BODIPY-17α-ethynylestradiol conjugates: Synthesis,fluorescence properties and receptor binding affinities

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.     

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+?breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.     

Evaluation of binding and antagonism/downregulation of brilanestrant molecule in estrogen receptor-α via quantum mechanics/molecular mechanics,molecular dynamics and binding free energy calculations

Abstract

The resistance to the endocrine therapy of breast cancer leads to the emergence of new class of drugs that downregulates the estrogen receptor action known as selective estrogen receptor downregulators (SERDs). The first approved SERD is fluvestrant; after this, there are several downregulators evolved and are in clinical trials, in which the brilanestrant (BRI) molecule shows nM range of binding affinity and efficacy. In the present study, to understand the binding nature of BRI molecule in the active site of ERα, the molecular docking analysis has been performed. Further, the QM/MM calculations were performed for the BRI–ERα complex to analyze the charge density distribution of intermolecular interactions. The molecular dynamics (MD) simulation was employed to understand the stability and binding mechanism of BRI molecule through root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF) and binding free energy calculations. From the MD simulation trajectory analysis, the alterations of Helix12 conformation and the key residue (Lys529), which is responsible for the ERα downregulation, have been identified. Further, the interaction between the H3 and H12 regions was identified for the antagonism of BRI molecule. The current study led us to understand the binding mechanism, antagonism and downregulation of BRI molecule, and this knowledge is essential to design novel SERDs for the treatment of endocrine-resistant positive breast cancer.

Communicated by Ramaswamy H. Sarma     

Structure-activity relationship study of estrogen receptor down-regulators with a diphenylmethane skeleton

Selective estrogen receptor (ER) down-regulators (SERDs) are pure ER antagonists that also induce ER degradation upon binding to the receptor. Although SERDs have been developed for the treatment of ER-positive breast cancers for nearly a decade, their precise mechanism(s) of action and structure-activity relationship are still unclear. Generally, Western blotting is used to examine the effects of SERDs on ER protein levels, but the methodology is low-throughput and not quantitative. Here, we describe a quantitative, high-throughput, luciferase-based assay for the evaluation of SERDs activity. For this purpose, we established stable recombinant HEK-293 cell lines expressing ERα fused with emerald luciferase. We also designed and synthesized new diphenylmethane derivatives as candidate SERDs, and evaluated their SERDs activity using the developed system in order to examine their structure-activity relationship, taking EC₅₀ as a measure of potency, and E_max as a measure of efficacy.     

The enhanced antiproliferative response to combined treatment of trichostatin A with raloxifene in MCF-7 breast cancer cells and its relevance to estrogen receptor β expression

Antiestrogen is one type of the endocrine therapeutic agents for estrogen receptor α (ERα)-positive breast cancer. Unfortunately, this treatment alone is insufficient. Here we reported a novel potential anticancer strategy by using histone deacetylase (HDAC) inhibitor to enhance the action of endocrine therapy in ERα-positive breast cancer cell. The well-described HDAC inhibitor, trichostatin A (TSA), and antiestrogen raloxifene were found to, respectively, inhibit E2-induced proliferation of MCF-7 breast cancer cell in a dose-responsive and time-dependent manner. TSA and raloxifene enhanced the antiproliferative activity of each other by promoting cell death via apoptosis and cell cycle arrest. Thus, they displayed better antiproliferative effects in combined treatment than that with either agent alone. The expression level of estrogen receptor β (ERβ) showed a marked increase after TSA or/and raloxifene treatment. Treatments with TSA or/and raloxifene resulting in the up-regulation of ERβ are in accordance with the antiproliferative effects of the two agents. Furthermore, the over-expression of ERβ by adenovirus delivery could inhibit the proliferation of MCF-7 tumor cells and drastically enhanced the antiproliferative effects of TSA and raloxifene. These results demonstrated that the interference of ERβ on the antiproliferative effects of HDAC inhibitor and antiestrogen constitutes a promising approach for breast cancer treatment.     

Selective estrogen receptor degraders with novel structural motifs induce regression in a tamoxifen-resistant breast cancer xenograft

Potent estrogen receptor ligands typically contain a phenolic hydrogen-bond donor. The indazole of the selective estrogen receptor degrader (SERD) ARN-810 is believed to mimic this. Disclosed herein is the discovery of ARN-810 analogs which lack this hydrogen-bond donor. These SERDs induced tumor regression in a tamoxifen-resistant breast cancer xenograft, demonstrating that the indazole NH is not necessary for robust ER-modulation and anti-tumor activity.     

Synthesis and biological evaluation of 4,6-diaryl-2-pyrimidinamine derivatives as anti-breast cancer agents

Breast cancer is the most frequently diagnosed cancers and the leading causes of cancer death among females worldwide. Estrogen receptor positive has been identified as the predominant internal reasons, involving in more than 70% breast cancer patients and SERMs which competes with estradiol for the binding to ERα in breast tissue are widely used in the treatment of ER+ breast cancer, such as tamoxifen, raloxifene. However, many SERMs may cause negative side effects due to their estrogenic activity in other tissues and approximate 50% of patients with ER-positive tumors either initially do not respond or become resistant to these drugs. Here, a series of designed 4,6-diaryl-2-pyrimidinamine derivatives had been synthesized to treat estrogen receptor positive breast cancer by simultaneously antagonizing ER and inhibiting VEGFR-2. Bioactivity evaluation showed that these compounds could significantly inhibit the proliferation of MCF-7, HUVEC and Ishikawa cells. Further studies identified compound III-3A could antagonize against estrogen action and inhibit the phosphorylation of VEGFR-2 as well as inhibit angiogenesis in vivo. The results indicated designed 4,6-diaryl-2-pyrimidinamine derivatives can be used to further study as anti-breast cancer drugs.     

TWIST represses estrogen receptor-alpha expression by recruiting the NuRD protein complex in breast cancer cells

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.     

Synthesis and biological evaluation of 2,3-diaryl isoquinolinone derivatives as anti-breast cancer agents targeting ERα and VEGFR-2

The estrogen receptor α is recognized as important pharmaceutical target for breast cancer therapy, and vascular endothelial growth factor receptors (VEGFRs) play important roles in tumor angiogenesis including breast cancer. A series of 2,3-diaryl isoquinolinone derivatives were designed and synthesized targeting both estrogen receptor α (ERα) and VEGFR-2. Bioactivity evaluation showed that compounds 7c, 7d and 7f exhibited significant anti-proliferative and anti-angiogenesis activities via ERα and VEGFR-2 dependent mechanisms.     

Identification of 10-dehydrooxyglycyuralin E as a selective human estrogen receptor alpha partial agonist

Selective estrogen receptor modulators (SERMs) act as either agonist or antagonist of estrogen receptor (ER) in a tissue selective manner and have been used in several diseases such as breast cancer, postmenopausal syndrome, osteoporosis, and cardiovascular diseases. However, current SERMs may also increase the risk of serious side effects and trigger drug resistance. Herein, a screening program, that was designed to search for novel SERMs, resulted in the identification of a series of 2-arylbenzofuran-containing compounds that are ligands for ERα, when applying the Gaussia-luciferase reporter assay. One of these compounds, 10-dehydrooxyglycyuralin E (T9) was chemically synthesized. T9 showed anti-estrogenic/proliferative activity in ERα-positive breast cancer cells. Pretreatment of T9 prevented the mRNA expression of GREB1, which is an estrogen response gene. Furthermore, by an in silico docking simulation study we demonstrated that T9 showed interactions directly to ERα. Taken together, these results demonstrated that T9 is a candidate of SERMs and a useful seed compound for the foundation of the selective activity of SERMs.     

Identification of selective estrogen receptor modulators by their gene expression fingerprints

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.     

Recent advances in peptidomimetics antagonists targeting estrogen receptor α-coactivator interaction in cancer therapy

Estrogen receptor α (ERα) is a crucial target for ERα positive breast cancer treatment. Previous drug discovery efforts were focused on developing inhibitors that targeted the canonical ligand binding pockets of the ligand binding domain (LBD) of ERα. However, significant percentage of patients developed cancer relapse with drug-resistance. ERα peptidomimetic modulators have been considered as promising treatments for drug resistant breast cancers as they are targeting ERα-coactivator interacting interface instead of the ligand binding pocket of ERα. Herein, we reviewed the recent development of ERα peptidomimetics antagonists.     

Synthesis and evaluation of 17α-triazolyl and 9α-cyano derivatives of estradiol

A variety of 17α-triazolyl and 9α-cyano derivatives of estradiol were prepared and evaluated for binding to human ERβ in both a TR-FRET assay, as well as ERβ and ERα agonism in cell-based functional assays. 9α-Cyanoestradiol (5) was nearly equipotent as estradiol as an agonist for both ERβ and ERα. The potency of the 17α-triazolylestradiol analogs is considerably more variable and depends on the nature of the 4-substituent of the triazole ring. While rigid protein docking simulations exhibited significant steric clashing, induced fit docking providing more protein flexibility revealed that the triazole linker of analogs 2d and 2e extends outside of the traditional ligand binding domain with the benzene ring located in the loop connecting helix 11 to helix 12.     

Synthesis of 17beta-estradiol platinum(II) complexes: biological evaluation on breast cancer cell lines

The synthesis of a novel series of 17beta-estradiol-linked platinum(II) complexes is described. The new molecules are linked with an alkyl chain at position 16alpha of the steroid nucleus and bear a 16beta-hydroxymethyl side chain. They are made from estrone in five chemical steps with an overall yield exceeding 28%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER+ and ER-) human breast cancers. The derivatives incorporating a 2-(2'-aminoethyl)pyridine ligand displayed good activity against the cell lines particularly when the connecting arm is 10 carbon atoms long.     

Design and synthesis of silicon-containing steroid sulfatase inhibitors possessing pro-estrogen antagonistic character

Steroid sulfatase (STS) is a potential target for treatment of postmenopausal hormone-dependent breast cancer. Several steroidal STS inhibitors have been reported, but steroidal compounds are difficult to optimize and may interact with other targets. On the other hand, we have shown that diphenylmethane (DPM) derivatives act as estrogen receptor (ER) agonists and antagonists. Here, we aimed to design and synthesize non-steroidal DPM-type STS inhibitors that would also serve as pro-estrogen antagonists, releasing a metabolite with ERα-antagonistic activity upon hydrolysis by STS. We synthesized a series of compounds and evaluated their biological activities by means of STS-inhibitory activity assay and ER reporter gene assay. Among them, silicon-containing compound 16a showed strong STS-inhibitory activity (IC₅₀ = 0.17 μM). Further, its putative metabolite (12a) exhibited potent ERα-antagonistic activity (IC₅₀ = 29.7 nM).     

Indole-3-carbinol and its N-alkoxy derivatives preferentially target ERα-positive breast cancer cells

Indole-3-carbinol (I3C) is a natural anti-carcinogenic compound found at high concentrations in Brassica vegetables. I3C was recently reported to inhibit neutrophil elastase (NE) activity, while consequently limiting the proteolytic processing of full length cyclin E into pro-tumorigenic low molecular weight cyclin E (LMW-E). In this study, we hypothesized that inhibition of NE activity and resultant LMW-E generation is critical to the anti-tumor effects of I3C. LMW-E was predominately expressed by ERα-negative breast cancer cell lines. However, ERα-positive cell lines demonstrated the greatest sensitivity to the anti-tumor effects of I3C and its more potent N-alkoxy derivatives. We found that I3C was incapable of inhibiting NE activity or the generation of LMW-E. Therefore, this pathway did not contribute to the anti-tumor activity of I3C. Gene expression analyzes identified ligand-activated aryl hydrocarbon receptor (AhR), which mediated sensitivity to the anti-tumor effects of I3C in ERα-positive MCF-7 cells. In this model system, the reactive oxygen species (ROS)-induced upregulation of ATF-3 and pro-apoptotic BH3-only proteins (e.g. NOXA) contributed to the sensitivity of ERα-positive breast cancer cells to the anti-tumor effects of I3C. Overexpression of ERα in MDA-MB-231 cells, which normally lack ERα expression, increased sensitivity to the anti-tumor effects of I3C, demonstrating a direct role for ERα in mediating the sensitivity of breast cancer cell lines to I3C. Our results suggest that ERα signaling amplified the pro-apoptotic effect of I3C-induced AhR signaling in luminal breast cancer cell lines, which was mediated in part through oxidative stress induced upregulation of ATF-3 and downstream BH3-only proteins.