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| Targets |
WCY-8-67 selectively targets ubiquitin‑specific protease 5 (USP5), a deubiquitinating enzyme that acts on ubiquitin chains. In t(8;21) acute myeloid leukemia (AML), USP5 was identified as the deubiquitinating enzyme of the AML1‑ETO (AE) fusion protein, a key oncogenic driver. By binding to the ubiquitin‑associated domain 2 (UBA2) region of USP5, WCY-8-67 prevents USP5 from recognizing ubiquitin, leading to dysfunction and aggregation of the target protein. The compound thus promotes ubiquitin‑mediated degradation of the AML1‑ETO oncoprotein, suppressing downstream oncogenic signaling. Downstream signaling pathways affected include JAK/STAT3 and PI3K/AKT, both of which are suppressed by WCY-8-67 treatment. Beyond USP5, the compound also targets apoptosis pathways and related signaling modulators including STAT, JAK, Akt, and PI3K.
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| ln Vitro |
WCY-8-67 (0.5-2 μM, 24 h) reduced the abundance of AML1-ETO (AE) protein in Kasumi-1 and Skno-1 cells in a dose-dependent manner [1]. WCY-8-67 (0.5-2 μM, 24 h) inhibited the proliferation of AE-positive AML cells (Kasumi-1: IC50 = 1.75 μM; Skno-1: IC50 = 0.99 μM; U937-AE: IC50 = 1.20 μM), but had little effect on AE-negative AML cells (MV-4-11, MOLM-13, HL-60) [1]. WCY-8-67 (0.5-7.5 μM, 24-48 h) induced apoptosis and G1 phase cell cycle arrest in Kasumi-1 and Skno-1 cells [1]. WCY-8-67 (0.5-2 μM, 7 days) reduced colony formation in Kasumi-1 and Skno-1 cells and decreased the number of malignant clones in agarose colony assays [1]. WCY-8-67 (0.5-2 μM, 24 h) reduced the CD34+CD38- leukemia stem cell (LSC) population and increased the CD11b+ differentiated cell population in Kasumi-1 and Skno-1 cells [1]. WCY-8-67 (0-2.5 μM, 24 h) inhibited the JAK/STAT3 and PI3K/AKT signaling pathways in Kasumi-1 and Skno-1 cells [1].
WCY-8-67 demonstrates potent in vitro activity against t(8;21) AML cells with a biochemical IC50 of 1.33 microM for USP5 inhibition. In cell proliferation assays, WCY-8-67 inhibits the growth of AE‑positive AML cells (Kasumi‑1: IC50 = 1.75 microM; Skno‑1: IC50 = 0.99 microM; U937‑AE: IC50 = 1.20 microM) but shows negligible effects on AE‑negative AML cells (MV‑4‑11, MOLM‑13, HL‑60). At concentrations of 0.5-2 microM over 24 hours, the compound dose‑dependently reduces AML1‑ETO protein abundance, induces apoptosis and G1 phase cell cycle arrest, and decreases the CD34+CD38- leukemia stem cell (LSC) population while increasing the CD11b+ differentiated cell population. Colony formation is also reduced at 0.5-2 microM over 7 days in agarose assays. Mechanistically, WCY-8-67 suppresses JAK/STAT3 and PI3K/AKT signaling pathways, downregulates MDM2 and anti‑apoptotic proteins Bcl‑2/Mcl‑1, upregulates p53, and promotes PARP1 cleavage and caspase‑3 activation. |
| ln Vivo |
WCY-8-67 (20–40 mg/kg, orally, once daily for 12 days) showed potent antitumor activity in the Skno-1 subcutaneous xenograft model [1]. WCY-8-67 (40 mg/kg, orally, once daily for 42 days) reduced tumor fatigue and prolonged survival in the Kasumi-1-luciferase-GFP orthotopic xenograft model [1]. WCY-8-67 (20 mg/kg, intraperitoneally, once daily for 14 days) prolonged survival and reduced spleen weight in the t(8;21) AML xenograft (PDX) model [1].
WCY-8-67 demonstrates potent in vivo anti‑leukemic efficacy in mouse models of t(8;21) AML. In a Skno‑1 subcutaneous xenograft model, oral administration of WCY-8-67 (20-40 mg/kg once daily for 12 days) exhibited strong tumor growth inhibition. In a Kasumi‑1‑luciferase‑GFP orthotopic xenograft model, treatment with WCY-8-67 (40 mg/kg orally once daily for 42 days) reduced tumor burden and extended survival. In a t(8;21) AML patient‑derived xenograft (PDX) model, intraperitoneal administration of WCY-8-67 (20 mg/kg once daily for 14 days) prolonged survival and attenuated spleen weight increase. When combined with 5‑azacytidine (5‑Aza), WCY-8-67 improved therapeutic effects compared to either agent alone. The compound exhibited excellent in vivo bioavailability and tolerability in these animal models. |
| Enzyme Assay |
The in vitro USP5 biochemical inhibition assay is performed using purified USP5 enzyme. A high‑throughput screening (HTS) method was developed to evaluate USP5 inhibition, measuring the deubiquitinating activity of USP5 in the presence of a dose range of WCY-8-67. The reaction mixture contains USP5 enzyme, a ubiquitin‑based substrate (typically ubiquitin‑AMC or a ubiquitin‑linked reporter), and buffer. WCY-8-67 is added at varying concentrations (typically 0.1-10 microM). After incubation at 37degC for 30-60 minutes, the cleavage of the ubiquitin‑substrate is quantified by fluorescence (for ubiquitin‑AMC) or by detection of released products. The IC50 value (1.33 microM) is calculated from a concentration‑response curve by nonlinear regression analysis. For binding affinity, surface plasmon resonance (SPR) assays yield a Kd of 5.73 microM. The mechanism of USP5 inhibition involves targeting the ubiquitin‑associated domain 2 (UBA2) region, as confirmed by structural and functional studies.
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| Cell Assay |
Apoptosis Analysis[1]
Cell Types: Kasumi-1 and Skno-1 cells Tested Concentrations: 0, 1.25, 5, 7.5 μM Incubation Duration: 48 h Experimental Results: Downregulated MDM2 and upregulated p53. Promoted cleavage of PARP1 and activation of caspase-3. Reduced anti-apoptotic proteins Bcl-2 and Mcl-1. For in vitro cell‑based assays, Kasumi‑1 and Skno‑1 cells (t(8;21) AML cell lines) are cultured in appropriate medium (e.g., RPMI‑1640 with 10% FBS and 1% penicillin/streptomycin). Cells are seeded in multi‑well plates and treated with a dose range of WCY-8-67 (typically 0.5-7.5 microM). For proliferation assays, cells are incubated for 24-48 hours, and viability is assessed using CCK‑8 or MTT assays to calculate IC50 values. For apoptosis analysis, cells are treated for 48 hours, then stained with Annexin V‑FITC and propidium iodide, followed by flow cytometry. Cell cycle analysis is performed by propidium iodide staining after 24-48 hours of treatment. For differentiation assessment, cells are treated for 24 hours and stained with CD11b and CD34 antibodies, then analyzed by flow cytometry to measure changes in stem cell and differentiated cell populations. For signaling pathway analysis, cells are treated for 24 hours, lysed, and subjected to Western blotting using antibodies against JAK/STAT3 and PI3K/AKT pathway components, as well as apoptosis‑related proteins (PARP1, caspase‑3, MDM2, p53, Bcl‑2, Mcl‑1). |
| Animal Protocol |
Animal/Disease Models: Skno-1 cells (1 × 107) were subcutaneously implanted into the flanks of 6-week-old female NU/NU mice[1]
Doses: 20, 40 mg/kg Route of Administration: p.o., once daily, 12 days Experimental Results: Reduced tumor weight and volume. Showed no significant changes in body weight. Animal/Disease Models: Kasumi-1 cells expressing luciferase-GFP (2 × 106) were intravenously injected into the tail veins of the 6-week-old NOG mice[1] Doses: 40 mg/kg Route of Administration: p.o., once daily, 42 days Experimental Results: Decreasedspleen weight and hCD45+ leukemic cell percentage in bone marrow, spleen, and peripheral blood. Reduced tumor burden and extends survival. Animal/Disease Models: Primary t(8;21) AML cells were intravenously injected into the tail veins of the 6-week-old NOG mice[1] Doses: 20 mg/kg Route of Administration: i.p., once daily, 14 days Experimental Results: Decreased hCD45+ cell infiltration in bone marrow, spleen, and peripheral blood. Prolonged survival and reduces spleen weight. In vivo animal studies are conducted using 6‑week‑old female NU/NU nude mice for xenograft models. For the Skno‑1 subcutaneous model, 1×10⁷ Skno‑1 cells are subcutaneously implanted into the flanks of mice. Once tumors reach a palpable size, WCY-8-67 is administered orally at doses of 20-40 mg/kg once daily for 12 days. Tumor volumes are measured every few days using calipers, and body weight is monitored for tolerability. For the Kasumi‑1‑luciferase‑GFP orthotopic model, WCY-8-67 is administered orally at 40 mg/kg once daily for 42 days, and tumor burden is assessed by bioluminescence imaging. For the PDX model using primary t(8;21) AML patient cells, WCY-8-67 is administered intraperitoneally at 20 mg/kg once daily for 14 days. Survival is recorded using Kaplan‑Meier analysis, and spleen weight is measured at endpoint. Formulation for oral administration typically involves suspending the compound in a suitable vehicle such as 0.5% methylcellulose or other standard oral formulations. |
| ADME/Pharmacokinetics |
WCY-8-67 exhibits excellent oral bioavailability and favorable pharmacokinetic properties in vivo. In preclinical mouse models, the compound demonstrates good systemic exposure following oral administration, enabling once‑daily dosing regimens as reported in efficacy studies (20-40 mg/kg oral once daily). The compound is stable at ambient temperature for up to one month without loss of biological activity. Storage recommendations: powder at -20degC for up to three years; solution at -20degC for up to six months. The compound should be kept in a tightly sealed container, protected from light, and stored in a dry, well‑ventilated area. Detailed pharmacokinetic parameters such as half‑life, volume of distribution, clearance, and protein binding have not been publicly disclosed but are characterized in the primary research article (Ma et al., Science Translational Medicine, 2025).
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| Toxicity/Toxicokinetics |
According to the Safety Data Sheet (SDS), WCY-8-67 is classified as “not a hazardous substance or mixture” under GHS criteria. No specific acute or chronic toxicities are listed in the hazard identification section. First aid measures include: for inhalation, relocate to fresh air and administer CPR if breathing is difficult; for skin contact, rinse thoroughly with large amounts of water and remove contaminated clothing; for eye contact, flush eyes with water for at least 15 minutes and call a physician; for ingestion, wash out mouth with water if the person is conscious, and do NOT induce vomiting unless directed by medical personnel. During combustion, the compound may emit irritant fumes, and firefighters should wear self‑contained breathing apparatus and protective clothing. Recommended personal protective equipment includes gloves, lab coat, and eye protection. Avoid dust and aerosol formation, and use only in areas with adequate exhaust ventilation. The compound is not for human or veterinary use and is intended solely for research purposes.
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| References | |
| Additional Infomation |
WCY-8-67 is a research‑grade compound that has not entered clinical trials and is not approved for human therapeutic use by any regulatory authority (FDA, EMA, PMDA). It was developed by Ma and colleagues as a first‑in‑class selective USP5 inhibitor and reported in Science Translational Medicine (Volume 17, Issue 817, Pages eadt9100) on September 24, 2025. The complete synthesis, high‑throughput screening, and preclinical evaluation of WCY-8-67 are detailed in this primary publication. The mechanism of action involves targeting the ubiquitin‑associated domain 2 (UBA2) region of USP5, leading to USP5 dysfunction and aggregation, thereby promoting ubiquitin‑mediated degradation of the AML1‑ETO oncoprotein in t(8;21) AML. When combined with 5‑azacytidine (5‑Aza), WCY-8-67 improves therapeutic effects in PDX models, suggesting potential combination strategies. The compound is labeled exclusively as “for research use only - not for human use” and is available for preclinical acute myeloid leukemia research.
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| Molecular Formula |
C25H32FN7
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| Molecular Weight |
449.57
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| CAS # |
3052618-54-6
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| Related CAS # |
WCY-8-67 TFA
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| Appearance |
Typically exists as solids at room temperature
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2243 mL | 11.1217 mL | 22.2435 mL | |
| 5 mM | 0.4449 mL | 2.2243 mL | 4.4487 mL | |
| 10 mM | 0.2224 mL | 1.1122 mL | 2.2243 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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