Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy

The pro-oncogenic activities of estrogen receptor alpha (ERα) drive breast cancer pathogenesis. Endocrine therapies that impair the production of estrogen or the action of the ERα are therefore used to prevent primary disease metastasis. Although recent successes with ERα degraders have been reported, there is still the need to develop further ERα antagonists with additional properties for breast cancer therapy. We have previously described a benzothiazole compound A4B17 that inhibits the proliferation of androgen receptor–positive prostate cancer cells by disrupting the interaction of the cochaperone BAG1 with the AR. A4B17 was also found to inhibit the proliferation of estrogen receptor—positive (ER+) breast cancer cells. Using a scaffold hopping approach, we report here a group of small molecules with imidazopyridine scaffolds that are more potent and efficacious than A4B17. The prototype molecule X15695 efficiently degraded ERα and attenuated estrogen-mediated target gene expression as well as transactivation by the AR. X15695 also disrupted key cellular protein–protein interactions such as BAG1–mortalin (GRP75) interaction as well as wild-type p53–mortalin or mutant p53–BAG2 interactions. These activities together reactivated p53 and resulted in cell-cycle block and the induction of apoptosis. When administered orally to in vivo tumor xenograft models, X15695 potently inhibited the growth of breast tumor cells but less efficiently the growth of prostate tumor cells. We therefore identify X15695 as an oral selective ER degrader and propose further development of this compound for therapy of ER+ breast cancers. Significance: An imidazopyridine that selectively degrades ERα and is orally bioavailable has been identified for the development of ER+ breast cancer therapeutics. This compound also activates wild-type p53 and disrupts the gain-of-function tumorigenic activity of mutant p53, resulting in cell-cycle arrest and the induction of apoptosis.


Introduction
Breast cancer is the most commonly diagnosed cancer worldwide with over 2.3 million new cases and 685,000 deaths in 2020. In 2040, the burden of breast cancer is predicted to increase to over 3 million new cases and 1 million deaths every year because of population growth and ageing (1). Approximately biosynthesis (e.g., aromatase inhibitors) or compounds that competitively modulate the action of ERα (e.g., selective ER modulators-SERM). Long-term treatment with the SERM tamoxifen is reported to promote endometrial carcinoma and venous thromboembolism due to partial ERα agonistic activity (3). Second-and third-generation SERMs have therefore been developed that show improved, though distinctly different, safety profiles compared with tamoxifen. However, the risk of venous thromboembolism remains a concern for most SERMs (4). In contrast, selective estrogen receptor degraders (SERD) are considered pure antagonists without agonist activity. They do not only antagonize ERα action but also downregulate ERα protein levels. One of the earliest examples of such a targeted protein degradation therapeutic is fulvestrant, that shows efficacy in tamoxifen-refractory patients and postmenopausal women who had progressed on prior hormone therapies (5) However, fulvestrant's major clinical limitations are its intramuscular route of administration and its low bioavailability (6)(7)(8). There is therefore the need for the development of orally bioavailable ER degraders (9). A number of such oral SERDs have been described that exhibit both preclinical and clinical antitumor activities (10,11) but despite these promising results, there is still an opportunity to develop further ERα antagonists with novel modes of action.
We aim to develop novel ERα inhibitors that do not competitively interact with the ligand-binding domain of the receptors where mutations mostly occur that block the action of the antagonists. Rather, we have embarked on the development of new antagonists that target molecular chaperones and cochaperones that assist the conformational folding or unfolding as well as the assembly of other macromolecular structures including steroid receptors. One such attractive group of proteins to target is the BCL2-associated athanogene protein 1 (BAG1), a nucleotide exchange factor that binds the ATPase binding domain of the molecular chaperone HSP70/HSC70. In addition to its co-chaperone activity, BAG1 interacts with a variety of other proteins to regulate diverse cellular processes including cell division, cell death and differentiation, and transcriptional activity of the ER and androgen receptor (AR). BAG1 is made up of different proteins translated from a single mRNA by alternative translation-initiation resulting in humans in four BAG1 isoforms (1L, 1M, 1, and 1S). These proteins have a similar carboxy-terminal BAG domain that is made up of three antiparallel alpha helical bundles of 30-40 amino acids (12) and a centrally located ubiquitin-like domain that directs HSP70 clients to the proteasome for degradation (13,14). The largest member of the family possesses a N-terminal nuclear localization sequence and is therefore localized to the nucleus where it interacts with and regulates the activity of transcription factors including ERα and the AR (15)(16)(17). The other BAG1 proteins are cytoplasmic and they regulate an array of molecular targets within the cytoplasm that control cell proliferation, apoptosis, and stress response (18).
We have recently described a benzothiazole-based compound A4B17 with antiproliferative action in ER + breast and AR-positive (AR + ) prostate cancer cells that is reported to dock into the BAG domain of BAG1 (19,20). This compound attenuated AR target gene expression by disrupting the interaction of BAG1 and the AR N-terminal domain that is otherwise required for transactivation by the AR. However, it required micromolar concentrations for its action. To improve on the efficacy of A4B17, we used scaffold hopping strategies to change the chemical scaffold of A4B17 and analyzed the resulting compounds for their antiproliferative action in breast and prostate cancer cells. We identified an imidazopyridine X15695 that potently inhibited proliferation of ER + breast and AR + prostate cancer cells. X15696 degraded ERα protein and potently inhibited ERα transactivation and in addition reactivated p53 by disrupting its interactions with members of the HSP70 chaperone family. In mouse tumor xenograft models, when administered orally, X15695 inhibited ER + breast cancer cell growth more efficiently than prostate cancer cell growth. These studies together identify X15695 as an oral SERD for further development for the treatment of breast cancer.

Cycloheximide Chase and MG132 Stability Assay
Unless otherwise stated, cycloheximide chase and MG132 stability experiments were performed as follows: 2 × 10 5 MCF-7 cells were seeded in 6 cm dishes and treated with 1 μmol/L X15695 or the equivalent volume of DMSO for 24 hours. Thereafter, cells were incubated with the protein synthesis inhibitor cycloheximide (100 μg/mL) and harvested at timepoints 0, 30, 60, 90, and 120 minutes. Cells were lysed and used for Western blotting to determine the ERα level at each timepoint. For MG132 experiments, MCF-7 cells were similarly treated but in the presence or absence of the proteasome inhibitor MG132 (0.5 μmol/L) for 48 hours. Cells were harvested and lysed for Western blot assay to detect the p53 level at each condition.

Co-immunoprecipitation
For co-immunoprecipitation experiments, 1 × 10 6 cells were seeded in 15 cm dishes and treated with 1 μmol/L X15695 or the equivalent volume of DMSO for 48 hours. Cells were harvested, washed with PBS and lysed using NP-40 lysis buffer. Protein A agarose beads were mixed with protein G agarose beads (4:1) and preincubated overnight at 4°C with anti-p53 or anti-BAG1 or con-trol IgG antibody. Cellular extracts from X15695 treated and untreated cells (1,000 μg) were incubated with antibody-preincubated protein A/G agarose beads for 3 to 4 hours at room temperature. The beads with immunoprecipitated proteins were collected at 4°C by centrifugation at 1,000 × g for 5 minutes and washed four to five times with NP-40 lysis buffer. The immunoprecipitated proteins were eluted by heating in SDS sample buffer for 5 minutes at 95°C and resolved on SDS-PAGE. Thereafter, they were subjected to Western blot analysis to detect mortalin, BAG1, BAG2, BAG5, and p53. Cellular extracts containing 50 to 70 μg protein from X15695 treated and untreated cells were used as the input control for the targeted proteins.

MTT Cell Viability Assay
Cells were seeded in duplicates at a density of 1 × 10 4 cells/well in a 24-well plate format and incubated for 6 days in culture medium. Cells transfected with siRNA were incubated for 72 hours after X15695 addition. Thereafter, the medium was exchanged for a fresh medium containing 0.5 mg/mL MTT and incubated at 37°C for 2 hours until intracellular purple formazan crystals were formed. The MTT solution was removed and the purple crystals were dissolved in isopropanol and the optical absorbance at 590 nm was recorded using SpectraMax iD3, Molecular Devices.

Clonogenic Assay
Cells were seeded at a density of 1-2 × 10 3 cells/well in a 6-well plate and treated with increasing concentrations of X15695 and cultured for 14-21 days. Medium and compounds were exchanged after 7 days. Cells were fixed with methanol/acetic acid mixture [3:1 volume for volume (v/v)] and the formation of colonies was visualized using 0.5% crystal violet (w/v) in 20% methanol (v/v). Plates were scanned in a conventional office scanner (Epson). The area covered by colonies was calculated using the ColonyArea Plugin for ImageJ (RRID:SCR_003070; ref. 24).

Reduced H2DCF-DA Oxidation Assay
MCF-7 and T47D cells were seeded at 1 × 10 3 cells and treated with 10 −9 to 10 −6 mol/L X15695 for 48 hours. Afterward, cells were loaded with 10 μmol/L H2DCF-DA in phenol red-free medium for 40 minutes. Cells were subsequently washed with medium and further incubated in medium for 120 minutes before analysis of fluorescence at 485 nm excitation and 530 nm emission using a multi-well fluorescence reader (SpectraMax iD3, Molecular Devices with SoftMax Pro 7 software). After the DCF assay, cells were frozen at −80°C overnight. After thawing, DNA was stained with a Hoechst dye 33258

Immunofluorescence Assay
For immunofluorescence assay, MCF-7, T47D, 22Rv.1, LAPC-4, and LNCaP cells (1 × 10 4 each) were seeded on coverslips in a 24-well plate and the desired treatment applied. After three times washing with PBS, cells were fixed with 4% paraformaldehyde solution for 10 minutes at room temperature. Next, the cells were washed three times with PBS and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature. Following three additional wash steps with PBS, cells were treated with 5% BSA (w/v) for 1 hour at room temperature and subjected to overnight incubation with the primary antibody p53 (DO1 clone or Sp5), ERα or mortalin/GRP75 and then washed three times with PBS followed by the incubation with goat anti-mouse IgG, Alexa Fluor 488 antibody and/or goat anti-rabbit IgG, Alexa Fluor 546 antibody for 1 hour at room temperature in the dark. All subsequent steps were performed in the dark. Cells were washed three times with PBS and stained with 0.1 μg/mL DAPI solution at 1:10,000 (v/v) dilution for 15 minutes at room temperature. After three times washing with PBS, the cells were mounted in Mowiol 4-88 on microscope slides and observed by laser-scanning confocal microscopy (Confocal Microscope platform STELLARIS6-LSM900, Leica). Induction of apoptosis in response to treatment with X15695 was analyzed in MCF-7 and T47D cells using PE Annexin V Apoptosis Detection Kit with 7-AAD (BD Biosciences, 556421) according to the manufacturers protocol. In short, cells were harvested by trypsinization and single-cell suspensions (1 × 10 6 cells) were prepared as described above in 100 μL PBS. Cells were stained with 5 μL PE Annexin V and 5 μL 7-AAD for 15 minutes at room temperature in the dark. Subsequently, 400 μL PBS were added to each sample and flow cytometry measurements were performed within 1 hour. Cells stained with PE Annexin V or 7-AAD alone were used for compensation. Flow cytometry analysis was performed using BD FACSAria Fusion. Flow cytometry data were analyzed using

Mouse Xenograft Experiments
All animal experiments were performed according to European and German

Graphing and Statistical Analysis
Experiments were performed with three or more replicates. Differences between two groups were analyzed by Student t test and multiple comparisons were determined by one-way ANOVA. If there were two factors (such as dose and time) investigated, data were analyzed by two-way ANOVA followed by a AACRJournals.org Cancer Res Commun; 3(7) July 2023 post hoc test. Data were expressed as means ± SEM, and P < 0.05 was considered significant. All analyses were performed using Microsoft Excel 2010 (RRID:SCR_016137) and GraphPad Prism 8.3.1 (RRID:SCR_002798) software.

Growth Inhibitory Properties of Imidazopyridine Derivatives
We have previously reported that the compound A4B17 derived from a screen of 47 benzothiazoles inhibited proliferation of ER + breast and AR + prostate cancer cells with half maximal inhibitory concentrations (IC 50 ) in the micromolar range (19,20). To improve on the potency of this compound, we used a scaffold hopping approach focusing on heterocycle replacement (26) to change the benzothiazole into an imidazopyridine (from core of type A to core of type B; Fig. 1A) to generate X20046 from A4B17 (Fig. 1B). This greatly improved the inhibitory properties of X20046 both in breast and prostate cancer cells (Supplementary Table S1).
We therefore synthesized a series of 27 imidazopyridines (Fig. 1C) based on the core structure of type B as given in Fig. 1A (top right), determined the Xray structure of X15695, X15696, and X19168 ( Fig. 1D; Supplementary Table S2) for rational drug design and analyzed the ability of all 27 compounds to inhibit the clonal expansion of a series of breast and prostate cancer cells. All the compounds outperformed A4B17 in the inhibition of clonal expansion of ER + breast and AR + prostate cancer cells and had very weak or no effect on receptor-negative cell lines (Supplementary Table S1). With only few exceptions, compounds that potently inhibited colony formation of ER + cells also potently inhibited the clonal expansion of the AR + prostate cancer cell lines. Six imidazopyridines, including X20046 (the imidazopyridine derivative of A4B17) were identified as particularly potent based on their IC 50 values in the two different cell types (Supplementary Table S1). These compounds share a striking resemblance in their core structures (Fig. 1B) and show a requirement for either a methyl group or a CF3 group at position C-6 of the imidazopyridine core similar to other derivatives that were only active against one of the two cancer cell types analyzed. From the six identified compounds, X15695 was chosen for further study.

Transcriptomics Analyses to Determine X15695 Action
To determine the mechanism of action of X15695, we first performed transcriptomics analyses in the ER + breast cancer cell lines MCF-7 and T47D after treating them with vehicle (DMSO), 17-β-estradiol (E 2 ), X15695, and a combination of E 2 and X15695. The RNA-seq datasets were analyzed using a fold  Fig. S1A and S1B). In the T47D cells, 487 DEGs (302 downregulated and 185 upregulated) were identified in response to X15695 in the absence of E 2 treatment while in the presence of E 2 , slightly less DEGs were seen (458; 280 downregulated and 176 genes upregulated; Supplementary Fig. S1C and S1D).
To investigate the pathways associated with the X15695-mediated changes in were the ERα and p53 signaling pathways. In the absence of E 2 , the p53 signaling pathway was also identified as the most regulated pathway ( Fig. 2A and B; Supplementary Fig. S1A and S1B, right). Among other signatures, the ERα and the p53 pathways were also the most significantly regulated pathways by X15695 in the T47D cells (Supplementary Fig. S1C and S1D, right). Therefore, a heat map was generated to compare E 2 response genes in MCF-7 and T47D cells and this showed a very strong overlap in the downregulation of expression of ERα target genes in the two cell lines (Supplementary Fig. S2A). Quantitative real-time polymerase chain reaction (RT-PCR) carried out with a select number of ERα response genes in the two cells showed indeed downregulation of E 2 -mediated gene expression by X15695 ( Supplementary Fig. S2B and S2C). On the contrary, the p53 pathway was upregulated by X15695 in the two cell lines as shown in the heat map but more strongly in the MCF-7 cells compared with the T47D cells ( Supplementary Fig. S2D). X15695-mediated upregulation of expression of p53 target genes in the absence and presence of E 2 was also confirmed in the qRT-PCR analyses, but the results in the MCF-7 cells were somewhat more prominent than in the T47D cells ( Supplementary Fig. S2E and S2F).
We detected that X15695 did not only potently inhibit E 2 -induced gene transcription but simultaneously increased the expression of genes negatively regulated by E 2 , including genes involved in cell-cycle arrest and in the induction of apoptosis. For example, E 2 -mediated downregulation of expression of CDKNA and BBC in the MCF-7 cells was dose-dependently upregulated by X15695 (Fig. 2C and D). In addition, the expression of CDKNA but not BBC was upregulated by X15695 in the absence of E 2 ( Fig. 2E and F), making CDKNA a gene under dual control mechanisms by E 2 and X15695. In contrast, the expression of the p53 downstream target gene BAX, was not regulated by E 2 but was transcriptionally enhanced by X15695 (Fig. 2G and H). These findings identified X15695 as a compound that targets both the ERα and p53 signaling pathways and can therefore control the actions of the p53-ER regulatory loop described in breast cancers (28).

Mechanism of Downregulation of ERα and Upregulation of p53
We sought to investigate the possible mechanisms of X15695-mediated regulation of ERα and p53 signaling pathways in the breast cancer cells. In immunoblotting studies, X15695 dose-dependently decreased ERα level in both cell lines (Fig. 3A and B). In the MCF-7 cells, ERα staining in the absence and presence of E 2 was strongly reduced by X15695 treatment (100 nmol/L and  1 μmol/L) in the cytoplasm and in the nucleus (Fig. 3C). A similar downregulation in ERα level was observed in immunofluorescence experiments in T47D cells (Fig. 3D), albeit in full medium without additional hormone.
To determine how X15695 downregulates ERα level, MCF-7 cells were treated with cycloheximide to inhibit de novo protein synthesis and the degradation kinetics of the steady state population of the receptor was determined. This showed that while ERα was relatively stable (>120 minutes) in the absence of X15695, its half-life was reduced to about 60 minutes in the presence of X15695 ( Fig. 3E and F), suggesting destabilization of ERα by X15695.
In contrast to the decrease in ERα levels in both MCF-7 and T47D cells, X15695 differentially regulated the level of p53 in the two cell lines. In Western blot experiments, p53 level was significantly increased by X15695 in MCF-7 but not in T47D cells (Fig. 4A and B). Note that MCF-7 cells express a wild-type p53 while T47D cells express a mutated p53 (L194F) which may account for the difference in regulation by X15695. The upregulation of p53 expression in the MCF-7 cells did not occur at the RNA level as demonstrated in qRT-PCR experiments ( Fig. 4C and D), suggesting that the regulation occurred at the protein level. As protein turnover is a dynamic process controlled by the rate of protein synthesis and degradation, inhibition of protein degradation should provide information on the contribution of the latter process to the accumulation of p53. X15695 treatment in the absence of the proteasomal inhibitor MG132 led to an accumulation of p53 in the MCF-7 cells but in the presence of MG132 (Fig. 4E), the basal level of p53 was increased and no further upregulation by X15695 was observed. Quantification of the effect of MG132 and X15695 on p53 level presented in Fig. 4F showed that X15695 functions by inhibiting proteasomal degradation of p53. In T47D cells where the proteasomal degradation pathway is reportedly nonfunctional (29,30), mutant p53 L194F accumulated in the non-treated cells and its level was unaltered by X15695 administration (Fig. 4B).
To further understand the mechanism leading to p53 accumulation, we analyzed the expression level of MDM2, an E3 ubiquitin ligase that directs p53 degradation by the proteasome machinery (31,32). Immunoblot analysis showed that in MCF-7 cells, X15695 slightly upregulated MDM2 protein level (Fig. 4G) rather than downregulating it to account of the negative effect of X15695 on p53 degradation. Therefore, X15695's action on p53 in MCF-7 cells is independent of MDM2. In T47D cells, neither MDM2 nor p53 level was affected by X15695 treatment (Fig. 4H). An alternative pathway for the upregulation of p53 level is through reactive oxygen species (ROS; ref. 33). This pathway is among the top processes identified in the GSEA plots of transcripts from both MCF-7 and T47D cells (Supplementary Fig. S1). Besides, increased ROS generation has been reported following BAG1 knockout in MCF-7 cells (34). We therefore measured ROS production in MCF-7 and T47D cells after X15695 treatment using the fluorescent probe H2DCF-DA and showed a significant dose-dependent ROS production in MCF-7 but not in T47D cells ( Fig. 4I and J). ROS production in MCF-7 cells possibly contributed in part to the upregulation of p53, as the ROS scavenger NAC at 100 μmol/L inhibited both the X15695-mediated increase in ROS production and upregulation of p53 level ( Fig. 4K and L).
In addition to increasing p53 expression level, X15695 also regulated the cellular localization of this protein. In immunofluorescence studies in MCF-7 cells, p53 was found mainly in the cytoplasm but accumulated in the nuclear compartment as early as 8 hours after X15695 treatment (Fig. 4M). In T47D cells, p53 was already nuclear in the absence of X15695 in agreement with published information (35) and its cellular localization was not further altered by X15695 treatment (Fig. 4N).

X15695 Disrupts p53-Mortalin Interaction
The cytoplasmic localization of wild-type p53 in tumor cells is reported to be due to sequestration by GRP75 (a.k.a mortalin), a member of the HSP70 molecular chaperone family (36,37). In immunofluorescence experiments in MCF-7 cells, we confirmed the cytoplasmic colocalization of p53 and mortalin but after X15695 treatment, p53 was translocated to the nucleus while mortalin assumed a perinuclear localization (Supplementary Fig. S3A). In contrast, p53 L194F was nuclear in T47D cells and its cellular localization was not altered by X15695 treatment (Supplementary Fig. S3B).
To account for the alteration in cellular localization of p53 in MCF-7 cells, we showed in co-immunoprecipitation experiments that in the absence of X15695, p53 was in a complex with mortalin but this interaction was decreased by X15695 treatment (Fig. 5A). Mortalin itself is reported to interact with BAG1 (38) and we showed that the interaction of all three isoforms of BAG1 with mortalin was also disrupted by X15695 treatment (Fig. 5B). In T47D cells that express a mutant p53, tumorigenesis is reported to proceed through a gain-offunction (GOF) mechanism involving nuclear interaction of mutant p53 with BAG2 and BAG5 (39,40). In co-immunoprecipitation experiments, we also showed that X15695 disrupted the interaction of BAG2 but not BAG5 with mutant p53 in the T47D cells (Fig. 5C), pointing to a possible action of X15695 in the disruption of BAG/p53 complexes in breast cancer cells.
To further confirm a role of p53 in X15695 action, we transfected MCF-7 and T47D cells with control and a mixture of siRNAs against p53 and analyzed the inhibitory effect of X15695 on cell survival in the p53 knockout cells. If p53 were to play a role in the action of X15695, the antiproliferation action of this compound is expected to be attenuated by the knockdown of p53. We showed a near complete knockdown of p53 in MCF-7 cells but not in the T47D cells (Fig. 5D). As a consequence, decreased cell survival mediated by X15695 was significantly attenuated in the p53 siRNA-transfected MCF-7 cells but this effect was somewhat compromised in the p53 knockdown T47D cells (Fig. 5E). These studies demonstrated the involvement of p53 in the inhibitory action of X15695.

X15695 Regulates Cell-cycle Progression and Apoptosis
One of the functions of p53 in the nucleus is to regulate the expression of downstream targets such as p21, GADD45A, PUMA, and BAX that control cell-cycle progression and apoptosis (28). As genes encoding these proteins are significantly activated by X15695 in the breast cancer cells (Supplementary Fig. S2E and S2F), X15695 would be expected to induce cell-cycle arrest and apoptosis. Flow cytometry experiments carried out with 7-AAD after treating MCF-7 and T47D cells with X15695 showed a G 1 -S-phase arrest in MCF-7 cells (Fig. 5F) while a G 2 -M cell-cycle arrest was observed in the T47D cells (Fig.   5G). Double staining with 7-AAD and Annexin V used for apoptosis evaluation revealed increased number of the cells at the late stages of apoptosis upon X15695 treatment in both MCF-7 and T47D cells (Fig. 5H and I).

X15695 Regulation of Tamoxifen-resistant MCF-7 Cells
The efficacy of X15695 as an ERα degrader was assessed by comparing it with fulvestrant, a FDA-approved pure ERα antagonist (9). ERα level was rapidly downregulated by fulvestrant within 4 to 8 hours of treatment recovering gradually thereafter while X15695 showed a rather slow but more sustained downregulation lasting over 24 to 48 hours (Fig. 6A). Furthermore, both compounds at 1 nmol/L were sufficient to destabilize ERα in 8 hours (Fig. 6B). The two compounds were also compared on the basis of their ability to decrease the viability of tamoxifen resistant MCF-7 cells (TRMCF-7). Here, X15695 and fulvestrant showed comparable activities as opposed to tamoxifen that was ineffective (Fig. 6C). In clonal expansion experiments with TRMCF-7 cells, AACRJournals.org Cancer Res Commun; 3(7) July 2023 tamoxifen was again inactive while X15695 and fulvestrant were both active, with fulvestrant being slightly more efficacious than X15695 (Fig. 6D and E).
Although oral administration is a more preferred route of drug administration, the low aqueous solubility of fulvestrant requires it to be administered intramuscularly (6)(7)(8). X15695 differs from fulvestrant in that it was found to significantly decrease tumor volume and weight within 2 weeks after oral application to a mouse xenograft tumor model (30 mg/kg body weight daily; Fig. 6F and H). In that study, X15695 was well tolerated and did not cause any weight loss or other signs of host toxicity (Fig. 6I). Western blot analysis of lysates from the tumors after the treatment period showed a significant decrease in ERα level and an increase in p53 expression, in line with the results in the cell culture experiments ( Fig. 6J and L).

Regulation of AR + Prostate Cancer Cells by Imidazopyridine Derivatives
As the imidazopyridines analyzed for the inhibition of clonal expansion of ER + breast cancer cells also inhibited proliferation of AR + prostate cancer cells (Supplementary Table S1), it is possibility that these compounds function through a common antitumor pathway. We therefore first investigated whether BAG1 played a role in the action of X15695 in the prostate tumor cells using BAG1 knockdown LNCaP prostate cancer cells. Two BAG1 shRNA clones that showed a clear reduction in BAG1 level compared with the control clone (Fig. 7A) showed a decrease in the IC 50 concentration for inhibition of clonal expansion (Fig. 7B), suggesting a contribution of BAG1 to proliferation of these cells. Unlike the breast cancer studies, treatment with X15695 did not strongly downregulate AR level in LNCaP prostate cancer cells nor in the castration-resistant prostate cancer (CRPC) cell line 22Rv.1 (Fig. 7C). Nonetheless, transcriptomics experiments carried out with LNCaP cells showed an X15695-mediated attenuation of androgen response. Specifically, GSEA showed that in the presence of DHT, androgen signaling pathway, G 2 -M cell-cycle arrest, and E2F pathways were attenuated by X15695, while only the latter two were attenuated in the absence of DHT (Fig. 7D and F). RT-PCR studies confirmed that X15695 downregulated a select number of classical AR target genes (KLK, FKBP, F) and androgen-induced ROS genes (MICAL, SAT, DUOX) previously identified as BAG1 sensitive targets (20) and also identified in the present RNA-seq study ( Fig. 7D and G). X15695 was found to be a better inhibitor of AR-target gene expression compared with the five other imidazopyridines identified previously (compare Fig. 7G with Supplementary  Fig. S4). However, in studies to determine the ability of the imidazopyridines to stabilize p53 expression in three different prostate cancer cell lines (22Rv.1 LNCaP, LAPC-4), none of the compounds strongly stabilized p53 (Supplementary Fig. S5A). However, we focused on X15695 for further analysis in the prostate cancer cells for better comparison with the studies in the breast cancer cells.  S5B). To determine whether p53 plays a role in the inhibitory action of X15695 in the prostate cancer cells, an attenuation of the X15695-mediated decrease in viability of the three prostate cancer cells was determined after p53 knockdown by siRNA transfection (Fig. 7H). Decreased viability observed after X15695 treatment was significantly compromised in all three cell lines transfected with p53 siRNA (Fig. 7I), indicating a contribution of p53 to the growth inhibitory action of X15695 in these cells. Flow cytometric analysis was also determined to assess the downstream effects of p53 on cell-cycle arrest or the induction of apoptosis in the prostate cancer cells following treatment with X15695. We found out that X15695 induced G 1 -S-phase arrest in the LNCaP cells and a G 2 -M arrest in the LAPC-4 cells ( Fig. 8A and B). No apoptotic effect of X15695 was identified in either prostate cancer cell type ( Supplementary Fig. S6), indicating an effect mediated primarily by the cell-cycle arrest.

X15695 Inhibits Prostate Tumor Growth in a Mouse Xenograft Model
To determine whether the cell-cycle block and the antiandrogen action of X15695 are sufficient to inhibit prostate tumor growth, we carried out mouse tumor xenograft experiments using LAPC-4 cells. We compared the action of X15695 (10 and 30 mg/kg) with the classical antiandrogen enzalutamide (10 mg/kg) over 42 days. X15695 effectively inhibited tumor growth over vehicle upon oral administration (30 mg/kg/day), albeit less effectively compared with enzalutamide (10 mg/kg/day; Fig. 8C and D), identifying X15695 as a weaker AR antagonist in this tumor model. As with the MCF-7 cell xenograft experiment, X15695 was well tolerated in the study with no signs of toxicity or weight loss (Fig. 8E).

X15695 is Selective for ER + Breast Cancer
The negative action of X15695 on proliferation of AR + prostate and ER + breast cancer, posed the question whether the proliferation of other tumor cells could be inhibited by this compound. X15695's effect on the clonal expansion of cervical, lung, and osteosarcoma (HeLa, A549, and U2OS) cells was compared with its effect on MCF-7 cells. While X15695 dose-dependently inhibited the clonal expansion of MCF-7 cells, it did not have any significant effect on the other tumors cell lines (Fig. 8F).
Collectively, these results demonstrate that X15695 inhibits proliferation of breast cancer cells and prostate cancer cells but its strong effect on the  degradation of ERα combined with the reactivation of p53 makes it a superior inhibitor of ER + breast cancer cells than AR + prostate cancer cells. These effects combined with its lack of action in inhibiting proliferation of other cancer cells warrants X15695 to be classified as an oral SERD.

Discussion
In this study, a compound with an imidazopyridine scaffold X15695 was shown to strongly downregulate ERα activity and to a lower extent AR action and to reactivate wild-type and mutant p53. As a consequence, X15695 inhibited proliferation of ER + breast cancer and AR + prostate cancer cells in vitro and in vivo in xenograft models. However, in the in vivo studies, X15695 was more efficacious in inhibiting breast cancer than prostate cancer cell proliferation. X15695 action was selective as no inhibition of proliferation was detected in steroid receptor negative breast or prostate cancer cells or other tumor cells analyzed that lack expression of gonadal steroid receptors.
Imidazopyridine-based compounds to which X15695 belongs have gained significant attention in medicinal chemistry due to their frequent occurrence in a large number of marketed drug formulations and drug candidates. These compounds have a wide variety of biological and pharmacologic activities such as antimycobacterial, antidiabetic, antiviral, and anticancer activities (41). For anticancer action, a library of amide derivatives of imidazopyridine has been shown to be highly potent in inhibiting the proliferation of breast (MCF-7, MDA MB-231), lung (A549), and prostate (DU145) cancer cell lines (42). Recently, imidazopyridine compounds that inhibit PI3K/Akt and the proliferation of AR + and AR -CRPC cells have been described (43,44).
However, the high concentrations of these compounds (up to 10 μmol/L) required for the inhibition of CRPC proliferation question their on-target mode of action.
Several in vitro experimental observations have shown that the anticancer effect of the imidazopyridine compounds results mainly from their inhibitory effects on six main molecular targets: PI3K/Akt, centromere-associated protein E (CENP-E), insulin-like growth factor-1 receptor (IGF-1R), cyclin-dependent kinases (CDK), tubulin polymerization, and hepatocyte growth factor receptor (45). So far, steroid receptors and p53 have not been identified as molecular targets for imidazopyridines.
We have shown that X15695 inhibits proliferation of tumor cells that express ERα and AR as well as the transactivation function of these two receptors but its effect is more profound on ERα compared with the AR-expressing to function as nucleotide exchange factors (46). BAG domain/HSP70/HSC70 complexes have also been reported to interact with several client proteins to mediate the biological action of these proteins and small molecules that disrupt these interactions have been suggested to be potential therapeutic drugs (47).
We have previously reported that A4B17 that is structurally related to X15695 disrupts interaction of BAG1L with the N-terminus of the AR, that is otherwise required for the activity of this receptor (19). A possibility exists that X15695 also disrupts interaction of BAG1 with ERα (16) to account for its inhibitory action on E 2 target gene expression in the ER + breast cancer cells. We have also shown that X15695 regulates p53 action by disrupting the interactions of mortalin with wild-type p53 and also of BAG1 with mortalin. Although the disruption of mortalin-p53 interaction by X15695 could be a result of its action on mortalin-BAG1 interaction, we cannot rule out an independent direct action of X15695 on mortalin-p53 interaction. We showed that treatment with X15695 disrupts the cytoplasmic retention of p53 by mortalin leading to nuclear translocation of p53 and the activation of the cell-cycle regulator p21 and the apoptotic action of p53. Reactivation of p53 has become a challenge in drug discovery programs for anticancer therapies. Several small-molecule inhibitors are reported to disrupt mortalin-p53 interaction to reactivate p53. Among them is Mortaparib plus that inhibits the proliferation of MCF-7 cells. However, Mortaparib plus reactivates only wild-type p53 and not mutant p53 in T47D cells (35,48). Other compounds such as PRIMA-1 and its methyl analog Eprenetapopt (APR-246) are reported to reactivate mutant p53 (49,50). APR-246, for example, is a first-in-class small molecule that restored wild-type p53 functions in TP-mutant cells and is currently in phase II clinical trials (51). Unlike Mortaparib plus and APR-246, we have shown in this study that X15695 reactivates both wild-type and mutant p53. With mutant p53, we have shown that it disrupts its interaction with BAG2 and the GOF action of mutant p53. These findings together speak in favor of X15695 being a regulator of AACRJournals.org Cancer Res Commun; 3(7) July 2023 protein-protein interactions involving the HSP70/HSC70 chaperone/cochaperone complex.
Two main types of mutant "hotspot" sites exist in p53: contact mutants (R273H, R248Q, and R248W) and conformational mutants (R175H, G245S, R249S, and R282H), both affecting the DNA-binding activity and the transcriptional downstream targets of p53 (52). Contact mutants generally produce structural changes in the p53 protein that directly affect DNA binding, while conformational mutants generate structural changes related to protein folding, but both types of mutants have GOF activities (53). So far BAG2 and BAG5 are reported to bind both types of mutant p53 (39,40) for their tumorigenic activities. From our results in this work on X15695-mediated disruption of BAG2-mutp53 interaction, the proliferation of a large number of tumor cells with mutant p53 status would be expected to be inhibited by X15695. This was evidently not the case as X15695 did not significantly alter the proliferation of the ER − breast cancer cell line MDA-MB231 that expresses mutp53 (R280K), or the AR − prostate tumor cell line DU145 that expresses mutp53 (P223L/V274F). These findings suggest that other regulatory factors in addition to p53 are required for X15695 to exert its maximum inhibitory action. These other regulatory factors could be the steroid receptors.
One of the important findings of this work is that X15695 downregulates ERα levels which puts it in the class of ER degraders to which the clinically approved antiestrogen fulvestrant belongs. However, unlike fulvestrant that requires intramuscular injection (9), X15695, when given orally, reduces ERα levels in two weeks in a xenograft tumor model. A further advantage of X15695 is that it induces cell-cycle arrest and apoptosis to enhance its antitumor action in breast cancer cells without the need for the combination drug therapy suggested for fulvestrant. To improve the anticancer action of fulvestrant, it is recommended to have it administered with other targeted therapies such as CDK4/CDK6 inhibitors (e.g., Palbociclib) that promote cell-cycle block at the G 1 -S phase (54). X15695 decreases ERα levels and induces cell cycle arrest and apoptosis on its own. The multi-targeting of factors by one compound characterizes the uniqueness of action of X15695 in this study. The actions of X15695 described here identify X15695 as a selective oral ERα degrader that warrants further development as an ER + breast cancer therapeutic.