ERRγ; ERRβ; DY131 selectively targets estrogen-related receptors ERRβ and ERRγ. It exhibits no significant agonistic activity on ERRα, ERα, or ERβ . The compound demonstrates an EC50 of approximately 130 nM for ERRβ/γ activation . Additionally, DY131 also inhibits Smo (Smoothened) signaling, with an IC50 of 0.8 μM for inhibiting Shh-induced Smo::EGFP accumulation, and approximately 2 μM for inhibiting SAG-induced Gli transcriptional activity in primary cilia .
DY131 is a selective agonist ligand for estrogen-related receptors ERRβ/γ; it has no effect on ERRα, estrogen receptors α and β (ERα/β) [1]
DY131 exerts antimitotic effects in breast cancer cells via ERRβ2 splice variant [2]

DY131 is a ligand that is unique to ERRβ/γ that exhibits preferential selectivity for ERRγ at lower concentrations. The related receptors ERα, ERβ, and ERRα are unaffected by it [1]. In a variety of breast cancer cell lines, DY131 suppresses growth, leading to a bimodal cell cycle arrest and the p38 stress kinase pathway being involved in cell death. DY131 postpones the transition from prophase to anaphase, while ERRβ2 facilitates the block in G2/M. Mitotic spindle defects are caused by DY131 treatment, which causes multi- and monopolar spindles to appear. ERRβ2, a cytosolic protein that also localizes to centrosomes, is also involved[2].

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1. DY131 effectively and selectively activates ERRβ/γ, with no agonistic or antagonistic effects on structurally related ERRα, ERα, or ERβ [1]
2. DY131 inhibits growth in a diverse panel of breast cancer cell lines (including MDA-MB-231, MCF7, HCC1806) in a concentration-dependent manner, while its effect on non-transformed breast epithelial cell lines was not specified; crystal violet staining confirmed growth inhibition over time (n=6, two-way ANOVA with Bonferroni post-tests, significant vs. DMSO control at Day 10/11) [2]
3. DY131 induces apoptotic cell death in breast cancer cells: flow cytometry showed increased subG1 DNA content (fragmented DNA) after 24 h exposure (n=3–5, one-way ANOVA with Tukey's post-test); Annexin V/PI staining confirmed dose-dependent increase in apoptotic cells after 12–24 h treatment (n=3–5, one-way ANOVA with Tukey's post-test); Western blot detected PARP cleavage and γH2AX upregulation (doxorubicin as positive control) [2]
4. DY131 induces bimodal cell cycle arrest (G1 and G2/M phases) in breast cancer cells: flow cytometry showed significant changes in G1, S, and G2/M phase distribution after 24 h exposure (n=3–5, one-way ANOVA with Tukey's post-test); Western blot confirmed upregulation of p21 and phosphorylation of Histone H3 (Serine 10) [2]
5. DY131-induced cell death is dependent on p38 MAPK activation: Western blot detected increased phosphorylated p38 (p-p38) in treated cells (densitometry: n=3, one-way ANOVA with Tukey's post-test); pre-treatment with p38 inhibitor SB203580 abrogated DY131-induced subG1 DNA content (n=3, two-way ANOVA with Bonferroni post-test) but did not affect G2/M arrest [2]
6. ERRβ2 facilitates DY131-induced G2/M arrest: transient transfection of MCF7 cells with ERRβ2 cDNA enhanced DY131-mediated Histone H3 phosphorylation (Serine 10); DY131 delays progression from prophase to anaphase in MCF7 cells stably expressing GFP-H2B (n=4–11 cells, one-way ANOVA with Tukey's post-test) [2]
7. DY131 causes mitotic spindle defects in breast cancer cells (HCC1806, MDA-MB-231): immunostaining for γ-tubulin/β-tubulin/DAPI showed increased monopolar and multipolar spindles after 24 h treatment with 5 μM DY131 (n=3, chi squared test) [2]
8. DY131 does not induce conventional DNA damage response or bind DNA directly: Western blot showed no activation of ATM signaling pathway; surface plasmon resonance (BIAcore) confirmed no binding of DY131 to dsDNA or ssDNA (mitoxantrone as positive control, experiment performed twice) [2]
9. DY131 has no transcription factor activity on ERRβ2 in breast cancer cells: luciferase reporter assay (ERRE-luciferase) showed no significant change in luciferase activity in MDA-MB-231/MCF7 cells treated with DY131 (n=3, two-way ANOVA with Bonferroni post-test) [2]
10. Clonogenic survival assay: MCF7 and MDA-MB-231 cells exposed to DY131 for 24 h showed reduced colony formation after 13 d culture[2]

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DY131 (0.1-30 μM; 5 days) treatment inhibits cell proliferation and reduces BrdUrd-positive cells in both LNCaP-ERRγ and LNCaP cells in a dose-dependent manner, with greater inhibition observed in LNCaP-ERRγ clones . In breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-468), all cell lines were completely growth-inhibited by 10 μM DY131, while MCF7 and MDA-MB-231 were significantly inhibited by 5 μM . In clonogenic survival assays, MDA-MB-231 showed a dose-dependent reduction in colony formation after a single 24-hour exposure to DY131 . Moreover, all breast cancer cell lines showed a significant, dose-dependent increase in the subG1 fraction, indicating apoptosis induction . In ECa109 and TE1 cells, DY131 (10 μM; 48 hours) inhibited cell proliferation and glycolysis activity . In placental villous explants, DY131 (20 μM or 50 μM; 48 hours) increased the expression of ESRRG signaling pathways and prevented abnormal cell turnover induced by hypoxia .

In a study using mature male C57BL/6 mice (8-10 weeks old; ~24.3 g), DY131 (5 μg/kg; subcutaneous injection; every other day for 12 days) treatment increased the expression of P450 side-chain cleavage enzyme (P450scc), steroidogenic acute regulatory protein (StAR), and HMG-CoA reductase (HMGCR), while decreasing hormone-sensitive lipase (HSL) expression . In a rat subarachnoid hemorrhage model, DY131 (6 mg/kg; intraperitoneal injection; administered 1 hour after SAH and additional doses on days 2 and 3) significantly improved neurological deficits and reduced oxidative stress and neuronal apoptosis . In a mouse model of LPS-induced acute liver injury (C57BL/6J mice), DY131 (5 mg/kg/day; intraperitoneal injection; once daily for 3 days) pretreatment ameliorated liver injury, oxidative stress, inflammation, and apoptosis . In a xenograft model of esophageal squamous cell carcinoma (BALB/c nude mice), DY131 (80 mg/kg; intraperitoneal injection; every 2 days for 25 days) inhibited tumor growth and enhanced anti-PD-1 therapy efficacy .

1. Receptor activation assay (ERRβ/γ, ERRα, ERα/β): Cells were transfected with receptor cDNA and relevant reporter constructs, then treated with DY131 (concentration not specified) or DMSO control for 18–20 h; luciferase activity was measured to assess receptor activation, with GSK4716 as a reference compound (n=3, two-way ANOVA with Bonferroni post-test) [1,2]
2. Surface plasmon resonance (BIAcore) assay: DY131, GSK4716, or mitoxantrone (positive control) were injected over dsDNA/ssDNA immobilized on sensor chips; peak Relative Unit (RU) values were recorded after 60 s injection to evaluate direct DNA binding (experiment performed twice, no binding of DY131 detected) [2]

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The specific non-cell-based enzyme/receptor binding assay protocol for DY131 is not detailed in the available literature. However, the compound has been evaluated for its metabolic profile using in vitro incubation with liver microsomes (rat liver microsomes, RLM; human liver microsomes, HLM) and S9 fractions (rat S9, RS9; human S9, HS9). These incubations were followed by high-resolution mass spectrometry analysis to identify and characterize metabolites. Nine unknown metabolites of GSK-9089 were identified, with most phase I metabolites formed after imine bond hydrolysis followed by deamidation, oxidation, and N-oxidation. The molecule underwent phase II metabolism to generate more polar metabolites mainly through glucuronide and sulfate conjugation biotransformation reactions .

In four wells of a 12-well plastic tissue culture dish, 150 (MDA-MB-231) or 200 (MCF7) cells were seeded per well on day 0. The next day, 18–24 hours were spent adding DY131 at the indicated concentrations. Day 2 involved removing the media that contained drugs, cleaning the wells with 1X PBS, and adding new media to them that contained no drugs. After a further 13 days of drug-free culture and two media changes, the cells were stained with crystal violet solution.

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1. Cell growth assay (crystal violet staining): Breast cancer (MDA-MB-231, MCF7, HCC1806) and non-transformed breast epithelial cell lines were cultured with varying concentrations of DY131 or DMSO control for up to 10/11 days; cells were fixed and stained with crystal violet, and staining intensity was quantified to assess cell growth (n=6, two-way ANOVA with Bonferroni post-tests) [2]
2. Apoptosis assay (flow cytometry): Breast cancer cells were treated with DY131 for 24 h, fixed, and stained with propidium iodide (PI) to detect subG1 DNA content (fragmented DNA); for live-cell apoptosis, cells were stained with Annexin V/PI and analyzed by flow cytometry (n=3–5, one-way ANOVA with Tukey's post-test) [2]
3. Western blot assay: Cells treated with DY131 (24 h) were lysed, and protein extracts were analyzed for PARP, γH2AX, total H2AX, p21, phosphorylated/total p38, phosphorylated/total Histone H3 (Ser10), ERRβ2, ERRβsf, and ERRγ; densitometry was performed for p-p38/total p38 ratio (normalized to β-actin, n=3, one-way ANOVA with Tukey's post-test) [2]
4. Cell cycle assay (flow cytometry): DY131-treated breast cancer cells (24 h) were fixed, stained with PI, and analyzed for cell cycle phase distribution (G1, S, G2/M); data were normalized to DMSO control (n=3–5, one-way ANOVA with Tukey's post-test) [2]
5. Clonogenic survival assay: MCF7 and MDA-MB-231 cells were seeded at low density, exposed to DY131 for 24 h, and cultured for 13 days; colonies were counted to evaluate clonogenic survival [2]
6. Live-cell confocal microscopy: MCF7 cells stably expressing GFP-H2B were arrested in G2 with nocodazole, then released into DY131 or DMSO control; time elapsed from chromatin condensation (prophase) to anaphase was quantified (n=4–11 cells, one-way ANOVA with Tukey's post-test) [2]
7. Immunofluorescence staining: HCC1806/MDA-MB-231 cells treated with 5 μM DY131 for 24 h were fixed and stained for γ-tubulin (centrosomal marker), β-tubulin (mitotic spindle), and DAPI (DNA); spindle morphology (monopolar/bipolar/multipolar) was scored (n=3, chi squared test) [2]
8. Subcellular fractionation (REAP assay): HCC1806 cells were fractionated into total cell lysate (TCL), nuclear (nuc), and cytoplasmic (cyto) fractions; Western blot was performed to detect subcellular localization of ERRβ2, ERRβsf, vinculin (cytoplasmic marker), and Histone H3 (nuclear marker) [2]
9. Luciferase reporter assay: MDA-MB-231/MCF7 cells were transiently co-transfected with promoter-reporter luciferase constructs (ERRE-luciferase) and receptor cDNA (ERRβ2, ERRβsf, AIB1); cells were treated with DY131, GSK4716, or DMSO for 18–20 h, and luciferase activity was measured (n=3, two-way ANOVA with Bonferroni post-test) [2]

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For cell proliferation assays, LNCaP-ERRγ and LNCaP cells were treated with DY131 at concentrations of 0.1, 1, 10, and 30 μM for 5 days. Cell proliferation was assessed by BrdUrd incorporation assay, and the number of BrdUrd-positive cells was quantified . For breast cancer cell studies, MCF7, MDA-MB-231, and MDA-MB-468 cells were treated with DY131 at various concentrations for specified durations, followed by assessment of cell viability, clonogenic survival, and subG1 fraction analysis by flow cytometry . In ECa109 and TE1 cells, DY131 (10 μM) was applied for 48 hours to evaluate cell proliferation and glycolysis activity . In primary cortical neurons, DY131 was used at 10 μM for 24, 48, or 72 hours to evaluate its protective effect against hemoglobin-induced neuronal toxicity . In AGS cells, DY131 was used at 5 μM as referenced in the literature .

Mouse study (steroidogenic gene expression): Mature male C57BL/6 mice (8-10 weeks old; approximately 24.3 g) were treated with DY131 (5 μg/kg) via subcutaneous injection every other day for 12 days . Rat study (subarachnoid hemorrhage): Sprague-Dawley rats were subjected to subarachnoid hemorrhage modeling, and DY131 (6 mg/kg) was administered via intraperitoneal injection 1 hour after SAH, with additional doses on days 2 and 3 after SAH . Mouse study (acute liver injury): C57BL/6J mice were pretreated with DY131 (5 mg/kg/day) via intraperitoneal injection once daily for 3 days before LPS challenge . Mouse study (xenograft tumor): BALB/c nude mice bearing ESCC xenografts were treated with DY131 (80 mg/kg) via intraperitoneal injection every 2 days for 25 days to evaluate tumor growth inhibition and anti-PD-1 therapy enhancement .

DY131 (GSK-9089) has a molecular weight of 311.38 g/mol and a molecular formula of C18H21N3O2 . The compound is soluble in DMSO (up to 30 mg/ml) and in ethanol (up to 8 mg/ml with warming) . For research storage, stock solutions are recommended to be stored at -20°C, where they are stable for up to 2 years . The compound can be stored in powder form at -20°C for up to 2 years . Metabolism studies using liver microsomes and S9 fractions have revealed that DY131 undergoes phase I metabolism via imine bond hydrolysis, deamidation, oxidation, and N-oxidation, as well as phase II metabolism via glucuronide and sulfate conjugation. Nine metabolites have been identified in rat biological samples (plasma, urine, faeces), though they were present only in trace amounts in vivo .

The available literature does not provide comprehensive toxicological data for DY131. However, DY131 has been reported to inhibit osteoclastogenesis and protect against inflammatory bone loss induced by LPS in vivo . In neuronal protection studies, DY131 significantly reduced neuronal apoptosis and oxidative stress without apparent toxicity in primary cortical neuron cultures . In LPS-induced acute liver injury studies, DY131 pretreatment ameliorated liver injury without adverse effects reported . In xenograft tumor studies, DY131 treatment at 80 mg/kg (intraperitoneal injection every 2 days for 25 days) was tolerated without significant toxicity noted . Detailed information regarding LD50, specific organ toxicity, or clinical safety data is not available in the current literature.

[1]. Bioorg Med Chem Lett. 2005 Mar 1;15(5):1311-3.

[2]. Oncotarget . 2016 Jul 26;7(30):47201-47220.

DY131 is a benzoic acid compound.

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1. DY131 (N'-{(1E)-[4-(diethylamino)phenyl]methylene}-4-hydroxybenzoylhydrazine) is a hydrazone compound with a 4-hydroxy group attached to one benzene ring and a 4-diethylamino group attached to the other benzene ring; it is a novel and selective pharmacological tool for studying the biological activity of ERRβ/γ[1]
2. DY131 exerts antimitotic activity in breast cancer (including triple-negative breast cancer, TNBC) by targeting ERRβ2 splice variants: ERRβ2 is located in the centrosome, and DY131 causes spindle defects in mitosis and delays chromosome separation (from prophase to anaphase)[2]
3. DY131-induced breast cancer cell death is mediated by the p38 stress kinase pathway, while cell cycle arrest (G2/M phase) is independent of p38 MAPK[2]
4. ERRβ2 has no transcription factor activity in breast cancer cells, and DY131 does not exert its effects through the transcriptional regulation of ERRβ2 [2]. 5. DY131 does not induce DNA damage or bind directly to DNA, which distinguishes its mechanism of action from traditional DNA-targeted chemotherapy drugs (e.g., doxorubicin, mitoxantrone) [2].

C18H21N3O2

311.38

311.163

C, 69.43; H, 6.80; N, 13.49; O, 10.28

95167-41-2

5497124

Light yellow to green yellow solid powder

1.1±0.1 g/cm3

1.574

3.17

2

4

6

23

380

0

O=C(C1C=CC(O)=CC=1)NN=CC1C=CC(N(CC)CC)=CC=1

WLKOCYWYAWBGKY-CPNJWEJPSA-N

InChI=1S/C18H21N3O2/c1-3-21(4-2)16-9-5-14(6-10-16)13-19-20-18(23)15-7-11-17(22)12-8-15/h5-13,22H,3-4H2,1-2H3,(H,20,23)/b19-13+

N-[(E)-[4-(diethylamino)phenyl]methylideneamino]-4-hydroxybenzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.03 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)