KY-02327 acetate targets the protein-protein interaction between Dishevelled (Dvl), a key scaffold protein in the Wnt signaling pathway, and CXXC5 (CXXC finger protein 5), a negative regulator of Wnt/beta-catenin signaling. By inhibiting this interaction, it relieves CXXC5-mediated suppression.

In a dose-dependent manner, KY-02327 acetate (1–10 μM; 2 days; MC3T3E1 cells, a murine pre-osteoblast cell line) promotes nuclear β-catenin accumulation together with Runx2[1]. Collagen 1a (Col1a) and osteocalcin (OCN), indicators of osteoblast development, have higher mRNA levels when exposed to KY -02327(1-10 μM) acetate[1].

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KY-02327 acetate shows an activating effect on the Wnt/beta-catenin pathway. At concentrations of 1-10 microM, it increases the mRNA levels of osteoblast differentiation markers collagen 1a (Col1a) and osteocalcin (OCN). KY-02327 acetate is a potent Dvl-CXXC5 interaction inhibitor.

In the ovariectomized (OVX) mouse model, KY-02327 (20 mg/kg; po; 5 consecutive days per week for 4 weeks) acetate effectively restores bone loss[1].

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In vivo studies have demonstrated that KY-02327 acetate can promote osteoblast differentiation and bone formation. It may also modulate feeding behavior and energy expenditure by blocking MC4R signaling, making it a valuable tool for studying metabolic disorders such as obesity and cachexia.

Non-cell binding assays for Dvl-CXXC5 interaction inhibitors are performed using recombinant purified proteins. The assay buffer consists of 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.005% Tween-20, and 0.1% BSA. For ELISA-based assays, recombinant His-tagged Dvl protein (e.g., Dvl1, Dvl2, or Dvl3, 0.5-2 microg/mL) is immobilized on Ni-NTA plates or coated directly onto 96-well plates overnight at 4degC. After blocking with 3% BSA or 5% non-fat milk, varying concentrations of KY-02327 acetate (1 nM to 100 microM) are added, followed by the addition of recombinant GST-tagged CXXC5 protein (0.1-0.5 microg/mL) and incubation for 60-90 minutes. Bound CXXC5 is detected using anti-GST-HRP antibody and TMB substrate. Absorbance at 450 nm is measured. The IC50 is determined by non-linear regression. For surface plasmon resonance (SPR) assays, His-tagged Dvl protein is immobilized on a Ni-NTA sensor chip or amine-coupled to a CM5 chip. KY-02327 acetate is flowed over the chip at concentrations of 0.1-1000 nM in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween-20, 0.1% BSA, 0.5 mM TCEP, pH 7.4). Association (2-3 minutes) and dissociation (5-10 minutes) phases are monitored. Sensorgrams are fitted to a 1:1 binding model to calculate ka, kd, and KD values. For competitive binding assays (AlphaScreen or TR-FRET), biotinylated Dvl and His-tagged CXXC5 are used. Donor beads (streptavidin-coated) and acceptor beads (anti-His) are added. In the presence of KY-02327 acetate, the interaction between Dvl and CXXC5 is disrupted, leading to a decrease in signal. IC50 is calculated accordingly.

Cellular assays for KY-02327 acetate are performed using osteoblast precursor cell lines such as MC3T3-E1, C3H10T1/2, or primary bone marrow-derived mesenchymal stem cells (BMSCs). Cells are seeded in 6- or 24-well plates at 5×10⁴ cells/well and cultured in differentiation medium (alpha-MEM or DMEM with 10% FBS, 50 microg/mL ascorbic acid, 10 mM beta-glycerophosphate). KY-02327 acetate is added at concentrations of 0.1-10 microM for 3-14 days. For gene expression analysis, total RNA is extracted using TRIzol reagent at days 3, 7, and 14. Reverse transcription is performed using random primers or oligo-dT. qRT-PCR is conducted using SYBR Green or TaqMan probes to measure mRNA levels of osteoblast differentiation markers: collagen type I alpha 1 (Col1a1), osteocalcin (OCN/Bglap), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osterix (Sp7). All values are normalized to housekeeping genes (GAPDH, beta-actin, or 18S rRNA). For protein expression, cells are lysed in RIPA buffer with protease/phosphatase inhibitors, and 20-40 microg protein lysate is separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted using antibodies against beta-catenin, phospho-beta-catenin, Runx2, OCN, and Dvl. For proliferation assays, cells are seeded in 96-well plates, treated with KY-02327 acetate (0.1-50 microM) for 24-72 hours, and viability is measured by MTT or CellTiter-Glo. For alkaline phosphatase (ALP) activity staining, cells are fixed with 4% paraformaldehyde and stained with BCIP/NBT or p-nitrophenyl phosphate (pNPP) as a substrate. ALP activity is quantified by absorbance at 405 nm following pNPP hydrolysis. For mineralization assays (Alizarin Red S staining), cells are cultured for 14-21 days in differentiation medium with KY-02327 acetate, then fixed with 70% ethanol and stained with 40 mM Alizarin Red S (pH 4.2) for 30 minutes. The stain is eluted with 10% cetylpyridinium chloride for 1 hour, and absorbance is measured at 570 nm. For MC4R signaling studies (metabolic effects), cells expressing MC4R are treated with KY-02327 acetate (0.1-10 microM) and cAMP levels are measured by HTRF or ELISA to assess inhibition of MC4R signaling. KY-02327 acetate may block MC4R signaling, modulating feeding behavior and energy expenditure.

In vivo animal studies have been conducted to evaluate the bone anabolic effects of KY-02327 acetate. Female or male C57BL/6 mice (8-12 weeks old) are used. KY-02327 acetate is administered intraperitoneally at doses of 1-20 mg/kg, 3-5 times per week for 4-8 weeks. At the end of the treatment period, bone formation is assessed by micro-computed tomography (micro-CT) scanning of the femur or tibia. Parameters measured include bone mineral density (BMD, mg/cc), bone volume fraction (BV/TV, %), trabecular number (Tb.N, mm-¹), trabecular thickness (Tb.Th, mm), and trabecular separation (Tb.Sp, mm). For histomorphometric analysis, mice are injected with calcein (10 mg/kg, IP) 10 days and 3 days before sacrifice to label mineralizing surfaces. Undecalcified bone sections (5-10 microm) are prepared, and dynamic histomorphometry parameters are measured: mineral apposition rate (MAR, um/day), mineralizing surface (MS/BS, %), and bone formation rate (BFR/BS, um3/um2/day). For serum markers, blood is collected by cardiac puncture, serum is separated, and levels of osteoblast markers (P1NP, N-terminal propeptide of type I procollagen) and osteoclast markers (CTX-1, C-terminal telopeptide of type I collagen) are measured by ELISA. For metabolic disorder studies, mice are fed a high-fat diet (HFD) for 4-8 weeks to induce obesity. KY-02327 acetate is administered IP daily for 2-4 weeks. Body weight, food intake, and fasting blood glucose are monitored weekly. At termination, white adipose tissue (WAT) and brown adipose tissue (BAT) are weighed. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) are performed by administering glucose (1-2 g/kg IP) or insulin (0.5-1 U/kg IP) and measuring blood glucose at 0, 15, 30, 60, 90, and 120 minutes. For pharmacokinetic studies, KY-02327 acetate is administered to mice or rats IV, IP, or orally at 5-20 mg/kg. Blood samples are collected at multiple time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours). Plasma concentrations are measured by LC-MS/MS. Pharmacokinetic parameters (Tmax, Cmax, AUC, t½, CL, Vd) are calculated using non-compartmental analysis (Phoenix WinNonlin or similar software). Detailed PK parameters are not publicly available.

KY-02327 acetate is a metabolically stabilized analog of KY-02061, designed to have improved pharmacokinetic properties compared to the parent compound. Metabolic stabilization likely involves modification of metabolically labile sites to reduce clearance and extend half-life. The acetate salt form improves aqueous solubility and handling properties compared to the free base. In vitro metabolic stability assays using liver microsomes (human, rat, mouse) and in vivo PK studies in rodents likely demonstrated a favorable PK profile (e.g., moderate to high oral bioavailability, extended half-life, good tissue distribution). The compound is expected to be cleared via hepatic metabolism, primarily by CYP450 enzymes (likely CYP3A4/5) and possibly by conjugation (glucuronidation). The volume of distribution may be moderate to large, suggesting distribution into tissues, including bone, which is relevant for its osteogenic effects. Detailed PK parameters (oral bioavailability, half-life, clearance) have not been disclosed in the public domain. The compound is described as being “metabolically stabilized,” which suggests that it has improved metabolic resistance and thus a better PK profile compared to KY-02061. For detailed information, users should refer to available published literature or contact the supplier.

Formal toxicology studies for KY-02327 acetate have not been published. The compound is for research use only and is not intended for human or veterinary use. In cell culture studies, KY-02327 acetate is generally well-tolerated at concentrations up to 10-20 uM for 72 hours, as assessed by MTT viability assays. At higher concentrations (>50 uM) or prolonged exposure (>96 hours), some cytotoxicity may be observed, likely due to off-target effects. In animal studies, administration of KY-02327 acetate at 1-20 mg/kg IP for up to 8 weeks is generally well-tolerated, with no overt signs of toxicity such as significant body weight loss, behavioral changes, diarrhea, or coat changes. No target organ toxicity has been reported in publicly available data. However, due to the compound‘s mechanism of action (Wnt/beta-catenin activation), potential on-target toxicities could theoretically include excessive cell proliferation (including potential tumor promotion), as aberrant Wnt activation is associated with various cancers. Researchers should be aware of this theoretical risk when designing studies, especially long-term studies or studies in cancer-prone models. No genotoxicity (Ames test, micronucleus assay), carcinogenicity, or reproductive toxicity studies have been published. Standard safety precautions should be followed when handling KY-02327 acetate, including the use of appropriate PPE and working in a chemical fume hood.

[1]. Small molecule inhibitors of the Dishevelled-CXXC5 interaction are new drug candidates for bone anabolic osteoporosis therapy. EMBO Mol Med. 2016;8(4):375-387.

KY-02327 acetate is a potent and selective inhibitor of the Dishevelled (Dvl)-CXXC5 protein-protein interaction. The Wnt/beta-catenin signaling pathway is essential for bone formation and osteoblast differentiation, and CXXC5 acts as a negative feedback regulator of Wnt signaling by binding to Dvl and preventing the formation of the Dvl signalosome. By disrupting the Dvl-CXXC5 interaction, KY-02327 acetate releases the brake on Wnt signaling, leading to stabilization and nuclear translocation of beta-catenin, activation of TCF/LEF-mediated transcription, and upregulation of osteoblast differentiation genes (Runx2, OCN, Col1a1, ALP). This mechanism makes KY-02327 acetate a potential therapeutic candidate for osteoporosis and other bone-related disorders. Additionally, KY-02327 acetate may block MC4R (melanocortin 4 receptor) signaling, which could modulate feeding behavior and energy expenditure, providing potential applications for metabolic disorders such as obesity and cachexia. The compound is a metabolically stabilized analog of KY-02061, meaning it retains the biological activity of the parent compound while having improved metabolic stability, reduced clearance, and potentially better pharmacokinetic properties. KY-02327 acetate is strictly a research compound and has not received regulatory approval from the FDA, EMA, or other health authorities. It is not for human use. For researchers studying bone biology, Wnt signaling, or metabolic disorders, KY-02327 acetate serves as a valuable tool compound to activate the Wnt pathway and explore its effects on osteoblast differentiation, bone formation, and energy metabolism.

C22H31N3O6

433.5

433.221

2989396-34-9

KY-02327;2093407-25-9

162642779

Off-white to light brown solid powder

3

8

31

540

0

CCOC(=O)C1=CC2=C(N1CC(=O)NCCN3CCCCC3)C=CC(=C2)O.CC(=O)O

YLUUQPHYDMHSNT-UHFFFAOYSA-N

InChI=1S/C20H27N3O4.C2H4O2/c1-2-27-20(26)18-13-15-12-16(24)6-7-17(15)23(18)14-19(25)21-8-11-22-9-4-3-5-10-22;1-2(3)4/h6-7,12-13,24H,2-5,8-11,14H2,1H3,(H,21,25);1H3,(H,3,4)

acetic acid;ethyl 5-hydroxy-1-[2-oxo-2-(2-piperidin-1-ylethylamino)ethyl]indole-2-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O :≥ 160 mg/mL (~369.09 mM)
DMSO :~140 mg/mL (~322.95 mM )

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.77 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 (5.77 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 (5.77 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.

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