DY131 (also known as GSK-9089) is a novel selective agonist at estrogen-related receptors ERRβ and ERRγ with minimal activity at ERRα, ERα and ERβ at concentrations up to 30 μM. DY131 stimulated the growth of ERα-negative endometrial cancer cells while inhibiting the growth of ERα-positive cancer cells. The structurally related receptors ERRα and the estrogen receptors alpha and beta (ERalpha/beta) were not affected by DY131. DY131 may be a cutting-edge treatment for prostate cancer since it targets ERRgamma.

Physicochemical Properties

Molecular Formula	C18H21N3O2
Molecular Weight	311.38
Exact Mass	311.163
Elemental Analysis	C, 69.43; H, 6.80; N, 13.49; O, 10.28
CAS #	95167-41-2
Related CAS #	95167-41-2
PubChem CID	5497124
Appearance	Light yellow to green yellow solid powder
Density	1.1±0.1 g/cm3
Index of Refraction	1.574
LogP	3.17
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	23
Complexity	380
Defined Atom Stereocenter Count	0
SMILES	O=C(C1C=CC(O)=CC=1)NN=CC1C=CC(N(CC)CC)=CC=1
InChi Key	WLKOCYWYAWBGKY-CPNJWEJPSA-N
InChi Code	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+
Chemical Name	N-[(E)-[4-(diethylamino)phenyl]methylideneamino]-4-hydroxybenzamide
Synonyms	DY-131; DY 131; DY131; GSK9089; GSK-9089; GSK 9089
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	ERRγ; ERRβ 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]
ln Vitro	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]. 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]
ln Vivo
Enzyme Assay	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]
Cell Assay	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. 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]
Animal Protocol
References	[1]. Bioorg Med Chem Lett. 2005 Mar 1;15(5):1311-3. [2]. Oncotarget . 2016 Jul 26;7(30):47201-47220.
Additional Infomation	DY131 is a member of benzoic acids. 1. DY131 (N'-{(1E)-[4-(diethylamino)phenyl]methylene}-4-hydroxybenzohydrazide) is a hydrazone compound synthesized with a 4-hydroxy group on one phenyl ring and a 4-diethylamino moiety on the other; it is a novel and selective pharmacologic tool for studying ERRβ/γ biological activities [1] 2. DY131 exerts antimitotic activity in breast cancer (including triple-negative breast cancer, TNBC) by targeting ERRβ2 splice variant: ERRβ2 localizes to centrosomes, and DY131 causes mitotic spindle defects and delays chromosome segregation (prophase to anaphase) [2] 3. DY131-induced cell death in breast cancer cells is mediated by the p38 stress kinase pathway, while cell cycle arrest (G2/M) is independent of p38 MAPK [2] 4. ERRβ2 has no transcription factor activity in breast cancer cells, and DY131 does not act via transcriptional regulation of ERRβ2 [2] 5. DY131 does not induce DNA damage or bind DNA directly, distinguishing its mechanism from conventional DNA-targeting chemotherapeutics (e.g., doxorubicin, mitoxantrone) [2]

Solubility Data

Solubility (In Vitro)

DMSO: ~10 mM Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.2115 mL	16.0576 mL	32.1151 mL
	5 mM	0.6423 mL	3.2115 mL	6.4230 mL
	10 mM	0.3212 mL	1.6058 mL	3.2115 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.