NR box in a helical structure.

The unbiased IPMS analyses suggest that ERX-11 interacts with ER and blocks the interactions of ER with multiple coregulators. While the broad effect of ERX-11 protein–protein interactions raises potential concerns about off-target activities, they are largely mitigated by the lack of overt signs of toxicity in cell culture models and multiple animal models tested to date. In addition, the multiplicity of targeted protein-protein interactions makes the development of resistance to the ERX-11 less likely. Thus, ERX-11 represents an exciting new mechanism to attenuate ER oncogenic functions.

Like tamoxifen, ERX-11 potently blocks the proliferation of therapy-sensitive cells. Unlike tamoxifen, ERX-11 has activity in multiple therapy-resistant models, including those driven by ER ligand-binding domain mutants. Unlike a classic SERD, ERX-11 does not cause immediate ER degradation, but appears to affect ESR1 levels over several days, by blocking its transcription.

Our data clearly indicate that ERX-11 binds to AF2 domain of ER. However, the exact interface between ERX-11 and ER has not been established. The ability of tamoxifen to compete efficiently for ERX-11 binding to purified ER in vitro and to ER within the cell suggests a significant overlap between the tamoxifen and ER-binding site on ER. In addition, the inability of the SERDs and ICI to disrupt the interaction between ER and ERX-11 suggests that ERX-11 may also interact with a secondary tamoxifen-binding site within ER. As shown for ER beta, tamoxifen has two distinct binding sites- one in the consensus ligand-binding pocket, and another in the hydrophobic groove of the coactivator recognition surface (Wang et al., 2006). Published studies using LXXLL peptide probes showed ER undergo distinct conformational changes as a result of binding with different ligands and such changes expose distinct surfaces on ER facilitating interaction with various coregulators (Paige et al., 1999). These studies also reported that tamoxifen binding can create unique surface on ER that facilitate binding of unique LXXLL-binding peptides (Tamrazi et al., 2002). Further investigations of the ERX-11- ER interface with co-crystallization studies are ongoing and are likely to clarify the precise nature of the interaction.

Our studies also suggest that ERX-11 can interact with ER, even in therapy-resistant cells. While therapy resistance can be attributed to multiple mechanisms, structural changes in ER via post-translational modifications or point mutations may create new binding surfaces on ER for coregulator interactions and potentially for ERX-11 interactions. The interaction between ERX-11 and ER-MTs in therapy-resistant cells supports a model for ERX-11 interaction with ER at an alternate secondary site distinct from the site of tamoxifen binding. These findings may explain the differences between ERX-11 and tamoxifen in their activity in both therapy-sensitive and therapy-resistant breast cancer cells.

The ability of ERX-11 to block ER dimerization may be responsible for its disruption of ER DNA binding and these findings are supported by published reports that LXXLL peptides may affect ER dimerization (Tamrazi et al., 2002). We have noted that ERX-11 has a weak affinity for the A-isoform of PGR but not for the B-isoform. We have also found that ERX-11 does not interact with other steroid receptors like GR or AR. In addition, ERX-11 failed to show activity on AR-expressing prostate cancer cells.

Disruption of multiple protein–protein interactions by ERXs may result in significant toxicity. To evaluate this, we have initially tested toxicity using a dose of 100 mg/kg in immune competent C57BL/6 mice for 14 days. We did not observe any uterotropic activity or immune effects. Further, no overt signs of toxicity was found in various E2-responsive organs including the liver, lung, heart, and kidney. Similarly, in five separate studies using a dose of 10 mg/kg in tumor-bearing mice, we did not find any overt signs of toxicity; however, ERX-11 treatment significantly limited ER expression in the tumors and reduced tumor growth. To address potential issues with immunogenicity of these tris-benzamides, we also evaluated ERX-11 treatment in syngeneic models and found no overt signs of toxicity.

While the ER coregulator protein levels are tightly regulated under normal conditions, many coregulators are over-expressed in breast cancer (Lonard et al., 2007), substantially contribute to ER-signaling, drive disease progression (Singh and Kumar, 2005) and correlate with a poor prognosis (List et al., 2001; Azorsa et al., 2001; Tamrazi et al., 2002). The differential coregulator milieu within breast tumors and the dependence of breast tumors on ER and coregulator-driven signaling may explain why ERX-11 has potent antiproliferative activity within the tumor but does not have any overt signs of toxicity. Since ERX-11 blocked some but not all ER coregulator interactions, ERX-11 may function as a polypharmacology agent, and its activity may depend on the concentration and repertoire of coregulators present in a tumor cell.

Importantly, using ex vivo culture of patient-derived tumor tissues, we demonstrated that ERX-11 is effective in limiting proliferation of ER-positive but not ER-negative tumors. We also discovered that ERX-11 treatment of primary tumors within their native microenvironment also reduced ER levels within the tumor. None of the primary ER-negative tumors responded to ERX-11 therapy. These explant studies represent the first evaluation of a drug effect using tissues from primary breast cancer patients and are likely to show biologically relevant outcomes.

In response to hormone binding, ER interacts with multiprotein complexes containing coregulators and transcriptional regulators to activate transcription (McKenna et al., 1999; Collingwood et al., 1999; Tsai and O'Malley, 1994; Torchia et al., 1998). Even though coregulators modulate ER functions, each coregulator protein appears to play an important but not overlapping function in vivo (Han et al., 2006; Xu et al., 1998). Accordingly, the ERX-mediated blockage of coregulator interactions with ER resulted in both inactivation and activation of unique sets of genes and pathways modulated by ER oncogenic signaling, leading to tumor suppression. The pathways/genes modulated by ERX-11 can be used to correlate with the outcome of its therapy, and they may serve as biomarkers that prognosticate response to these agents.

The biology of E2- ER signaling is complex and context dependent. Elegant studies have shown that ER, depending on the ligand and presence of unique set of coregulators can promote apoptosis (Jordan, 2015). In that scenario, antiestrogen and SERM are shown to inhibit apoptosis, hence, many of these drugs exhibit cytostatic response. Estrogen-induced apoptosis is shown to cause an increase in Fas receptor associated with the extrinsic pathway of apoptosis (Lewis-Wambi and Jordan, 2009). Similarly, a recent study found that inhibition of SRC family coregulators using small molecule inhibitor promote significant apoptosis (Song et al., 2016). We consistently observed activation of apoptosis by ERX-11 but failed to observe any apoptosis by tamoxifen or ICI in our assays. We believe that activation of apoptosis is not due to elimination of ER rather due to unique mechanism of action of ERX-11. Specifically, we predict that changes in the ER signaling is due to alterations in coregulator binding to ER. Accordingly, our RNAseq data showed that alterations in genes that contribute activation of apoptosis. However, further studies are needed to clearly identify the mechanisms by which ERX-11 promotes apoptosis.

Currently used drugs (AEs and AIs) are associated with an initial period of clinical response; however, most patients develop resistance with cancer progression. Recent studies suggested that selective estrogen receptor downregulators (SERDs), molecules that eliminate ER expression, may have utility for treating breast cancers that have progressed on AE and/or AIs (McDonnell et al., 2015). Several orally available SERDs (GDC-0810, AZD9496) were recently developed and shown to have utility in treating resistant tumors using preclinical models (Lai et al., 2015; Weir et al., 2016). However, it is important to note that each of the SERDs are only in phase I clinical trials (clinical trials.gov) and none of them are either FDA approved or have proven efficacy in patients. The only FDA-approved agent is Fulvestrant (ICI), which has known pharmacologic limitations as evidenced by its dosing regimen of intramuscular injections every 14 days. Recent studies also showed ESR1 mutations lead to constitutive activity and reduced sensitivity to ER antagonists, and mutations such as Y537S contribute to fulvestrant resistance in vivo (Toy et al., 2017). Since ERXs are small, stable, orally bioavailable small molecular inhibitors, their use as an alternative therapeutic approach may decrease therapy resistance and reduce side effects, which are the current limitations of AEs or AIs.

In summary, we have developed and tested the utility of ERX-11 as a novel therapeutic agent for ER-positive, therapy-sensitive and therapy-resistant breast cancers. Since ERX-11 is orally available and is well tolerated with fewer side effects, ERX-11 can be readily extended to clinical use as a therapeutic agent, and may enhance the survival of advanced breast cancer patients.

Materials and methods

Cell lines

Human breast cancer cells MCF-7, ZR-75, T-47D, MDA-MB-231, BT474, BT549, BT453, SUM159, 4T1, MM468, HCC1937, HCC1187, HCC70, MDA-MB-157, MDA-MB-453, MDA-MB-468 and HEK293T cells were either obtained from American Type Culture Collection (ATCC, Manassas, VA) or a kind gift from Dr. John Minna at UT Southwestern. Ishikawa cells were purchased from Sigma (St. Louis, MO). All of these cells were passaged in the user's laboratory for fewer than 6 months after receipt or resuscitation. Validation experiments were performed using a number of luminal, basal and TNBC cells with extensive prior available molecular profiles were a kind gift of Dr. John Minna. All the model cells utilized are free of mycoplasma contamination. Additionally, STR DNA profiling of the cells was used to confirm the identity using UTHSA and UT Southwestern core facilities. MCF-7-PELP1 cells (Vallabhaneni et al., 2011), MCF-7-HER2 cells (Nabha et al., 2005), MCF-7-TamR cells (Nabha et al., 2005), MCF-7-LTLTca cells (Macedo et al., 2008) and D2A1 cells (Tekmal and Durgam, 1997) were described earlier. MCF-7-LTLTca and MCF-7-TamR cells were cultured in Phenol red-free RPMI medium containing 10% dextran charcoal-treated serum supplemented with either 1 μmol/L of letrozole or 1 μmol/L of tamoxifen, respectively.

Reagents

17-β-Estradiol (cat#E2257), and (Z)−4-Hydroxytamoxifen (cat# H7904) were purchased from Sigma (St. Louis, MO). CRISPR/Cas9 plasmids targeting ESR1 gene were obtained from Horizon Discovery (Cambridge, MA). The anti-PELP1 (cat# 300-180A) and anti-AIB1 (cat# A300-347A) antibodies were purchased from Bethyl Laboratories (Montgomery, TX). Cleaved caspase 3 antibody was purchased from Cell signaling technology (cat# 9661S, Danvers, MA). TUNEL kit (cat# 11684795910) for apoptosis detection was purchased from Roche (Mannheim, Germany) and Ki-67 (1:150) anti-human clone MIB-1 antibody (cat#M7240) was purchased from Dako (Carpinteria, CA).

ERX-11 synthesis

The designed tris-benzamides were constructed by iterative amide bond formation of a 3-alkoxy-4-nitrobenzoic acid with a 3-alkoxy-4-aminobenzamide (Figure 1—figure supplement 3). A 4-nitrobenzoic acid containing a trityl-protected hydroxyethoxy group 3 g was coupled to bis-benzamide 8 that was synthesized by following the previously reported procedure (Ravindranathan et al., 2013), making tris-benzamide 9 (Figure 1—figure supplement 3C). See the chemistry supplement for detailed synthetic procedures and characterization.

Cell viability assays

The effects of ERX analogues on cell viability were measured using the MTT Cell Viability Assay in 96-well plates. Breast cancer cells were seeded in 96-well plates (1 × 103 cells/well) in phenol red-free RPMI medium containing 5% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS) serum. After an overnight incubation, cells were treated with varying concentrations of the ERX analogues in the presence or absence of E2 (1 × 10−8 M) for 7 days. For some experiments viability was also measured using Cell Titer-Glo Luminescent Cell Viability Assay (Promega) in 96-well, flat, clear-bottom, opaque-wall micro plates according to manufacturer’s protocol. For some experiments, apoptosis was measured using Caspase-Glo 3/7 Assay (Promega) using manufacturer’s protocol.

Immunoprecipitation and western blotting

Western blotting and immunoprecipitation were performed as described previously (Nair et al., 2010). Biotin-ERX-11 pull-down assays were done using a previously established protocol using avidin beads (Mann et al., 2013). Pull-down assays using ER-AF2-GST were performed as described previously (Nair et al., 2010). Purified ER full-length proteins and ER LBD (AF2) protein was purchased from Thermo Fisher Scientific, Waltham, MA. The sequences of LXXLL peptides used in the competition assays. SRC1- LXXLL peptide: LTARHKILHRLLQEGSPSD; SRC2- LXXLL peptide: DSKGQTKLLQLLTTKSDQM; SRC3/AIB1- LXXLL peptide: ESKGHKKLLQLLTCSSDDR; PELP1-LXXLL-1 peptide: GLSAVSSGPRLRLLLLESVSG; PELP1-3 LXXLL peptide: SIKTRFEGLCLLSLLVGESPT

Animal studies

All animal experiments were performed after obtaining UTHSA IACUC approval and using methods in the approved protocol. For xenograft tumor assays, 2 × 106 ZR-75, or ZR-75- ER MT-Y537S, or MCF-7- PELP1 cells were mixed with an equal volume of matrigel and implanted in the mammary fat pads of 6-week-old female athymic nude mice as described (Cortez et al., 2012). Based on our previous data as well as published findings, the number of mice needed were chosen to demonstrate differences in tumor incidence or treatment effect. Calculations are based on a model of unpaired data power = 0.8; p<0.05. Once tumors reached measurable size, mice were divided into control and treatment groups (n = 5–8 tumors per group). The control group received vehicle and the treatment groups received ERX-11 (10 mg/kg/day) in 30% Captisol orally. Dose were selected based on pilot MTD study of 10, 50 and 100 mg/kg of ERX-11 for 14 days using C57BL/6 mice. The mice were monitored daily for adverse toxic effects. For MCF-7-LTLT xenograft studies, MCF-7-LTLT model cells were first injected into the mammary glands of nude mice implanted with androstenedione pellets. When the tumor was established, it was dissected into small pieces and they were again implanted subcutaneously into nude mice implanted with androstenedione pellets. Fulvestrant treatment was used as a positive control and MCF-7-LTLT xenografts were treated with 200 mg/kg/ 2 days a week/sc. For syngeneic mice studies, D2A1 cells were first injected into the mammary glands of BALB/c mice. When the tumor was established, it was dissected into small pieces and they were again implanted subcutaneously into the BALB/c mice. After three days of tumor tissue implantation, mice were randomly selected to receive control (n = 7–8) and treatment (n = 7–8) with 20 mg/kg/day of ERX-11 orally. Tumor growth was measured with a caliper at 3–4 day intervals. At the end of each experiment, the mice were euthanized, and the tumors were removed, weighed and processed for IHC staining.

Patient-derived explant (PDEx) studies

UTSW Patients provided written consent allowing the use of discarded surgical samples for research purposes according to an institutional board-approved protocol. De-identified patient tumors were obtained from the UTSW Tissue Repository after institutional review board approval (STU-032011–187). Excised tissue samples were processed and cultured ex vivo as previously described (Mohammed et al., 2015). Briefly, tissue samples were incubated on gelatin sponges for 48 hr in culture medium containing 10% FCS, followed by treatment with either vehicle or E2 (10 nM) in the absence or presence of 10 μM ERX-11 for 48 hr (see Table 4 for clinicopathologic characteristics of these tumors). Representative tissues were fixed in 10% formalin at 4°C overnight and subsequently processed into paraffin blocks. Sections were stained with hematoxylin and eosin and examined to confirm and quantify the presence/proportion of tumor cells. Immunohistochemistry was then performed.

Protein interaction analyses

String analyses were performed for human PELP1 using the http://string-db.org website, with the evidence view at the highest stringency for no more five interactors.

Conformational analysis of ERX-11

A Monte Carlo conformational search was performed using the torsional sampling method (MCMM) implemented in MacroModel (version 9.0, Schrödinger, New York, NY) with automatic setup options. The calculation was done with the maximum number of steps set to 5000 using 100 steps per rotatable bond and an energy cutoff of 21 kJ/mol above the global energy minimum. The searches were done using MM3 force field (chosen for its accuracy with organic molecules) combined with the GB/SA water solvation model with standard settings and the following cut-offs: van der Waals, 8.0 Å; electrostatic, 20.0 Å; and hydrogen bond, 4.0 Å. The observed conformations were minimized by 500 iterations of Polak-Ribiere Conjugate Gradient (PRCG) algorithm (a conjugate gradient minimization scheme that uses the Polak-Ribiere first derivative method with restarts every 3N iterations) (0.05 kJ/mol).

Molecular docking of ERX-11 to ER

AutoDock 4.2 software package, as implemented through the graphical user interface called AutoDockTools (ADT), was used to create input PDBQT files of a receptor and a ligand. The input file of ER was prepared using the published coordinates (PDB 1L2I). Water molecules were removed from the protein structure and hydrogen was added. All other atom values were generated automatically by ADT. The docking area was assigned visually around the peptide ligand. A grid box of 24 Å x 20 Å x 24 Å was calculated around the docking area using AutoGrid. The x,y,z coordinates of the center of the grid box were set to x = −9.0, y = 14.0 and z = 26.0, respectively. The input file of ERX-11 was created from its energy-minimized conformation using ADT. Docking calculations were performed with AutoDock Vina 1.1.2. A search exhaustiveness of 16 was used and all other parameters were left as default values.

Reporter gene assays

Briefly, cells were transiently co-transfected with 200 ng of ERE-Luc reporter with 100 ng of ER-WT, ER-MT, PELP1, SRC1, SRC2, SRC3 or control vectors using Turbofect transfection reagent (Thermo Scientific, Waltham, MA). After 24 hr, cells were treated with either vehicle or ERX-11 for an additional 24 hr. β-galactosidase reporter (50 ng) plasmid was co-transfected and used for data normalization. Cells were lysed in Passive Lysis Buffer, and luciferase activity was measured using the luciferase assay system (Promega, Madison, WI) in a luminometer.

RNA sequencing and RT-qPCR

RNA-seq was performed using the UTHSA core–established protocol. Briefly, ZR-75 cells were treated with either vehicle or ERX-11 for 48 hr, and total RNA was isolated using RNAesy mini kit (Qiagen) according to the manufacturer’s instructions. Differential expression analysis was performed by DEseq and significant genes with at least 1.5-fold change with p<0.01 were chosen for analysis. The interpretation of biological pathways using RNA-seq data was performed with IPA software using all significant and differentially expressed genes. RNA-seq data have been deposited in the GEO database under accession number GSE75664. To validate the selected genes, reverse transcription (RT) reactions were performed by using SuperScript III First Strand kit (Invitrogen, Carlsbad), according to manufacturer’s protocol. Real-time PCR was done using SybrGreen on an Illumina Real-Time PCR system, using primers listed in Table 5.

Table 5.
Primer sequences used for RTqPCR

ChIP

Chromatin immunoprecipitation (ChIP) analysis was performed using antibodies specific for the ER (Santa Cruz). Briefly, MCF-7 (7 × 106) or T-47D (2 × 107) cells were plated in 150 mm dishes, starved in unsupplemented phenol red-free DMEM for 24 hr and then treated for 2 hr with either ethanol or E2 after prior incubation with either DMSO or ERX-11. Relative recruitment was determined by qPCR of purified ChIP and input DNA in triplicate. The results presented are representative of two independent experiments.

NanoBiT luciferase studies

The NanoBiT assay utilizes a structural complementation-based approach to monitor protein–protein interactions within living cells. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 1 kDa) subunits of NanoLuc Luciferase were optimized for the analysis of protein interaction dynamics. When LgBiT and SmBiT subunits are separated, the Large BiT part loses the majority of luciferase activity. However, when the direct interaction between fusion proteins on LgBiT and SmBiT occurred, the interaction promotes structural complementation between LgBiT and SmBiT and results in full luciferase activity. Protein–protein interaction are then monitored in living cells following addition of the Nano-Glo Live Cell Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate and observed luminescent signals.

To generate different NanoBiT fusion constructs, human ER and PELP1 coding sequences were amplified by PCR and separately subcloned into NB-MCS vectors (Promega). To test the protein–protein interaction between ER and PELP1 by using the NanoBiT assay, C-LgBit-ESR1 paired with C-SmBit-PELP1 or C-LgBit-PELP1 with C-SmBit-ESR1 constructs were transiently transfected into HEK-293T cells by using Fugene HD transfection reagent (Promega). To test the ER dimerization, C-SmBit-ESR1 were cotransfected with either N-LgBit-ESR1 or C-LgBit-ESR1 constructs. On the day after transfection, the medium for the HEK-293T cells was changed to phenol red-free DMEM containing 1% charcoal-stripped FBS. After a 24 hr incubation, the cells were treated with DMSO or ERX-11 (10 μM) for 2 hr and then treated cells with EtOH or E2 (10 nM) for 30 mins. After treatment, Nano-Glo live cell reagents were added into cells and luminscence was measured after 10 mins.

Proximity ligation assays

MCF-7 cells were cultured on collagen-coated cover slips. After treated with vehicle or 10 µM ERX-11 for 30 min, cells are treated with vehicle or 10 nM E2 for 30 min. After the treatment, cells were washed with PBS and then fixed with 10% buffered formalin for 20 min and permeabilized with ice cold methanol at −20°C for 5 min. The cells were then blocked with blocking solution (provided with Duolink In Situ PLA probe, Sigma) for 30 min at 37°C followed by incubating with primary antibodies for ER from different species at 37°C for 2 hr. After washing twice with Wash Buffer A (Duolink In Situ Reagents) at room temperature, cells were then incubated with the appropriate anti-species secondary antibodies to which oligonucleotides had been conjugated (anti-rabbit PLA probe PLUS and anti-mouse PLA probe MINUS) for 1 hr at 37°C, followed by treatment with Duolink ligation-ligase solution for 30 min at 37°C. Finally, the cells were incubated with the Duolink amplification-polymerase solution for 60 min at 37°C, followed by washing and mounting on slides with Duolink Mounting Medium with DAPI. Images were taken using a Nikon Fluorescence Microscope.

IPMS

MCF-7 or ZR-75 cells were grown in RPMI-1640 medium supplemented with 10% FBS. After cells reach 90% confluence, the medium was changed to phenol red-free RPMI1640 containing 1.5% charcoal stripped serum for 48 hr to starve the cells. After starvation, the cells were treated with vehicle or 10 µM ERX-11 for 2 hr followed by a 2 hr treatment with vehicle or 10 nM E2. Then, the cells were washed using PBS and incubated with Pierce IP Lysis Buffer (Thermo Fisher Scientific Inc.) at 4°C for 20 min. The cell lysates were centrifuged at 14,000 rpm at 4°C for 10 min, and the supernatants were used for IPMS analysis.

Dynabeads Protein G (Invitrogen) are pre-coupled with anti- ER antibody (Santa Cruz, sc-8002) and then incubated with 1.5 mg cell lysate over night at 4°C. Then the Dynabeads were washed using PBS and PBST (0.01% Tween 20) and eluted in 30 µL NuPAGE LDS sample buffer at 95°C for 15 min. The final eluents containing ER and associated proteins were separated by using SDS-PAGE. The obtained proteins were proteolytically digested and subjected to mass spectrometry (MS) analysis.

Gel band samples were digested overnight with trypsin (Promega) following reduction and alkylation with DTT and iodoacetamide (Sigma). The samples then underwent solid-phase extraction cleanup with Oasis HLB plates (Waters) and the resulting samples were analyzed by LC/MS/MS, using an Orbitrap Fusion Lumos (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex). Samples were injected onto a 75 μm i.d., 50 cm long Easy Spray column (Thermo) and eluted with a gradient from 0% to 28% buffer B over 60 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.08% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.2 kV and capillary temperature of 275°C. MS scans were acquired at 120,000 resolution and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charge 2–7. Dynamic exclusion was set for 25 s.

Raw MS data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3. Peptide identification was performed using the X!Tandem and open MS search algorithm (OMSSA) search engines against the human protein database from Uniprot, with common contaminants and reversed decoy sequences appended. Fragment and precursor tolerances of 20 ppm and 0.1 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification and oxidation of Met was set as a variable modification. Label-free quantitation of proteins across samples was performed using SINQ normalized spectral index Software.

Immunohistochemistry

For immunohistochemical analysis sections were incubated overnight with the ER (1:50) or PELP1 (1:200) or Ki67 (1:100) antibody in conjunction with proper controls. The sections were then washed three times with 0.05% Tween in PBS for 10 min, incubated with secondary antibody for 1 hr, washed three times with 0.05% Tween in PBS for 10 min, visualized by DAB substrate and counterstained with hematoxylin QS (Vector Lab, Burlingame, CA). A proliferative index was calculated as the percentage of Ki-67-positive cells in five randomly selected microscopic fields at 40X per slide. TUNEL analysis was done using the in situ Cell Death Detection Kit (Roche, Indianapolis, IN) as per the manufacturer’s protocol, and five randomly selected microscopic fields in each group were used to calculate the relative ratio of TUNEL-positive cells.

For the PDEx samples, 5-µm sections were de-waxed, rehydrated and endogenous peroxidases were blocked with hydrogen peroxide. Sections were then boiled in citrate and blocked in 5% serum for 1 hr. Primary antibodies were incubated overnight at 4°C at 1:200 for Ki67 (Vector Laboratories Burlingame, CA) and at 1:400 for ER (Santa Cruz Biotechnology, CA). Biotinylated anti-rabbit secondary antibodies (DAKO Carpentaria, CA) were incubated for 60 min at room temperature after slides were washed for 1 hr in PBS. Slides were incubated in ABC-HRP complex (Vector Laboratories Burlingame, CA) for 30 min. Bound antibodies were then visualized by incubation with 3,3’ diaminobenzidine tetrahydrochloride (liquid DAB, DAKO). Slides were then rinsed in tap water and counterstained with hematoxylin, and then cover slide were mounted. Tumor cells with nuclear staining were recorded as positive manually per tissue core, by a reviewer who was blinded to the clinical data. The numbers of Ki67-positive tumor cells were counted in three high-power fields (x40). The ER immunostaining was registered semi-quantitatively in two ways. Staining intensity (0, no staining; 1, weak staining; 2, moderate staining; and 3, intense staining) and the proportion of stained cells (0, no staining; 1, 1–25% staining; 2, 26–50%; 3, 51–75%; and 4, if more than 75% of the tumor cells were positive.

Statistics

GraphPad Prism 6 software (GraphPad Software, SanDiego, CA) was used to analyze all data. Data represented in the bar graphs is shown as mean ± SEM. t-test was performed for all pairwise comparisons. A value of p<0.05 was considered as statistically significant. The multiple groups’ statistical data were analyzed with one-way ANOVA. RNA-seq data were analyzed using IPA software.

Table 6.
Analyses of the amino acids at the flanking sequences of top ER binders whose interactions are blocked by ERX-11 in MCF-7 and ZR-75 cells, as determined by unbiased IP-MS.

Acknowledgements

This study is supported by the CPRIT grant # DP150096 (RV, JA, GVR), DOD grant # W81XWH-12-1-0288 and W81XWH-13-2-0093 (GVR) funding from the Dorothy and James Cleo Thompson foundation (GVR), the John A Cole foundation (GVR), the Welch Foundation (AT-1595, JA), UTH Cancer Center grant P30 CA-54174 (RV) and NIH grant 1R01CA179120-01 (RV). We acknowledge the invaluable insights into ER structural modeling provided by Dr. Geoff Greene of the University of Chicago.

Appendix 1

Supplementary information on synthetic procedures and characterization of compounds

Chemicals and general synthetic procedures

Nα-Fmoc-protected amino acids, aminomethylated polystyrene resin (100–200 mesh), Rink amide MBHA resin (100–200 mesh), and Rink amide linker were purchased from EMD Chemicals (Gibbstown, NJ). All amino acids used were of the L-configuration. Other chemicals and solvents were purchased from the following sources: benzotriazol-1-yloxytri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), and 6-chloro-1-hydroxybenzotriazole (Cl-HOBt), 1-[Bis(dimethylamino)methylene]−1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA), and trifluoroacetic acid (TFA) (Oakwood Products, West Columbia. SC); acetonitrile (CAN), ethyl acetate (EtOAc), hexanes, N,N-dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran (THF), and methanol (Fisher Scientific, Pittsburgh, PA); 4-amino-3-hydroxybenzoic acid, tin(II) chloride dehydrate, tetrakis(triphenyphosphine)palladium, triphenylsilane, and triisopropylsilane (TIS), N,N-Di-Boc-1H-pyrazole-1-carboxamide, biotin, α-cyano-4-hydroxycinnamic acid (CHCA), piperidine, anisole, acetic anhydride, ninhydrin, dimethyl sulfide (DMS), 1,2-ethanedithiol, iodomethane, 1-bromobutane, 2-bromopropane, 1-bromo-2-methylpropane, benzyl bromide, allyl bromide, 2-bromoethanol, 4-bromobutyric acid, and 4-amino-1-propanol (Aldrich, Milwaukee, WI). DMF, DCM, THF, and chloroform were dried over activated 4 Å molecular sieves before use. Other solvents were used without further purification. Thin-layer chromatography (TLC) was performed on silica gel plates (250 mm, Sorbent Technologies, Atlanta, GA) and the plates were visualized under UV at 254 nm. Standard grade silica gel (230–400 mesh, Sorbent Technologies, Atlanta, GA) was used for flash column chromatography. HPLC analyses were carried out on Agilent 1100 series HPLC system (Foster City, CA) equipped with a diode-array UV detector and a C18-bounded HPLC column (Vydac 218TP104, 4.6 mm ×250 mm, 10 μm) by using a 40 min-gradient elution from 10% to 90% acetonitrile in water (0.1% TFA) and a flow rate of 1.0 mL/min. Eluents were monitored at 220 nm. 1H and 13C NMR spectra were recorded on Bruker AVANCE III 500 (500 MHz) NMR spectrometer. Chemical shifts were reported in ppm from tetramethylsilane (TMS) as an internal standard. Data were expressed as follows: chemical shift (d), multiplicity (s, singlet; d, doublet; dd, doublet of doublet; t, triplet; dt, doublet of triplet, q, quartet; brs, broad singlet; m, multiplet), coupling constants (Hz). Solid-phase reactions were carried out in 12 mL polypropylene cartridges with 20 m PE frit (Applied Separations, Allentown, PA) and a labquake tube shaker (Fisher Scientific, Pittsburgh, PA) was used for mixing. MALDI-TOF MS was measured on Shimadzu AXIMA Confidence MALDI-TOF mass spectrometer (nitrogen UV laser, 50 Hz, 337 nm) by using α-cyano-4-hydroxycinnamic acid (CHCA) as matrix.

General procedure for the alkylation of phenol

A 500 mL round-bottomed flask was charged with methyl 3-hydroxy-4-nitrobenzoate 1 (31.4 mmol), K2CO3 (62.8 mmol), DMF (300 mL), and alkyl halide (94.2 mmol). The reaction mixture was heated at 90°C for 12 hr. Alternatively, the reaction mixture was stirred at room temperature for the synthesis of 2a. The reaction mixture was cooled to room temperature, and then partitioned between EtOAc (400 mL) and brine (300 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (200 mL). The combined organic layers were washed brine (200 mL ×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the compound two as yellow solid. The product was used in the next reaction without further purification.

General procedure for the hydrolysis of esters

A 500 mL round-bottomed flask was charged with methyl 3-alkoxy-4-nitrobenzoate 2 (31 mmol), MeOH (200 mL), THF (200 mL) and 10% NaOH (30 mL). Alternatively, 1N LiOH (60 mL) was used for the synthesis of 3 f. The reaction mixture was stirred at room temperature for 12 hr. The volatile was removed under reduced pressure, and the resulting residue was acidified with 1 N HCl (200 mL) and then extracted with EtOAc (300 mL). The organic layer was separated and washed with 1 N HCl (100 mL) and brine (100 mL). The organic layer was then dried over Na2SO4, filtered, and concentrated under reduced pressure to give the compound three as yellow solid.

General procedure for amide bond formation

Fmoc-Rink amide MBHA resin (0.40 g, 0.20 mmol, 0.5 mmol/g) was swollen in DMF for 2 hr and washed with DMF (3 × 2 min). The Fmoc protecting group was removed by treating with piperidine (20% in DMF, 1 × 5 min and 1 × 30 min), and washed with DMF (3 × 2 min). 3-Alkoxy-4-nitrobenzoic acid three was introduced by using a preactivated HOAt ester which was prepared by mixing a 3-alkoxy-4-nitrobenzoic acid 3 (4 equiv), HATU (four equiv), and DIEA (eight equiv) in DMF (8 mL) for 1 hr. The solution was added to the resin and shaken at room temperature for 24 hr. The resin was then filtered and washed with DMF (3 × 2 min).

General procedure for reduction of nitro group

Resin-bound nitro group (0.20 mmol) was swollen in 50% AcOH/0.5N HCl/THF (1:1:6, 8 mL) for 20 min and treated with SnCl2[center dot]2H2O (five equiv). The reaction mixture was shaken at room temperature for 24 hr. The resin was then filtered, and washed with 0.5N HCl/DMF (1:6) (3 × 5 min), H2O/DMF (1:6) (3 × 5 min), and DMF (3 × 5 min).

Procedure for guanidinylation 

Resin-bound N-Fmoc group 7i was swollen in DMF for 2 hr and washed with DMF (3 × 2 min). The Fmoc protecting group was removed by treating with piperidine (20% in DMF, 1 × 5 min and 1 × 30 min), and washed with DMF (3 × 2 min). Then, the resulting resin was swollen in DMF and treated with N,N’-di-Boc-1H-pyrazole-1-carboxamidine (three equiv.) and DIEA (six equiv.). The reaction mixture was shaken at room temperature for 24 hr. The resin was then filtered, and washed with DMF (3 × 2 min) giving resin-bound N,N’-di-Boc-guanidine 7 j.

General procedure for cleavage of tris-benzamide

Resin-bound tris-benzamide seven was washed with DMF (3 × 2 min) and DCM (3 × 2 min), and dried in vacuo. The dried resin was treated with a cleavage mixture of TFA/TIS/H2O (95:2.5:2.5, 8 mL) for 90 min. The TFA solution was then filtered, and the resin was washed with TFA (2 mL). The combined TFA solution was concentrated to a volume of approximately 0.5 mL with a gentle stream of nitrogen, and the tris-benzamide was precipitated with cold diethyl ether (10 mL). The resulting precipitate was collected by centrifugation and the ether solution was decanted. Washing with cold diethyl ether was repeated and the tris-benzamide was dried in vacuo.

Appendix 1—chemical structure 1.

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The title compound was obtained as a yellow solid (5.1 g, 84% yield over two steps) using iodomethane (13.4 g, 94.2 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 13.66 (br s, 1 hr), 7.98 (d, J = 8.2 Hz, 1 hr), 7.78 (d, J = 1.5 Hz, 1 hr), 7.66 (dd, J = 8.2, 1.5 Hz, 1 hr), 4.00 (s, 3 hr). 13C NMR (DMSO-d6, 125 MHz): δ 166.3, 152.0, 142.4, 136.2, 125.5, 121.8, 15.0, 57.3.

Appendix 1—chemical structure 2.

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The title compound was obtained as a yellow solid (6.4 g, 91% yield over two steps) using 2-bromopropane (11.8 g, 94.2 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 13.62 (br s, 1 hr), 7.93 (d, J = 8.3 Hz, 1 hr), 7.78 (d, J = 1.5 Hz, 1 hr), 7.63 (dd, J = 8.3, 1.5 Hz, 1 hr), 4.92 (septet, J = 6.1 Hz, 1 hr), 1.32 (d, J = 6.1 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 166.3, 150.1, 143.7, 135.9, 125.3, 121.7, 117.0, 72.9, 22.0.

Appendix 1—chemical structure 3.

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The title compound was obtained as a yellow solid (7.7 g, 93% yield over two steps) using 1-bromobutane (12.9 g, 94.2 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 13.63 (br s, 1 hr), 7.96 (d, J = 8.2 Hz, 1 hr), 7.77 (d, J = 1.5 Hz, 1 hr), 7.64 (dd, J = 8.2, 1.5 Hz, 1 hr), 4.24 (t, J = 6.4 Hz, 2 hr), 1.75–1.69 (m, 2 hr), 1.48–1.41 (m, 2 hr), 0.94 (t, J = 7.3 Hz, 3 hr). 13C NMR (DMSO-d6, 125 MHz): δ 166.3, 151.3, 142.6, 136.1, 125.4, 121.7, 115.7, 69.6, 30.8, 19.0, 14.0.

Appendix 1—chemical structure 4.

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The title compound was prepared according to the published procedure.

Appendix 1—chemical structure 5.

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The title compound was obtained as a yellow solid (7.5 g, 89% yield over two steps) using benzyl bromide (8.2 g, 47.1 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 13.66 (br s, 1 hr), 8.01 (d, J = 8.2 Hz, 1 hr), 7.89 (d, J = 1.5 Hz, 1 hr), 7.68 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.48–7.35 (m, 5 hr), 5.41 (s, 2 hr). 13C NMR (DMSO-d6, 125 MHz): δ 166.2, 151.0, 142.8, 136.3, 136.1, 129.0, 128.6, 127.9, 125.5, 122.1, 116.3, 71.1.

Appendix 1—chemical structure 6.

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The title compound was obtained as a yellow solid (8.7 g, 85% yield over two steps) using tert-butyl 4-bromobutyrate [5] (10.5 g, 47.1 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 13.65 (br s, 1 hr), 7.98 (d, J = 8.2 Hz, 1 hr), 7.76 (d, J = 1.5 Hz, 1 hr), 7.65 (dd, J = 8.2, 1.5 Hz, 1 hr), 4.25 (t, J = 6.3 Hz, 2 hr), 2.38 (t, J = 7.5 Hz, 2 hr), 1.99–1.92 (m, 2 hr), 1.41 (s, 9 hr). 13C NMR (DMSO-d6, 125 MHz): δ 172.2, 166.2, 151.3, 142.6, 136.2, 125.5, 121.9, 115.8, 80.3, 69.0, 31.4, 28.2, 24.5.

Appendix 1—chemical structure 7.

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A 500 mL round-bottomed flask was charged with methyl 3-hydroxy-4-nitrobenzoate 1 (5.0 g, 25.4 mmol), K2CO3 (7.0 g, 50.6 mmol), TBAB (0.42 g, 1.3 mmol), DMF (200 mL), and 2-bromoethyl trityl ether [6] (14.0 g, 38.1 mmol). The reaction mixture was heated at 90°C for 12 hr and then cooled to room temperature. The solution was then poured into brine (200 mL) and extracted with EtOAc (200 mL ×2). The combined organic extracts were washed with brine (100 mL ×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 2 g as a yellow solid. The product was used in the next reaction without further purification.

A 1 L round-bottomed flask was charged with compound 2 g, MeOH (200 mL), THF (600 mL) and 10% NaOH (40 mL). The reaction mixture was stirred at room temperature for 24 hr and then concentrated under reduced pressure. The reaction mixture was acidified with 5% citric acid (200 mL) and extracted with extracted with EtOAc (300 mL ×2). The combined organic extracts were washed with brine (200 mL ×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. Recrystallization from EtOAc/hexanes (1:1) gave compound 3 g as a light yellow solid (11.2 g, 94% yield over two steps). Rf = 0.33 (EtOAc). 1H NMR (DMSO-d6, 500 MHz): δ 13.65 (br s, 1 hr), 8.01 (d, J = 8.2 Hz, 1 hr), 7.89 (d, J = 1.5 Hz, 1 hr), 7.69 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.40–7.38 (m, 6 hr), 7.35–7.32 (m, 6 hr), 7.29–7.26 (m, 3 hr), 4.49–4.47 (m, 2 hr), 3.31–3.29 (m, 2 hr). 13C NMR (DMSO-d6, 125 MHz): δ 166.3, 151.22, 143.9, 142.9, 136.0, 128.7, 128.4, 127.5, 125.3, 122.0, 116.1, 86.5, 69.3, 62.7.

Appendix 1—chemical structure 8.

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A 250 mL round-bottomed flask was charged with methyl 3-hydroxy-4-nitrobenzoate 1 (2.7 g, 13.6 mmol), K2CO3 (3.8 g, 27.2 mmol), TBAB (0.23 g, 0.68 mmol), DMF (100 mL), and 3-(tritylamino)propyl methanesulfonate (7.0 g, 17.7 mmol). The reaction mixture was heated at 90°C for 12 hr and then cooled to room temperature. The solution was then poured into brine (200 mL) and extracted with EtOAc (200 mL ×2). The combined organic extracts were washed with brine (100 mL ×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give compound 2 hr as a yellow solid. The product was used in the next reaction without further purification.

A 500 mL round-bottomed flask was charged with compound 2 hr, MeOH (100 mL), THF (100 mL) and 1N LiOH (28 mL). The reaction mixture was stirred at room temperature for 24 hr and then concentrated under reduced pressure. The reaction mixture was acidified with 5% citric acid (50 mL) and extracted with extracted with EtOAc (100 mL ×2). The combined organic extracts were washed with brine (100 mL ×3), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude product. Purification by flash chromatography using EtOAc gave compound 3 hr as a white solid (3.0 g, 46% yield over two steps).

The compound 3 hr (3.0 g, 6.2 mmol) was treated with a cleavage mixture of TFA/TIS/DCM (10:5:85, 50 mL) for 30 min. The excess of TFA were removed under reduced pressure and precipitation with diethyl ether gave the crude product, which was directly used for the next reaction without further purification.

A 250 mL round-bottomed flask was charged with the crude product, NaHCO3 (2.7 g, 31.1 mmol), THF (100 mL), and H2O (50 mL). Then, Fmoc-OSu (3.2 g, 9.5 mmol) was added to the solution and the reaction mixture was stirred at room temperature for 12 hr. The volatile was removed under reduced pressure and the aqueous phase was washed with diethyl ether. The aqueous phase was acidified with 1 N HCl (50 mL) and then extracted with EtOAc (100 mL ×2). The combined organic layers were washed with 1 N HCl (50 mL) and brine (50 mL). The organic layer was then dried over Na2SO4, filtered, and concentrated under reduced pressure to give the compound 3i as a yellow solid (2.5 g, 87% yield over two steps). 1H NMR (DMSO-d6, 500 MHz): δ 13.67 (br s, 1 hr), 7.99 (d, J = 8.3 Hz, 1 hr), 7.89 (d, J = 7.6 Hz, 2 hr), 7.77 (d, J = 1.4 Hz, 1 hr), 7.69 (d, J = 7.6 Hz, 2 hr), 7.66 (dd, J = 8.3, 1.5 Hz, 1 hr), 7.41 (t, J = 7.6 Hz, 2 hr), 7.37 (t, J = 5.6 Hz, 1 hr), 7.33 (dt, J = 0.9, 7.5 Hz, 2 hr), 4.33 (d, J = 6.7 Hz, 2 hr), 4.24–4.20 (m, 3 hr), 3.16 (q, J = 6.4 Hz, 2 hr), 1.88 (quin, J = 6.4 Hz, 2 hr). 13C NMR (DMSO-d6, 125 MHz): δ 166.29, 156.6, 151.5, 144.4, 142.5, 141.2, 136.2, 128.1, 127.5, 125.60, 125.55, 121.7, 120.6, 115.8, 67.5, 65.6, 47.3, 37.3, 29.2.

Appendix 1—chemical structure 9.

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The title compound was obtained as a yellow solid (64 mg, 53% overall yield) using compound 3a and Fmoc-Rink amide MBHA resin (0.70 mmol/g, 0.30 g, 0.21 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 9.88 (br s, 1 hr), 9.46 (br s, 1 hr), 8.06 (d, J = 8.7 Hz, 1 hr), 7.99 (br s, 1 hr), 7.99 (d, J = 8.3 Hz, 1 hr), 7.95 (d, J = 8.2 Hz, 1 hr), 7.85 (d, J = 1.5 Hz, 1 hr), 7.66 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.65 (d, J = 1.5 Hz, 1 hr), 7.62 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.59 (d, J = 1.8 Hz, 1 hr), 7.55 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.36 (br s, 1 hr), 4.04 (s, 2.2 hr), 4.03 (s, 0.8 hr), 3.93 (d, J = 6.7 Hz, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 2.17–2.07 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.1, 152.1, 151.31, 150.30, 141.5, 139.9, 132.4, 131.5, 130.29, 130.27, 125.6, 124.5, 122.8, 120.4, 120.3, 120.1, 113.8, 111.7, 111.6, 75.1, 75.0, 57.3, 28.34, 28.26, 19.6, 19.5. HRMS-ESI (m/z): [M-H]- calcd for C30H34N4O8: 577.2304, found: 577.2318.

Appendix 1—chemical structure 10.

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The title compound was obtained as a yellow solid (65 mg, 49% overall yield) using compound 3b and Fmoc-Rink amide MBHA resin (0.70 mmol/g, 0.30 g, 0.21 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 9.84 (br s, 1 hr), 9.46 (br s, 1 hr), 8.00 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.96 (d, J = 8.2 Hz, 1 hr), 7.85 (d, J = 1.5 Hz, 1 hr), 7.65 (d, J = 1.8 Hz, 1 hr), 7.63 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.61 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.59 (d, J = 1.8 Hz, 1 hr), 7.55 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.36 (br s, 1 hr), 4.96 (septet, J = 6.1 Hz, 1 hr), 3.93 (d, J = 6.4 Hz, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 2.17–2.08 (m, 2 hr), 1.35 (d, J = 6.1 Hz, 6 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.1, 151.3, 150.30, 150.25, 142.9, 139.6, 132.4, 131.5, 130.3, 125.5, 124.5, 122.7, 120.4, 120.2, 120.1, 115.6, 111.7, 111.6, 75.1, 75.0, 73.0, 28.34, 28.28, 22.1, 19.6, 19.5. HRMS-ESI (m/z): [M-H]- calcd for C32H38N4O8: 605.2617, found: 605.2640.

Appendix 1—chemical structure 11.

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The title compound was obtained as a yellow solid (83 mg, 48% overall yield) using compound 3 c and Fmoc-Rink amide MBHA resin (0.70 mmol/g, 0.40 g, 0.28 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 9.85 (br s, 1 hr), 9.46 (br s, 1 hr), 8.04 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.96 (d, J = 8.2 Hz, 1 hr), 7.84 (d, J = 1.5 Hz, 1 hr), 7.65 (d, J = 1.8 Hz, 1 hr), 7.64 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.61 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.59 (d, J = 1.8 Hz, 1 hr), 7.55 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.36 (br s, 1 hr), 4.28 (t, J = 6.3 Hz, 2 hr), 3.93 (d, J = 6.7 Hz, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 2.16–2.10 (m, 2 hr), 1.79–1.73 (m, 2 hr), 1.51–1.43 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr), 0.96 (d, J = 7.3 Hz, 3 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.1, 151.5, 151.3, 150.3, 141.8, 139.8, 132.4, 131.5, 130.30, 130.29, 125.5, 124.4, 122.7, 120.4, 120.3, 120.0, 114.4, 111.7, 111.6, 75.1, 75.0, 69.6, 30.8, 28.34, 28.28, 19.61, 19.55, 19.0, 14.0. HRMS-ESI (m/z): [M-H]- calcd for C33H40N4O8: 619.2773, found: 619.2791.

Appendix 1—chemical structure 12.

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The title compound was obtained as a yellow solid (83 mg, 53% overall yield) using compound 3d and Fmoc-Rink amide MBHA resin (0.50 mmol/g, 0.50 g, 0.25 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 9.85 (br s, 1 hr), 9.46 (br s, 1 hr), 8.05 (d, J = 8.2 Hz, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.97 (d, J = 8.2 Hz, 1 hr), 7.83 (d, J = 1.5 Hz, 1 hr), 7.65 (d, J = 1.8 Hz, 1 hr), 7.64 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.62 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.59 (d, J = 1.8 Hz, 1 hr), 7.55 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.36 (br s, 1 hr), 4.05 (d, J = 6.4 Hz, 2 hr), 3.94 (d, J = 6.4 Hz, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 2.17–2.07 (m, 3 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.03 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.1, 151.5, 151.2, 150.3, 141.7, 139.9, 132.4, 131.5, 130.29, 130.28, 125.6, 124.4, 122.7, 120.4, 120.3, 120.1, 114.3, 111.7, 111.5, 75.8, 75.1, 75.0, 28.34, 28.29, 28.1, 19.61, 19.58, 19.2. HRMS-ESI (m/z): [M-H]- calcd for C33H40N4O8: 619.2773, found: 619.2794.

Appendix 1—chemical structure 13.

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The title compound was obtained as a yellow solid (56 mg, 57% overall yield) using compound 3e and Fmoc-Rink amide MBHA resin (0.50 mmol/g, 0.30 g, 0.15 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 9.88 (br s, 1 hr), 9.46 (br s, 1 hr), 8.09 (d, J = 8.3 Hz, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.97 (d, J = 1.8 Hz, 1 hr), 7.94 (d, J = 8.2 Hz, 1 hr), 7.69 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.65 (d, J = 1.8 Hz, 1 hr), 7.62 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.59 (d, J = 1.8 Hz, 1 hr), 7.56 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.50–7.38 (m, 5 hr), 7.36 (br s, 1 hr), 5.43 (s, 2 hr), 3.93 (d, J = 6.4 Hz, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 2.17–2.09 (m, 3 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.01 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.0, 151.3, 151.2, 150.3, 141.9, 139.8, 136.2, 132.5, 131.5, 130.30, 130.27, 129.1, 128.7, 127.9, 125.7, 124.6, 122.8, 120.4, 120.34, 120.26, 115.1, 111.7, 111.6, 75.1, 75.0, 71.3, 28.34, 28.26, 19.61, 19.56. HRMS-ESI (m/z): [M-H]- calcd for C36H38N4O8: 653.2617, found: 653.2632.

Appendix 1—chemical structure 14.

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The title compound was obtained as a yellow solid (90 mg, 49% overall yield) using compound 3 f and Fmoc-Rink amide MBHA resin (0.70 mmol/g, 0.40 g, 0.28 mmol). 1H NMR (DMSO-d6, 500 MHz): δ 12.20 (br s, 1 hr), 9.87 (br s, 1 hr), 9.46 (br s, 1 hr), 8.06 (d, J = 8.2 Hz, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.94 (d, J = 8.2 Hz, 1 hr), 7.84 (d, J = 1.5 Hz, 1 hr), 7.65 (d, J = 1.8 Hz, 1 hr), 7.65 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.61 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.59 (d, J = 1.8 Hz, 1 hr), 7.55 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.36 (br s, 1 hr), 4.30 (t, J = 6.3 Hz, 2 hr), 3.93 (d, J = 6.7 Hz, 2 hr), 3.92 (d, J = 6.7 Hz, 2 hr), 2.43 (t, J = 7.3 Hz, 2 hr), 2.16–2.09 (m, 2 hr), 2.03–1.98 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 174.4, 167.8, 164.7, 164.1, 151.4, 151.3, 150.3, 141.7, 139.9, 132.4, 131.5, 130.30, 130.28, 125.7, 124.6, 122.7, 120.4, 120.24, 120.16, 114.4, 111.7, 111.6, 75.1, 75.0, 69.0, 30.2, 28.34, 28.28, 24.4, 19.6, 19.5. HRMS-ESI (m/z): [M-H]- calcd for C33H38N4O10: 649.2515, found: 649.2535.

Appendix 1—chemical structure 15.

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The title compound was prepared using compound 3i and Fmoc-Rink amide MBHA resin (0.50 mmol/g, 0.40 g, 0.20 mmol). Following the general procedure, the resin-bound N-Fmoc group 7i was converted to the resin-bound N, N’-di-Boc-guanindine 7 j by the removal of Fmoc group with piperidine followed by reaction with N,N’-di-Boc-1H-pyrazole-1-carboxamidine. After cleavage from the resin and HPLC purification, ERX-12 was obtained as a yellow solid (31 mg, 20% overall yield). 1H NMR (DMSO-d6, 500 MHz): δ 9.88 (br s, 1 hr), 9.46 (br s, 1 hr), 8.09 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.98 (d, J = 8.2 Hz, 1 hr), 7.92 (d, J = 8.2 Hz, 1 hr), 7.85 (d, J = 1.5 Hz, 1 hr), 7.69 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.66 (d, J = 1.8 Hz, 1 hr), 7.64 (br s, 1 hr), 7.62 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.60 (d, J = 1.8 Hz, 1 hr), 7.56 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.36 (br s, 1 hr), 4.32 (t, J = 6.1 Hz, 2 hr), 3.93 (d, J = 6.7 Hz, 2 hr), 3.92 (d, J = 6.6 Hz, 2 hr), 3.30 (q, J = 6.5 Hz, 2 hr), 2.16–2.08 (m, 2 hr), 2.03–1.98 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.0, 157.3, 151.5, 151.40, 150.35, 141.6, 140.0, 132.6, 131.6, 130.3, 130.2, 125.8, 124.7, 122.8, 120.4, 120.3, 114.6, 111.69, 111.65, 75.1, 75.0, 67.1, 38.0, 28.4, 28.34, 28.26, 19.6, 19.5. HRMS-ESI (m/z): [M + H]+calcd for C33H41N7O8: 664.3089, found: 664.3094.

Appendix 1—chemical structure 16.

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This compound was prepared according to the published procedure (Ravindranathan et al., 2013).

Appendix 1—chemical structure 17.

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To a solution of compound 3 g (2.82 g, 6.01 mmol) and DIEA (2.0 mL, 11.3 mmol) in DMF (30 mL) was added HATU (2.94 g, 7.50 mmol) and the mixture was stirred for 1 hr. Compound 8 (1.5 g, 3.75 mmol) was added and the resulting mixture was stirred at room temperature for 24 hr. The solution was then poured into 5% citric acid (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic extracts were washed with saturated NaHCO3 (50 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. Recrystallization from EtOAc gave compound nine as a light yellow solid (2.3 g, 72%). 1H NMR (DMSO-d6, 500 MHz): δ 9.87 (br s, 1 hr), 9.41 (br s, 1 hr), 8.07 (d, J = 8.2 Hz, 1 hr), 8.02 (d, J = 8.2 Hz, 1 hr), 7.98 (d, J = 8.2 Hz, 1 hr), 7.97 (br s, 2 hr), 7.71 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.61 (br s, 1 hr), 7.60–7.58 (m, 2 hr), 7.55 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.42–7.40 (m, 6 hr), 7.36–7.33 (m, 7 hr), 7.29–7.26 (m, 3 hr), 4.52–4.50 (m, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 3.91 (d, J = 6.4 Hz, 2 hr), 3.33–3.32 (m, 2 hr, overlap with H2O peak), 2.18–2.05 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 0.99 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.1, 151.3, 150.2, 144.0, 141.9, 140.1, 132.1, 131.4, 130.4, 128.7, 128.4, 127.6, 125.5, 124.4, 122.6, 120.4, 120.29, 120.26, 114.7, 111.7, 86.5, 75.1, 75.0, 69.3, 62.5, 28.34, 28.26, 19.61, 19.55.

Appendix 1—chemical structure 18.

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A solution of compound 9 (1.0 g, 1.18 mmol) in TFA (20 mL) was stirred at room temperature for 30 min. The excess of TFA were removed under reduced pressure and precipitation with diethyl ether gave ERX-11 as a white solid (0.61 g, 85%). 1H NMR (DMSO-d6, 500 MHz): δ 9.86 (br s, 1 hr), 9.46 (br s, 1 hr), 8.03 (d, J = 8.2 Hz, 1 hr), 7.99 (br s, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.94 (d, J = 8.2 Hz, 1 hr), 7.87 (d, J = 1.9 Hz, 1 hr), 7.65 (d, J = 1.9 Hz, 1 hr), 7.64 (dd, J = 8.5, 1.6 Hz, 1 hr), 7.61 (dd, J = 8.2, 1.9 Hz, 1 hr), 7.59 (d, J = 1.9 Hz, 1 hr), 7.55 (dd, J = 8.2, 1.9 Hz, 1 hr), 7.36 (br s, 1 hr), 4.30 (t, J = 5.0 Hz, 2 hr), 3.93 (d, J = 6.6 Hz, 2 hr), 3.92 (d, J = 6.6 Hz, 2 hr), 3.77 (t, J = 5.0 Hz, 2 hr), 2.17–2.08 (m, 2 hr), 1.05 (d, J = 6.6 Hz, 6 hr), 1.02 (d, J = 6.6 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 167.8, 164.7, 164.1, 151.6, 151.34, 150.30, 141.9, 139.7, 132.4, 131.5, 130.30, 130.29, 125.6, 124.6, 122.7, 120.4, 120.3, 120.1, 114.73, 111.66, 111.58, 75.1, 75.0, 72.0, 59.7, 28.34, 28.27, 19.61, 19.55. HRMS-ESI (m/z): [M-H]- calcd for C31 H36 N4 O9: 607.2410, found: 607.2435.

Appendix 1—chemical structure 19.

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This compound was prepared according to the published procedure (Ravindranathan et al., 2013).

Appendix 1—chemical structure 20.

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To a solution of compound 3 g (1.5 g, 3.2 mmol) and DIEA (1.1 mL, 6.1 mmol) in DMF (30 mL) was added HATU (1.5 g, 4.1 mmol) and the mixture was stirred at room temperature for 1 hr. Compound 10 (0.90 g, 2.0 mmol) was added and the resulting mixture was stirred at room temperature for 24 hr. The solution was then poured into 5% citric acid (50 mL) and extracted with EtOAc (50 mL x 2). The combined organic extracts were washed with saturated NaHCO3 (50 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. Recrystallization from EtOAc/hexanes (1:1) gave compound 11 as a light yellow solid (1.5 g, 83%). Rf = 0.33 (hexanes/EtOAc 4:1). 1H NMR (DMSO-d6, 500 MHz): δ 9.80 (br s, 1 hr), 9.53 (br s, 1 hr), 8.18 (d, J = 8.2 Hz, 1 hr), 8.07 (d, J = 8.2 Hz, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.97 (br s, 1 hr), 7.71 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.69 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.62–7.59 (m, 3 hr), 7.42–7.40 (m, 6 hr), 7.36–7.32 (m, 6 hr), 7.29–7.26 (m, 3 hr), 6.12–6.04 (m, 1 hr), 5.43 (ddd, J = 17.2, 3.1, 1.7 Hz, 1 hr), 5.31 (ddd, J = 10.3, 2.9, 1.4 Hz, 1 hr), 4.83 (dt, J = 5.4, 1.4 Hz, 2 hr), 4.52–4.50 (m, 2 hr), 3.95 (d, J = 6.4 Hz, 2 hr), 3.91 (d, J = 6.4 Hz, 2 hr), 3.33–3.32 (m, 2 hr, overlap with H2O peak), 2.18–2.07 (m, 2 hr), 1.06 (d, J = 6.7 Hz, 6 hr), 0.99 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 165.5, 164.8, 164.1, 151.34, 151.29, 145.0, 144.0, 141.9, 140.1, 133.2, 132.5, 128.7, 128.4, 127.6, 126.2, 125.5, 124.4, 122.6, 122.3, 120.4, 120.3, 118.4, 114.7, 112.5, 111.6, 86.5, 75.1, 75.0, 69.3, 65.6, 62.5, 28.32, 28.26, 19.56, 19.54.

Appendix 1—chemical structure 21.

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To a solution of compound 11 (1.5 g, 1.7 mmol) in DCM (50 mL) were added Pd(PPh3)4 (196 mg, 0.17 mmol) and phenylsilane (0.37 mL, 3.4 mmol), and the mixture was stirred at room temperature for 1 hr. The reaction mixture was then concentrated under reduced pressure and the resulting solid was washed with ether. The corresponding carboxylic acid was obtained as a white solid (1.45 g, quantitative yield) and used in the next reaction without further purification.

To a solution of the carboxylic acid (300 mg, 0.35 mmol) and DIEA (0.24 mL, 1.4 mmol) in DMF (10 mL) was added HATU (179 mg, 0.46 mmol) and the mixture was stirred at room temperature for 1 hr. N-Boc-ethylenediamine (112 mg, 0.70 mmol) was added and the resulting mixture was stirred at room temperature for 24 hr. The solution was then poured into 5% citric acid (20 mL) and extracted with EtOAc (20 mL x 2). The combined organic extracts were washed with saturated NaHCO3 (20 mL x 2), dried over Na2SO4, filtered, and concentrated under reduced pressure. Recrystallization from EtOAc/hexanes (1:1) gave compound 12 as a light yellow solid (152 mg, 44%). 1H NMR (DMSO-d6, 500 MHz): δ 9.86 (br s, 1 hr), 9.41 (br s, 1 hr), 8.48 (t, J = 5.6 Hz, 1 hr), 8.06 (d, J = 8.2 Hz, 1 hr), 8.03 (d, J = 8.2 Hz, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.98 (br s, 1 hr), 7.71 (dd, J = 8.2, 1.5 Hz, 1 hr), 7.61–7.58 (m, 2 hr), 7.55–7.51 (m, 2 hr), 7.42–7.40 (m, 6 hr), 7.36–7.32 (m, 6 hr), 7.29–7.26 (m, 3 hr), 6.95 (t, J = 5.6 Hz, 1 hr), 4.51–4.50 (m, 2 hr), 3.92 (d, J = 6.2 Hz, 2 hr), 3.91 (d, J = 6.2 Hz, 2 hr), 3.33–3.30 (m, 4 hr), 3.15–3.11 (m, 2 hr), 2.17–2.06 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 0.99 (d, J = 6.7 Hz, 6 hr).

Appendix 1—chemical structure 22.

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A solution of compound 12 (50 mg, 0.050 mmol) in TFA/TIS/H2O (95:2.5:2.5, 5 mL) was stirred at room temperature for 30 min. The excess of TFA were removed with a gentle stream of nitrogen, and a white solid was precipitated with cold diethyl ether. The precipitate was filtered, washed with diethyl ether and dried in vacuo. The resulting yellow solid was used in the next reaction without further purification.

To a solution of the yellow solid, biotin (24 mg, 0.10 mmol) and DIEA (0.035 mL, 0.20 mmol) in DMF (5 mL) was added HATU (43 mg, 0.11 mmol), and the resulting mixture was stirred at room temperature for 12 hr. The reaction solution was then poured into 1N HCl (20 mL) and extracted with EtOAc (20 mL x 2). The combined organic extracts were washed with saturated NaHCO3 (20 mL x 2) and concentrated under reduced pressure. The resulting solid was washed with EtOAc (10 mL x 5) and dried in vacuo giving ERX-11-biotin as a light yellow solid (32 mg, 73%). 1H NMR (DMSO-d6, 500 MHz): δ 9.87 (br s, 1 hr), 9.47 (br s, 1 hr), 8.53 (t, J = 5.5 Hz, 1 hr), 8.03 (d, J = 8.2 Hz, 1 hr), 7.99 (d, J = 8.2 Hz, 1 hr), 7.97 (br s, 1 hr), 7.94 (d, J = 8.2 Hz, 1 hr), 7.87 (d, J = 1.5 Hz, 1 hr), 7.65–7.64 (m, 2 hr), 7.62 (dd, J = 8.2, 1.8 Hz, 1 hr), 7.56 (d, J = 1.5 Hz, 1 hr), 7.52 (dd, J = 8.2, 1.7 Hz, 1 hr), 6.43 (br s, 1 hr), 6.36 (br s, 1 hr), 4.98 (t, J = 5.6 Hz, 1 hr), 4.32–4.29 (m, 3 hr), 4.13–4.10 (m, 1 hr), 3.93 (d, J = 6.4 Hz, 2 hr), 3.92 (d, J = 6.4 Hz, 2 hr), 3.79–3.76 (m, 2 hr), 3.33–3.31 (m, 2 hr, overlap with H2O peak), 3.27–3.23 (m, 2 hr), 3.09–3.05 (m, 2 hr), 2.82 (dd, J = 12.5, 5.2 Hz, 1 hr), 2.58 (d, J = 12.2 Hz, 2 hr), 2.16–2.08 (m, 4 hr), 1.64–1.43 (m, 4 hr), 1.35–1.28 (m, 2 hr), 1.05 (d, J = 6.7 Hz, 6 hr), 1.02 (d, J = 6.7 Hz, 6 hr). 13C NMR (DMSO-d6, 125 MHz): δ 173.0, 166.2, 164.7, 163.2, 162.8, 151.6, 151.4, 150.4, 141.9, 139.7, 132.4, 131.7, 130.3, 130.2, 125.5, 124.6, 122.9, 120.3, 120.13, 120.11, 114.8, 111.6, 111.4, 75.1, 75.0, 72.0, 61.5, 59.70, 59.68, 55.9, 38.8, 36.3, 28.7, 28.5, 28.4, 28.3, 25.7, 19.63, 19.55. HRMS-ESI (m/z): [M-H]- calcd for C43H55N7O11S: 876.3608, found: 876.3602.

General procedure for peptide synthesis

The peptides were synthesized manually using standard Nα-Fmoc/tBu solid-phase peptide synthesis protocol. Aminomethylated polystyrene resin (0.25 mmol, 0.4 mmol/g) was swollen in DMF for 2 hr and washed with DMF (3 × 1 min). Fmoc-Rink amide linker (1.5 equiv), HBTU (1.5 equiv), Cl-HOBt (1.5 equiv), and DIEA (three equiv) were dissolved in DMF (6 mL). The resulting solution was then added to the resin and shaken for 12 hr. The coupling reaction was followed by Kaiser ninhydrin test, and unreacted amines were capped by using acetic anhydride (20 equiv) in DMF (6 mL) for 20 min. The Fmoc protecting group of the Rink amide linker was removed via treatment with 20% piperidine in DMF (6 mL, 1 × 5 min and 1 × 30 min) and washed with DMF (3 × 1 min). The first amino acid was introduced by using a preactivated Fmoc amino acid that was prepared by mixing a Fmoc amino acid (four equiv), HBTU (four equiv), Cl-HOBt (four equiv), and DIEA (eight equiv) in DMF (6 mL) for 5 min. The coupling reaction was conducted for 4 hr or until Kaiser ninhydrin test became negative. When a coupling reaction was found to be incomplete, the resin was washed with DMF (3 × 1 min) and the amino acid was coupled again with a freshly prepared preactivated Fmoc amino acid. When the second coupling reaction did not result in negative Kaiser ninhydrin test, the resin was washed with DMF (3 × 1 min) and the unreacted amines were capped by being treated with acetic anhydride (20 equiv) in DMF for 20 min. These steps (removal of a Fmoc group and coupling of a Fmoc amino acid) were repeated until all amino acids in the sequence of a peptide were coupled. The resin was then washed with DCM (5 × 1 min) and dried in vacuo.

General procedure for cleavage and final deprotection of peptides

A cleavage mixture of trifluoroacetic acid (TFA), dimethyl sulfide, 1,2-ethanedithiol, and anisole (8 mL, 90:5:3:2) was added to a peptide on dried resin (0.25 mmol) in a disposable 50 mL polypropylene tube, and the mixture was stirred for 90 min at room temperature in the dark. Then, the TFA solution was filtered, and the resin was washed with TFA (3 mL) and DCM (3 mL). The combined TFA solution was concentrated to a volume of approximately 3 mL with a gentle stream of nitrogen, and the peptide was precipitated with cold diethyl ether (40 mL). The precipitated peptide was centrifuged, and the ether solution was decanted to remove the scavengers. Washing with cold diethyl ether was repeated, and the precipitated peptide was centrifuged, decanted, and dried under vacuum. The resulting peptide was characterized via RP-HPLC and MALDI-TOF MS.

General procedure for the purification of peptides

A crude peptide was dissolved in 50% aqueous acetic acid, and the insoluble was removed by centrifugation. The acetic acid solution containing peptide was purified with HPLC by using a reverse phase semipreparative Vydac column (C4-bonded, 214TP1010, 10 mm × 250 mm, 10 μm) with gradient elution at a flow rate of 3.0 mL/min. A fraction containing the peptide was collected and lyophilized. The purity of all of the synthesized peptides was checked by analytical HPLC and found to be greater than 95%. The molecular mass of the purified peptides was confirmed by MALDI-TOF MS (Appendix 1—Table 1).

Appendix 1—Table 1.

Characterization of the peptides

DOI: http://dx.doi.org/10.7554/eLife.26857.054

LXXLL PeptideSequencemolecular mass (MALDI-TOF-MS)
calculatedobserved
SRC1Ac-LTARHKILHRLLQEGSPSD-NH2[M + H]+ for C96H162N32O28: 2212.22212.4
SRC2Ac-DSKGQTKLLQLLTTKSDQM-NH2[M + H]+ for C92H162N26O32: 2176.22176.6
AIB1Ac-ESKGHKKLLQLLTCSSDDR-NH2[M + H]+ for C92H159N29O31: 2199.22199.8
PELP1Ac-SIKTRFEGLCLLSLLVGESPT-NH2[M + H]+ for C103H174N26O31: 2304.32304.6

Appendix 1—figure 1.

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1H and 13C NMR of compound 3a.

DOI: http://dx.doi.org/10.7554/eLife.26857.055

Appendix 1—figure 2.

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1H and 13C NMR of compound 3b.

DOI: http://dx.doi.org/10.7554/eLife.26857.056

Appendix 1—figure 3.

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1H and 13C NMR of compound 3c.

DOI: http://dx.doi.org/10.7554/eLife.26857.057

Appendix 1—figure 4.

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1H and 13C NMR of compound 3e.

DOI: http://dx.doi.org/10.7554/eLife.26857.058

Appendix 1—figure 5.

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1H and 13C NMR of compound 3 f.

DOI: http://dx.doi.org/10.7554/eLife.26857.059

Appendix 1—figure 6.

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1H and 13C NMR of compound 3 g.

DOI: http://dx.doi.org/10.7554/eLife.26857.060

Appendix 1—figure 7.

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1H and 13C NMR of compound 3i.

DOI: http://dx.doi.org/10.7554/eLife.26857.061

Appendix 1—figure 8.

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1H and 13C NMR of ERX-5.

DOI: http://dx.doi.org/10.7554/eLife.26857.062

Appendix 1—figure 9.

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1H and 13C NMR of ERX-7.

DOI: http://dx.doi.org/10.7554/eLife.26857.063

Appendix 1—figure 10.

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1H and 13C NMR of ERX-8.

DOI: http://dx.doi.org/10.7554/eLife.26857.064

Appendix 1—figure 11.

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1H and 13C NMR of ERX-9.

DOI: http://dx.doi.org/10.7554/eLife.26857.065

Appendix 1—figure 12.

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1H and 13C NMR of ERX-10.

DOI: http://dx.doi.org/10.7554/eLife.26857.066

Appendix 1—figure 13.

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1H and 13C NMR of ERX-12.

DOI: http://dx.doi.org/10.7554/eLife.26857.067

Appendix 1—figure 14.

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1H and 13C NMR of ERX-13.

DOI: http://dx.doi.org/10.7554/eLife.26857.068

Appendix 1—figure 15.

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1H and 13C NMR of compound 9.

DOI: http://dx.doi.org/10.7554/eLife.26857.069

Appendix 1—figure 16.

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1H and 13C NMR of ERX-11.

DOI: http://dx.doi.org/10.7554/eLife.26857.070

Appendix 1—figure 17.

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1H and 13C NMR of compound 11.

DOI: http://dx.doi.org/10.7554/eLife.26857.071

Appendix 1—figure 18.

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Appendix 1—figure 19.

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1H and 13C NMR of compound ERX-11-biotin.

DOI: http://dx.doi.org/10.7554/eLife.26857.073

Appendix 1—figure 20.

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Characterization of SRC1-LXXLL peptide.

(a) HPLC chromatogram of SRC1-LXXLL peptide. (b) MALDI-TOF of SRC1-LXXLL peptide.

DOI: http://dx.doi.org/10.7554/eLife.26857.074

Appendix 1—figure 21.

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Characterization of SRC2-LXXLL peptide.

(a) HPLC chromatogram of SRC2-LXXLL peptide. (b) MALDI-TOF of SRC2-LXXLL peptide.

DOI: http://dx.doi.org/10.7554/eLife.26857.075

Appendix 1—figure 22.

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Characterization of AIB1-LXXLL peptide.

(a) HPLC chromatogram of AIB1-LXXLL peptide. (b) MALDI-TOF of AIB1-LXXLL peptide.

DOI: http://dx.doi.org/10.7554/eLife.26857.076

Appendix 1—figure 23.

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Object name is elife-26857-app1-fig45.jpg
Characterization of PELP1-LXXLL peptide.

(a) HPLC chromatogram of PELP1-LXXLL peptide. (b) MALDI-TOF of PELP1-LXXLL peptide.

DOI: http://dx.doi.org/10.7554/eLife.26857.077

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Funding Information

This paper was supported by the following grants:

  • http://dx.doi.org/10.13039/100004917Cancer Prevention and Research Institute of Texas DP150096 to Ganesh V Raj, Ratna K Vadlamudi.
  • http://dx.doi.org/10.13039/100000090Congressionally Directed Medical Research Programs W81XWH-12-1-0288 to Ganesh V Raj.
  • http://dx.doi.org/10.13039/100000090Congressionally Directed Medical Research Programs W81XWH-13-2-0093 to Ganesh V Raj.
  • http://dx.doi.org/10.13039/100000090Congressionally Directed Medical Research Programs W81XWH-16-1-0294 to Rajeshwar Rao Tekmal.
  • http://dx.doi.org/10.13039/100000928Welch Foundation AT-1595 to Jung-Mo Ahn.
  • http://dx.doi.org/10.13039/100000002National Institutes of Health CA179120-01 to Ratna K Vadlamudi.
  • http://dx.doi.org/10.13039/100000002National Institutes of Health P30 CA-54174 to Ratna K Vadlamudi.
  • UTHSA , School of Medicine Briscoe Women Health Scholar to Ratna K Vadlamudi.

Additional information

Competing interests

The authors declare that no competing interests exist.

Author contributions

GVR, Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing.

GRS, Data curation, Software, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing, Co Second author.

SMa, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing, Co Second author.

T-KL, Software, Investigation, Methodology, Co Second author.

SV, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing, Co Second author.

RL, Investigation, Methodology.

XL, Validation, Investigation, Methodology.

SMu, Data curation, Investigation.

C-CC, Investigation, Methodology.

W-RL, Investigation, Methodology.

MM, Investigation, Methodology.

SRK, Investigation, Methodology.

BM, Investigation, Methodology.

VKG, Investigation, Methodology.

DS, Investigation, Methodology.

RRT, Resources, Supervision, Investigation.

J-MA, Conceptualization, Data curation, Software, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing.

RKV, Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing.

Ethics

Animal experimentation: All animal experiments were performed after obtaining UTHSCSA IACUC approval and using methods in the approved protocol (15039X).

Additional files

Major datasets

The following dataset was generated:

Sareddy GR,Chen Y,Vadlamudi RK,2016,Estrogenreceptor coregulator binding modulators (ERXs) effectively target estrogenreceptor positive human breast cancers,http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE75664,Publicly available at the NCBI Gene Expression Omnibus (accession no. GSE75664)

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2017; 6: e26857.

Decision letter

Jeffrey Settleman, Reviewing editor
Jeffrey Settleman, Calico Life Sciences, United States;

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Coregulator Binding Inhibitors effectively target Estrogen receptor positive Breast Cancers" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Kendall W Nettles (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As you will see below, the reviewers have a number of concerns that require considerable work and explanation.

The estrogen receptor-alpha (ESR1) continues to be an important target in luminal breast cancer. Whereas the currently used endocrine therapies (e.g. aromatase inhibitors, tamoxifen and more recently fulvestrant) have had considerable success, the effectiveness of these therapies is limited by de novo and acquired resistance, and in the case of fulvestrant, suboptimal pharmaceutical properties. These newly described findings strongly support a novel first in class therapeutic for breast cancer, and a design strategy that can be readily applied to other nuclear receptors. However, the reviewers agree that some of these findings have been misinterpreted and additional information is required to support some of the key claims.

Specific comments:

1) The focus in the manuscript is on blocking the active conformation of the receptor, and especially the constitutively active mutants found in metastatic treatment resistant disease, but it Ignores the role of corepressors that are required for efficacy of tamoxifen (see PMID:12482846, 12145334, 17283072). It also ignores that their compound binds better to an antagonist conformation of the receptor, upon deletion of helix 12. There is a literature on CBI's as coactivator binding inhibitors. This group is now calling them coregulator binding inhibitors, which is confusing. The authors are encouraged to propose an alternative terminology. Perhaps coregulator binding modulator? Such a change would highlight that these compounds are distinct from previous CBIs in binding with higher affinity to an antagonist conformation surface in addition to the AF2 surface, which only exists with the agonist conformer, and that ECBI-11 uniquely activates apoptotic genes.

2) Crystal structures have shown that the LBD can adopt three major conformations, with helix 12 docked to form one side of the AF2 coactivator binding surface (diethylstilbesterol, 3ERD.pdb); 2) docked into the coregulator binding surface, blocking both coactivator and corepressor binding (tamoxifen, 3ERT.pdb); 3) or in solution (fulvestrant analog, 1HJ1.pdb), allowing binding of a corepressor peptide to a longer groove than seen in the AF2 surface (2JFA.pdb). Another important consideration is that the ligands can also directly contact corepressors (visualized in PMID 17283072, 2JFA.pdb). The identification of binding sites and discussion of the mechanism needs further clarification.

3) If ECBI-11 blocks corepressor binding one would expect AF1 activity. The coactivator binding data in Figure 2—figure supplement 2D suggests some residual binding with ECBI-11, similar to tamoxifen, so it will be important to determine the degree of AF1 activity of the compound, compared to tamoxifen. ECBI vs tamoxifen should be tested with an ERE-luc reporter assay in HepG2 cells, which show high tamoxifen agonist activity, or some other cell line with tamoxifen agonist activity.

4) In Figure 1B-C, even though ER+ cell lines are more sensitive to ECBI-11 than ER- cell lines, the maximum reduction of viability varies dramatically. The authors should show a graph displaying the% maximum inhibition, and can they also include data comparing activity of ECBI-11 in these ER+ lines to fulvestrant or tamoxifen.

5) Figure 1D: This model ignores the fact that the compound binds more strongly to an antagonist conformer, when helix 12 is deleted. Please model ECBI-11 to the LBD in the absence of helix 12, with tamoxifen versus SERD bound LBD. Use pdb files 3ERT and 5ACC and delete residues 531+. Please also describe whether the compound is interacting with helix 12 in the current model. Please make another figure panel showing side-chains and identify key molecular interactions, which could be supplemental.

6) The data in Figure 1H are confusing. Firstly, a control to evaluate the interaction of ECBI-11 with apo-ESR1 is missing. However, more puzzling is the fact that GDC-810 and AZD9494, both of which disrupt the AF-2 packet of ESR1, do not block the interaction of ECBI-11 with ESR-1. Tamoxifen does this, as expected. The authors note this discrepancy and suggest that maybe the activity of tamoxifen relates to a second tamoxifen binding site ESR1, as suggested several years ago by Elwood Jensen. This may be the case but if so it puts a significant hole in the mechanistic model that ECBI-11 is working through the canonical AF-2 pocket. Resolution of this issue is needed in light of the authors' proposed model.

7) Figure 2B. The analysis is entirely focused on which coregulators are dismissed, and ignores the potential coregulators required for induction of apoptosis or transcriptional repression. Please provide additional analyses of the ECBI-recruited coregulators. This is one area where focusing on "binding inhibitor" is misrepresenting the results, and in doing so underselling the significance. By treating it as a binding inhibitor, the data reveals only a partial inhibition. By focusing on coregulator binding modulation, the authors can point out the novel ensemble of proteins that are driving new biology such as efficacy in treatment resistance models.

8) Figure 2K. This is a key experiment that addresses whether tamoxifen competition derives from its positioning of helix 12, its side chain interaction with ECPI-11 or whether tamoxifen displaces ECBI-11 through a putative second binding site, overlapping with the corepressor binding site (PMID: 16782818). These data suggest that it is one of the latter two explanations. This needs to be further clarified using either transfected or recombinant protein. Tamoxifen binds in the pocket with single digit nanomolar affinity, and to the second site with "greatly reduced" affinity. The authors should perform a dose curve to determine which is more likely.

9) The Discussion needs revision to reflect the fact that the compound binds more tightly to an antagonist conformer, but also the agonist AF2 surface. This is a feature that is significantly different from previous CBIs, and likely contributes to the improved efficacy. The authors should include a more balanced Discussion that is less focused on inhibition of binding. There appear to be several contributions to the unique mechanism of action, including altered coregulator binding, inhibition of dimerization and DNA binding, induction of apoptotic genes, and a reduction in ESR1 expression.

10) It is not entirely convincing that the observed anti-tumor activities are on-target. The central premise of the work is that the ESR1 coregulator binding pocket that forms upon binding 17-β estradiol (E2) allows the interaction of specific LXXLL motifs within coregulators to engage the receptor and that this interaction can be targeted with peptidomimetics. In support of this mechanism the authors show that their lead compound ECBI-11 inhibits E2-dependent transcriptional activity, ESR1-coregulator binding, and the growth of ESR1 expressing cell lines. However, there are several pieces of inconsistent data that the authors themselves acknowledge (somewhat) that they cannot reconcile with this simple model. The authors should modify the messaging of the manuscript to present a new, potentially very important molecule (ECBI-11), whose activities are partially ESR1-dependent.

11) Does ECBI-11 inhibit the activity of an ERα-VP16 chimera? This receptor derivative does not require AF-2 as the VP16 activator overrides the activity of AF1 and AF-2. If the drug does indeed inhibit this chimera then the primary mechanistic hypothesis is unlikely to be correct. This, or a similar experiment that is specifically designed to "disprove" the authors mechanistic hypothesis, is needed.

12) Whereas it is interesting that the activity of ECBI-11 was restricted to ESR1 positive cells it cannot be concluded that the inhibition observed results from the inhibition of ESR1. Not all of the cells tested require E2/ESR1 for growth. Maybe it's just being a luminal breast cancer that defines responsively.

13) It is not clear why ZR75 cells were used for the in vivo studies when MCF-7 cell derived tumors are the gold standard. Did ECBI-11 not work in MCF-7 cells in vivo? There is data showing efficacy in the MCF-7 LTLT model although it's not clear how these cells were derived and a positive control (fulvestrant) was not included. Thus, although ECBI-11 works in this model, the role of ESR1 is not clear.

14) The data generated in the D2A1 model are problematic. The authors present this as a model of ESR-1 positive luminal cancer. However, a literature search revealed that it is used as a model of TNBC! Is the subline the authors using different from that used by others? Have they shown that ESR1 is expressed in these cells/tumors? This data is only of value (with respect to implicating ESR1) if they show that the tumors can be inhibited by more standard drugs (tamoxifen and/or fulvestrant).

15) The authors note that ECBI-11 treatment induces apoptosis in cells in which fulvestrant does not. Given that fulvestrant eliminates ESR1, expression it is unclear why ECBI-11 can induce this activity unless the apoptosis is due to an off-target activity.

16) The authors should include additional controls to demonstrate the specificity of ECBI-11 in cells.

a) In Figure 2H authors should include western analysis of more proteins that are immuno-precipitated by ER (e.g. SRC's, MED, TIF1, p300).

b) A negative control (a protein whose binding to ER is not reduced by ECBI-11 e.g. TIF1) should be included in their proximity ligation assays shown in Figure 2I and Figure 2—figure supplement 2D. A similar negative control needs to be included in Figures 4C-D.

17) If the authors want to examine effects of ECBI-11 on AR binding (Figure 4—figure supplement 1A), why not include an AR agonist to stimulate AR activity? E2 is not an AR agonist and will not induce AR activity. In the absence of this control it is difficult to conclude that ECBI-11 does not modulate AR activity. This experiment should also include an AR antagonist to demonstrate that disruption of AR binding can be detected in their assay.

Specific comments:

1) The focus in the manuscript is on blocking the active conformation of the receptor, and especially the constitutively active mutants found in metastatic treatment resistant disease, but it Ignores the role of corepressors that are required for efficacy of tamoxifen (see PMID:12482846, 12145334, 17283072). It also ignores that their compound binds better to an antagonist conformation of the receptor, upon deletion of helix 12. There is a literature on CBI's as coactivator binding inhibitors. This group is now calling them coregulator binding inhibitors, which is confusing. The authors are encouraged to propose an alternative terminology. Perhaps coregulator binding modulator? Such a change would highlight that these compounds are distinct from previous CBIs in binding with higher affinity to an antagonist conformation surface in addition to the AF2 surface, which only exists with the agonist conformer, and that ECBI-11 uniquely activates apoptotic genes.

We agree with reviewer’s suggestions. Our mechanistic studies do indeed confirm that the activity of our compound is due to multiple mechanisms of action,including its abilityto bind to ER,modulate coregulator binding to ER, inhibit ER dimerization and induce apoptotic genes. To reflect its multiple mechanisms of action, we will use the nomenclature ERX, for Estrogen Receptor coregulator binding modulators where X refers to multiple mode of actions of ERX-11 on ER functions. We have modified the figures and the manuscript to reflect the new terminology.

2) Crystal structures have shown that the LBD can adopt three major conformations, with helix 12 docked to form one side of the AF2 coactivator binding surface (diethylstilbesterol, 3ERD.pdb); 2) docked into the coregulator binding surface, blocking both coactivator and corepressor binding (tamoxifen, 3ERT.pdb); 3) or in solution (fulvestrant analog, 1HJ1.pdb), allowing binding of a corepressor peptide to a longer groove than seen in the AF2 surface (2JFA.pdb). Another important consideration is that the ligands can also directly contact corepressors (visualized in PMID 17283072, 2JFA.pdb). The identification of binding sites and discussion of the mechanism needs further clarification.

As suggested by the reviewer, we have now performed docking simulations of ERX-11 on four crystal structures corresponding to 3ERD, 3ERT, 1HJ1, and 2JFA (Figure 2—figure supplement 6).

In 3ERD, binding of the agonist leads to a rearrangement of helix 12 and forms a hydrophobic cleft (i.e., AF2 binding site) that is surrounded by helix 3, 4, 5, and 12: we found that ERX-11 makes hydrophobic contact with the AF2 site with its two isobutyl side chain groups (Figure 2—figure supplement 6AA). In addition, the hydroxyl group of ERX-11 interacts with a residue near AF2 domain. These data indicate that ERX-11 interacts with ER LBD differently than the agonist.

The crystal structure of 3ERT shows that 4-hydroxytamoxifen (antagonist) induces conformational change and makes helix 12 occupy the AF2 binding site, blocking both coactivator and corepressor binding. This makes ERX-11 change its binding site to a nearby pocket formed by helix 5, 11, and 12, as shown in the Figure 2—figure supplement 6AB. These data show that ERX-11 could only interact with ER in the presence of tamoxifen, through an alternate binding site. These data could explain why the interaction between ERX-11 and purified ER could be blocked by tamoxifen. However, within the cell, tamoxifen cannot block ERX-11 binding to ER:, suggesting that the secondary binding site of ERX-11 on ER may be stabilized by coregulators.

Docking simulation of ERX-11 on human ERα with affinity tagged corepressor peptide (Figure 2—figure supplement 6AC) (2JFA.pdb) or rat ERβ crystal structure with ICI boundFigure 2—figure supplement 6AD (1HJ1.pdb) showed that ECBI-11 can still bind to the AF2 domain, in a similar manner as it does when the ligand is bound. These data support our biochemical findings that ICI does not block ERX-11 interaction with ER.

3) If ECBI-11 blocks corepressor binding one would expect AF1 activity. The coactivator binding data in Figure 2—figure supplement 2D suggests some residual binding with ECBI-11, similar to tamoxifen, so it will be important to determine the degree of AF1 activity of the compound, compared to tamoxifen. ECBI vs tamoxifen should be tested with an ERE-luc reporter assay in HepG2 cells, which show high tamoxifen agonist activity, or some other cell line with tamoxifen agonist activity.

We have now tested whether ERX-11 has any residual activity via AF1 domain using endometrial Ishikawa cell line, which exhibit agonist activity via AF1. As expected, tamoxifen treatment promoted agonist activity in this model. However, in this assay we failed to detect any agonist activity of ERX-11. These results suggest that ERX-11 lacks AF1 activity. We have included this data as Figure 2—figure supplement 4D.

4) In Figure 1B-C, even though ER+ cell lines are more sensitive to ECBI-11 than ER- cell lines, the maximum reduction of viability varies dramatically. The authors should show a graph displaying the% maximum inhibition, and can they also include data comparing activity of ECBI-11 in these ER+ lines to fulvestrant or tamoxifen.

We have included a graph displaying the% maximum inhibition mediated by ERX-11 (1μM) in a number ER+ and ER- cell lines as a waterfall graph in Figure 1—figure supplement 1D. We have also included data comparing the activity of ERX-11 in ER+ cell lines to fulvestrant or tamoxifen in Figure 1—figure supplement 1D, E.

5) Figure 1D: This model ignores the fact that the compound binds more strongly to an antagonist conformer, when helix 12 is deleted. Please model ECBI-11 to the LBD in the absence of helix 12, with tamoxifen versus SERD bound LBD. Use pdb files 3ERT and 5ACC and delete residues 531+. Please also describe whether the compound is interacting with helix 12 in the current model. Please make another figure panel showing side-chains and identify key molecular interactions, which could be supplemental.

We have created a model showing the side chains and interactions of ERX-11 with residues in the ER protein using 1L2I.pdb structure (Figure 2—figure supplement 6B).

We also have carried out additional docking experiment without helix 12 with the crystal structures of 3ERT.pdb and 5ACC.pdb as suggested by the reviewer. The deletion of the helix 12 destroys the AF2 binding site and this makes ERX-11 look for another binding site. It is interesting to note that ERX-11 was found to bind to the tamoxifen binding site when the helix 12 was deleted in the crystal structure of 3ERT.pdb (Figure 2—figure supplement 6CA). The superimposition of tamoxifen (red) and ERX-11 (green) clearly shows the overlap of their binding sites (Figure 2—figure supplement 6CB). This may explain our experimental results showing the competition of tamoxifen with ERX-11 on ER-▲12. On the other hand, the deletion of the helix 12 of the crystal structure of 5ACC.pdb still allows ERX-11 to bind to the AF2 domain (Figure 2—figure supplement 6C), which does not overlap with the binding site of SERD in the crystal structure (Figure 2—figure supplement 6D 2—figure supplement C). This also confirms our experimental results of the inability of the SERDS to compete with ERX-11 binding to ER-▲12.

6) The data in Figure 1H are confusing. Firstly, a control to evaluate the interaction of ECBI-11 with apo-ESR1 is missing. However, more puzzling is the fact that GDC-810 and AZD9494, both of which disrupt the AF-2 packet of ESR1, do not block the interaction of ECBI-11 with ESR-1. Tamoxifen does this, as expected. The authors note this discrepancy and suggest that maybe the activity of tamoxifen relates to a second tamoxifen binding site ESR1, as suggested several years ago by Elwood Jensen. This may be the case but if so it puts a significant hole in the mechanistic model that ECBI-11 is working through the canonical AF-2 pocket. Resolution of this issue is needed in light of the authors' proposed model.

We have now repeated this experiment and included apo-ER control. The results showed that ERX-11 has weak interaction with apo-ER and addition of E2 significantly increased ERX-11 ability to interact with ER. Further, addition of tamoxifen interfered with ERX-11 interaction with ER, while SERDs only slightly reduced ERX-11 binding. New data replaced old Figure 1H. Further, using crystal structures, we have refined our models, that explain why ERX-11 interaction with ER could be affected by tamoxifen but not by SERD. We thank the reviewers for their suggestions to model the interaction using existing structures, as it potentially explains our findings(Figure 2—figure supplements 6,,77).

7) Figure 2B. The analysis is entirely focused on which coregulators are dismissed, and ignores the potential coregulators required for induction of apoptosis or transcriptional repression. Please provide additional analyses of the ECBI-recruited coregulators. This is one area where focusing on "binding inhibitor" is misrepresenting the results, and in doing so underselling the significance. By treating it as a binding inhibitor, the data reveals only a partial inhibition. By focusing on coregulator binding modulation, the authors can point out the novel ensemble of proteins that are driving new biology such as efficacy in treatment resistance models.

Based on the reviewer’s suggestions, we have now reanalyzed the ERX-11-recruited binding proteins (Figure 2B). Pathways analysis in terms of either biological processes or molecular functions revealed that ERX-11 binding proteins were involved in the activation of multiple pathways leading to transcriptional regulation (Figure 2—figure supplement 1). Based on these studies, and the finding that our compound mediates apoptosis, we have revised the terminology of our compounds to ERX to reflect its biological activity.

8) Figure 2K. This is a key experiment that addresses whether tamoxifen competition derives from its positioning of helix 12, its side chain interaction with ECPI-11 or whether tamoxifen displaces ECBI-11 through a putative second binding site, overlapping with the corepressor binding site (PMID: 16782818). These data suggest that it is one of the latter two explanations. This needs to be further clarified using either transfected or recombinant protein. Tamoxifen binds in the pocket with single digit nanomolar affinity, and to the second site with "greatly reduced" affinity. The authors should perform a dose curve to determine which is more likely.

We have added a dose response experiment that suggests that ERX-11 binding to a putative seconding binding site on ER in the presence of tamoxifen. Increasing concentrations of tamoxifen fail to dislodge ERX-11 from ER (Figure 2—figure supplement 5C). However, for the ER▲12 mutant, increasing concentrations of tamoxifen is only able to dislodge ERX-11 from ER at higher concentrations (Figure 2—figure supplement 5D), suggesting that ERX-11 interaction with the primary binding site of ER is through the second binding site of tamoxifen with greatly reduced affinity. We have also added a model to explain the potential interaction between ER and ERX-11 or between ER▲12 mutant and ERX-11 in the absence or presence of agonist, SERDs or tamoxifen (Figure 2—figure supplement 7). Importantly, these data indicate that tamoxifen displaces ERX-11 from its binding site.

9) The Discussion needs revision to reflect the fact that the compound binds more tightly to an antagonist conformer, but also the agonist AF2 surface. This is a feature that is significantly different from previous CBIs, and likely contributes to the improved efficacy. The authors should include a more balanced Discussion that is less focused on inhibition of binding. There appear to be several contributions to the unique mechanism of action, including altered coregulator binding, inhibition of dimerization and DNA binding, induction of apoptotic genes, and a reduction in ESR1 expression.

We agree with reviewer’s suggestion. We have now modified the Discussion and renamed our compound as ERX-11 to reflect several contributions to its unique mode of action.

10) It is not entirely convincing that the observed anti-tumor activities are on-target. The central premise of the work is that the ESR1 coregulator binding pocket that forms upon binding 17-β estradiol (E2) allows the interaction of specific LXXLL motifs within coregulators to engage the receptor and that this interaction can be targeted with peptidomimetics. In support of this mechanism the authors show that their lead compound ECBI-11 inhibits E2-dependent transcriptional activity, ESR1-coregulator binding, and the growth of ESR1 expressing cell lines. However, there are several pieces of inconsistent data that the authors themselves acknowledge (somewhat) that they cannot reconcile with this simple model. The authors should modify the messaging of the manuscript to present a new, potentially very important molecule (ECBI-11), whose activities are partially ESR1-dependent.

We agree with reviewers’ suggestions. We have now modified our model and softened the discussion reflecting ERX-11 has complex mode of activity on ER and that its effects are only partially ER-dependent.

11) Does ECBI-11 inhibit the activity of an ERα-VP16 chimera? This receptor derivative does not require AF-2 as the VP16 activator overrides the activity of AF1 and AF-2. If the drug does indeed inhibit this chimera then the primary mechanistic hypothesis is unlikely to be correct. This, or a similar experiment that is specifically designed to "disprove" the authors mechanistic hypothesis, is needed.

We have now conducted suggested experiment usingERα-VP16 chimera receptor that does not require AF-2 as the VP16 activator overrides the activity of AF1 and AF-2. In these reporter-based assays, ERX-11 failed to reduce the ERE-luc reporter activity driven by ERα-VP16 chimera. In this assay, we have used tamoxifen and ICI as controls (Figure 2—figure supplement 4C). As expected tamoxifen that signals via AF2, and AF1 also did not affected the ERα-VP16 chimera reporter activity, while ICI that degrades ER significantly reduced the reporter activity.

Collectively, these results confirm that the ERX-11 block signaling specifically driven by AF2 domain.

12) Whereas it is interesting that the activity of ECBI-11 was restricted to ESR1 positive cells it cannot be concluded that the inhibition observed results from the inhibition of ESR1. Not all of the cells tested require E2/ESR1 for growth. Maybe it's just being a luminal breast cancer that defines responsively.

As suggested by the reviewer, it is possible that ERX-11 target will be luminal breast cancer cells. However, our mechanistic studies support that ER as the primary target for this ERX-11 activity. To further support the importance of ER in ERX-11 activity, we have used restoration model cells where ER was introduced into ER-negative breast cancer model MDA-MB-231. MTT assays revealed that introduction of WT ER into MDA-MB-231 cells, restored ERX-11 growth inhibitory activity in non-responsive MDA-MB-231 cells. Similarly, introduction of the ER▲12 mutant into these cells restored responsiveness to ERX-11. These results further underscore the importance of ER in ERX-11 mode of action(Figure 2—figure supplement 5E).

13) It is not clear why ZR75 cells were used for the in vivo studies when MCF-7 cell derived tumors are the gold standard. Did ECBI-11 not work in MCF-7 cells in vivo? There is data showing efficacy in the MCF-7 LTLT model although it's not clear how these cells were derived and a positive control (fulvestrant) was not included. Thus, although ECBI-11 works in this model, the role of ESR1 is not clear.

We have used two different models (MCF-7 and ZR-75) for in vivo studies to avoid any artifacts due to genetic background. Further, ERX-11 showed efficacy in both MCF-7 based xenografts tested in this study (Figure 5E, MCF-7-PELP1, and 6E, MCF-7-LTLT). MCF-7-LTLT cells were widely used cells we received from Dr. Angela Brodie lab, exhibit-resistance to letrozole. MCF-7-LTLT cells were developed by continuous exposure of letrozole over a long period of time. We have now conduced an in vivo experiment showing the efficacy of fulvestrant on this xenograft. Further, we have also treated these tumors with ERX-11 as comparison with fulvestrant. Results showed that fulvestrant was able to reduce the growth of MCF-7-LTLT tumors. Further, ERX-11 exhibited similar potency in this model as fulvestrant. New data was included as Figure 6—figure supplement 1A.

14) The data generated in the D2A1 model are problematic. The authors present this as a model of ESR-1 positive luminal cancer. However, a literature search revealed that it is used as a model of TNBC! Is the subline the authors using different from that used by others? Have they shown that ESR1 is expressed in these cells/tumors? This data is only of value (with respect to implicating ESR1) if they show that the tumors can be inhibited by more standard drugs (tamoxifen and/or fulvestrant).

D2A1 cells were initially characterized by Dr. Tekmal (coauthor of this manuscript) for hormonal therapy response (PMID 9310256). In D2A1 model cells, cellular gene int-5/aromatase in BALB/c mammary alveolar hyperplastic nodule (D2 HAN/D2 tumor cells) is activated as a result of mouse mammary tumor virus integration within the 3' untranslated region of the aromatase gene. Thus, these models also have ability to synthesize local estrogen via aromatase induction. Further, this model expresses estrogen receptor (ER) and represents a model of intra-tumoral estrogen driven mammary cancer. D2A1 cells are shown to be useful model for evaluating the effects of aromatase inhibitors and antiestrogens. To address the reviewers’ concerns, we have now conducted western blot analysis of D2A1 cells. Results showed that D2A1 cells express ER. Mammary gland lysates as well as murine ER positive cells E0771 were used as positive controls. Tamoxifen was able to reduce the growth of D2A1 cells. Further, IHC analysis confirmed that D2A1 tumors express ER and ERX-11 treatment substantially reduced ER expression (Figure 5—figure supplement 1B).

15) The authors note that ECBI-11 treatment induces apoptosis in cells in which fulvestrant does not. Given that fulvestrant eliminates ESR1, expression it is unclear why ECBI-11 can induce this activity unless the apoptosis is due to an off-target activity.

We consistently observed activation of apoptosis by ERX-11 but failed to observe any apoptosis by tamoxifen or ICI in our assays. We believe that activation of apoptosis is not due to elimination of ER rather due to unique mechanism of action of ERX-11. Specifically, we predict that changes in the ER signaling due to alterations in coregulator binding to ER. Accordingly, our RNAseq data showed that alterations in genes that contribute activation of apoptosis. However, further studies are needed to clearly identify the mechanisms by which ERX-11 promotes apoptosis.

16) The authors should include additional controls to demonstrate the specificity of ECBI-11 in cells.

a) In Figure 2H authors should include western analysis of more proteins that are immuno-precipitated by ER (e.g. SRC's, MED, TIF1, p300).

b) A negative control (a protein whose binding to ER is not reduced by ECBI-11 e.g. TIF1) should be included in their proximity ligation assays shown in Figure 2I and Figure 2—figure supplement 2D. A similar negative control needs to be included in Figures 4C-D.

We have modified our figures to reflect the IP western blot analysis of additional proteins affected by ERX-11 as well as proteins that are not. We have also included a negative control for the PLA in Figure 2I. We have also added negative control quantification in Figure 2—figure supplement 4B.

17) If the authors want to examine effects of ECBI-11 on AR binding (Figure 4—figure supplement 1A), why not include an AR agonist to stimulate AR activity? E2 is not an AR agonist and will not induce AR activity. In the absence of this control it is difficult to conclude that ECBI-11 does not modulate AR activity. This experiment should also include an AR antagonist to demonstrate that disruption of AR binding can be detected in their assay.

We have now conducted requested experiment with control AR agonist. Results showed ERX-11 has no activity on AR binding induced by DHT.


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