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| Targets |
XY101 targets retinoic acid receptor-related orphan receptor γ (RORγ) (Ki = 1.2 nM; GAL4 reporter gene assay IC50 = 2.5 nM) [1]
XY101 shows no significant binding to other nuclear receptors (e.g., RORα, RORβ, AR, ERα) with Ki > 10 μM [1] |
|---|---|
| ln Vitro |
Prostate-specific antigen (PSA), AR-V7, and androgen receptor (AR) expression are all efficiently inhibited by XY101 [1].
1. RORγ transcriptional activity inhibition: - XY101 acts as a potent RORγ inverse agonist, inhibiting RORγ-mediated transcriptional activation in GAL4-RORγ LBD transfected HEK293T cells with an IC50 of 2.5 nM [1] - It suppresses RORγ binding to its response element (RORE) in electrophoretic mobility shift assay (EMSA) at concentrations ≥5 nM [1] 2. Antiproliferative activity in CRPC cells: - XY101 inhibits proliferation of castration-resistant prostate cancer (CRPC) cell lines in a concentration-dependent manner: C4-2B (IC50 = 3.8 nM), 22Rv1 (IC50 = 5.2 nM), DU145 (IC50 = 7.1 nM) (72-hour CCK-8 assay) [1] - It exhibits minimal effect on normal prostate epithelial cells (PrEC) with IC50 > 10 μM [1] - Long-term clonogenic assay shows >85% inhibition of colony formation in C4-2B cells at 10 nM XY101 [1] 3. Downregulation of AR signaling pathway: - XY101 (5 nM, 24 hours) reduces mRNA levels of AR target genes (AR, PSA, TMPRSS2) by 65%, 72%, and 68% in C4-2B cells (RT-qPCR) [1] - Western blot analysis confirms 70% reduction in AR protein level and 80% reduction in PSA protein level in C4-2B cells treated with 10 nM XY101 for 48 hours [1] - It inhibits RORγ-AR protein-protein interaction, as demonstrated by co-immunoprecipitation (Co-IP) assay [1] 4. Induction of apoptosis and cell cycle arrest: - XY101 (10 nM) induces apoptosis in C4-2B cells, with apoptotic rate increasing from 4.5% (control) to 32.6% (48 hours, Annexin V/PI staining) [1] - Flow cytometry shows G0/G1 cell cycle arrest, with G0/G1 population increasing from 41.2% to 68.3% in C4-2B cells treated with 5 nM XY101 [1] - It upregulates pro-apoptotic proteins (Bax, cleaved caspase-3/9) and downregulates anti-apoptotic protein Bcl-2 (Western blot) [1] |
| ln Vivo |
XY101 exhibited substantial anti-tumor effectiveness during therapy, reduced tumor growth, and was well tolerated without significant weight loss [1].
1. Antitumor activity in C4-2B CRPC xenograft model: - XY101 (10, 30 mg/kg, p.o., once daily for 21 days) inhibits C4-2B tumor growth in nude mice by 52% and 75%, respectively, compared to vehicle control [1] - The 30 mg/kg group shows significant reduction in tumor weight (from 0.82 g to 0.21 g) and no obvious body weight loss [1] - Tumor tissue analysis reveals 68% reduction in AR mRNA level, 72% reduction in PSA protein level, and 55% reduction in Ki-67 (proliferation marker) positive cells [1] 2. Antitumor activity in 22Rv1 CRPC xenograft model: - XY101 (30 mg/kg, p.o., once daily for 21 days) reduces 22Rv1 tumor volume by 70% and prolongs median survival from 35 days (control) to 58 days (drug-treated group) [1] - Immunohistochemical staining of tumor tissues shows increased apoptotic cells (TUNEL assay) and decreased RORγ/AR protein expression [1] - Serum PSA level (tumor marker) is reduced by 65% in drug-treated mice [1] |
| Enzyme Assay |
1. Fluorescence polarization (FP) binding assay:
- Recombinant human RORγ ligand-binding domain (LBD) is diluted in binding buffer to a final concentration of 20 nM [1] - Fluorescein-labeled RORγ agonist ligand is added to the RORγ LBD solution at a final concentration of 5 nM [1] - XY101 is serially diluted (0.1 nM to 100 nM) and mixed with the above mixture, incubated at room temperature for 1 hour [1] - Fluorescence polarization is measured at excitation 485 nm and emission 535 nm, and Ki value is calculated using a competitive binding model [1] 2. GAL4-RORγ reporter gene assay: - HEK293T cells are co-transfected with GAL4-RORγ LBD expression plasmid and UAS-luciferase reporter plasmid [1] - Transfected cells are seeded in 96-well plates and cultured overnight, then treated with XY101 (0.1 nM to 50 nM) plus RORγ agonist (1 μM) for 24 hours [1] - Luciferase activity is measured using a luminometer, and IC50 is calculated by fitting the dose-response curve of activity inhibition [1] 3. Electrophoretic Mobility Shift Assay (EMSA): - Biotin-labeled RORE (ROR response element) oligonucleotide is synthesized and annealed [1] - Recombinant RORγ protein (50 ng) is incubated with XY101 (0.5-20 nM) for 30 minutes at room temperature [1] - Biotin-labeled RORE is added to the mixture and incubated for another 20 minutes, then separated by non-denaturing polyacrylamide gel electrophoresis [1] - The gel is transferred to a nylon membrane, and biotin-labeled DNA is detected using streptavidin-HRP conjugate to assess RORγ-RORE binding inhibition [1] |
| Cell Assay |
1. Cell proliferation assay (CCK-8):
- CRPC cell lines (C4-2B, 22Rv1, DU145) and normal PrEC cells are seeded in 96-well plates at 3×10^3 cells per well and cultured overnight [1] - XY101 is serially diluted (0.1 nM to 20 μM) and added to the cells, incubated at 37°C with 5% CO2 for 72 hours [1] - CCK-8 reagent is added to each well, incubated for 2 hours, and absorbance at 450 nm is measured to calculate cell viability and IC50 values [1] 2. RT-qPCR for AR target genes: - C4-2B cells are seeded in 6-well plates at 2×10^5 cells per well and treated with XY101 (1, 5, 10 nM) for 24 hours [1] - Total RNA is extracted, reverse-transcribed into cDNA, and qPCR is performed using specific primers for AR, PSA, TMPRSS2, and GAPDH (internal control) [1] - Relative mRNA levels are calculated using the 2^(-ΔΔCt) method, and fold changes compared to the control group are reported [1] 3. Apoptosis and cell cycle analysis: - C4-2B cells are treated with XY101 (5, 10 nM) for 48 hours, harvested, and washed with cold PBS [1] - Apoptosis assay: Cells are stained with Annexin V-FITC and PI for 15 minutes in the dark, then analyzed by flow cytometry [1] - Cell cycle assay: Cells are fixed in 70% ethanol at -20°C overnight, stained with PI solution containing RNase A for 30 minutes, and analyzed by flow cytometry [1] 4. Western blot and Co-IP assay: - XY101-treated C4-2B cells are lysed with RIPA buffer containing protease and phosphatase inhibitors [1] - For Western blot: Equal amounts of protein are separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against AR, PSA, Bax, Bcl-2, cleaved caspase-3/9, and GAPDH [1] - For Co-IP: Cell lysates are incubated with anti-RORγ antibody overnight, then mixed with protein A/G beads for 4 hours, and the immunoprecipitates are analyzed by Western blot with anti-AR antibody [1] |
| Animal Protocol |
1. C4-2B CRPC subcutaneous xenograft model:
- Male BALB/c nude mice (6-8 weeks old) are subcutaneously injected with 5×10^6 C4-2B cells suspended in Matrigel (1:1 with PBS) [1] - When tumors reach a volume of ~100 mm³, mice are randomized into vehicle control and XY101 treatment groups (n=8 per group) [1] - XY101 is dissolved in 0.5% carboxymethyl cellulose (CMC) + 0.1% Tween 80, administered orally at 10 or 30 mg/kg once daily for 21 days [1] - Tumor volume is measured every 3 days using calipers, and body weight is monitored weekly [1] - At the end of treatment, mice are euthanized, tumors are excised, weighed, and stored for mRNA, protein, and immunohistochemical analysis [1] 2. 22Rv1 CRPC xenograft model: - Male NOD-SCID mice (6-8 weeks old) are subcutaneously injected with 1×10^7 22Rv1 cells [1] - When tumors reach ~150 mm³, mice are randomized into control and XY101 groups (n=10 per group) [1] - XY101 is administered orally at 30 mg/kg once daily for 21 days, with the same vehicle as the C4-2B model [1] - Survival time is recorded daily, and serum PSA level is measured by ELISA every 7 days [1] - Mice are euthanized when tumors exceed 1500 mm³ or show distress, and tumor tissues are collected for TUNEL and immunohistochemical staining [1] |
| ADME/Pharmacokinetics |
XY101 had oral bioavailability of 45% and 42% in rats (oral 10 mg/kg) and mice (oral 30 mg/kg), respectively[1]
- In rats, the plasma half-life (t1/2) after intravenous injection (5 mg/kg) was 4.8 h, the volume of distribution (Vd) was 1.5 L/kg, and the total clearance (CL) was 210 ml/kg/h[1] - In mice, the peak plasma concentration (Cmax) of 2.1 μg/ml was reached 1 h after oral administration of 30 mg/kg, and the AUC₀ at 24 hours was 18.6 μg·h/ml[1] - The plasma protein binding rates were 93% (human plasma) and 91% (mouse plasma), respectively[1] - It showed good stability in human liver microsomes (t1/2 > 2 h) and was mainly metabolized by CYP3A4[1] |
| Toxicity/Toxicokinetics |
Acute toxicity: No deaths or adverse reactions were observed in mice when administered a single oral dose of up to 200 mg/kg [1]
- Subacute toxicity: No significant changes were observed in body weight, food intake, or hematological parameters (white blood cells, red blood cells, platelets) in mice after administration of XY101 once daily (30 mg/kg) for 28 days [1] - Serum ALT, AST, creatinine, and blood urea nitrogen levels in treated mice were within the normal range [1] - No histopathological abnormalities were detected in the liver, kidneys, heart, lungs, or prostate tissue of treated mice [1] |
| References | |
| Additional Infomation |
XY101 is a potent, selective, and orally bioavailable RORγ inverse agonist developed specifically for the treatment of castration-resistant prostate cancer (CRPC) [1]. Its binding mode involves occupying the ligand-binding pocket of RORγ, forming hydrogen bonds with key residues (Tyr502, His479), and engaging in hydrophobic interactions with hydrophobic residues in the pocket [1]. XY101 exerts its antitumor effect by inhibiting the transcriptional activation of RORγ-mediated androgen receptor (AR) target genes, thereby inhibiting CRPC cell proliferation and inducing apoptosis [1]. CRPC is characterized by the persistence of the AR signaling pathway despite androgen deprivation therapy, and XY101 targets this key pathway, providing a potential new therapeutic strategy for CRPC [1].
|
| Molecular Formula |
C25H20F7NO4S
|
|---|---|
| Molecular Weight |
563.48443031311
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| Exact Mass |
563.1
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| CAS # |
2349368-16-5
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| PubChem CID |
138105957
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
682.4±55.0 °C at 760 mmHg
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| Flash Point |
366.5±31.5 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.544
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| LogP |
5.56
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
38
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| Complexity |
888
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
AUIAOCHKUNGZHV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H20F7NO4S/c1-2-38(36,37)19-10-3-15(4-11-19)13-22(34)33-18-8-5-16(6-9-18)20-12-7-17(14-21(20)26)23(35,24(27,28)29)25(30,31)32/h3-12,14,35H,2,13H2,1H3,(H,33,34)
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| Chemical Name |
2-(4-ethylsulfonylphenyl)-N-[4-[2-fluoro-4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]phenyl]acetamide
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~443.67 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.69 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 20.8 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: ≥ 2.08 mg/mL (3.69 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (3.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7747 mL | 8.8734 mL | 17.7469 mL | |
| 5 mM | 0.3549 mL | 1.7747 mL | 3.5494 mL | |
| 10 mM | 0.1775 mL | 0.8873 mL | 1.7747 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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