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| Targets |
The METTL3 and METTL14 proteins, which are the core components of the N6-methyladenosine (m6A) methyltransferase complex (also known as the "writer" complex).
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| ln Vitro |
WD6305 TFA is a selective degrader, not an inhibitor. It induces the degradation of METTL3 and METTL14 via the ubiquitin-proteasome pathway. It has DC50 values (concentration for 50% degradation) of 140 nM for METTL3 and 194 nM for METTL14, with a Dmax (maximal degradation) of 91.9% for METTL3.
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| ln Vivo |
WD6305 TFA potently reduces METTL3 and METTL14 protein levels in AML cells, such as Mono-Mac-6 cells. This leads to a decrease in the overall m6A modification levels on mRNA. Consequently, it inhibits cell proliferation and induces apoptosis in AML cells, showing its potential as an anticancer agent.
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| Enzyme Assay |
A general protocol for a cell-free (or biochemical) assay for a PROTAC involves assessing the formation of the ternary complex (PROTAC-E3 ligase-target protein). The target protein (e.g., METTL3) and the E3 ligase (e.g., VHL) are purified. The formation of the METTL3-VHL complex in the presence of various concentrations of WD6305 TFA can be detected using a TR-FRET (Time-Resolved Fluorescence Energy Transfer) assay. One protein is tagged with a donor (e.g., GST with a terbium-labeled anti-GST antibody) and the other with an acceptor (e.g., His with a d2-labeled anti-His antibody). When the PROTAC brings the two proteins into close proximity, a FRET signal is generated. The EC50 for ternary complex formation is calculated from the dose-response curve.
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| Cell Assay |
A general protocol for assessing the degradation activity of WD6305 TFA involves treating cells with the compound and then measuring target protein levels by Western blot. Mono-Mac-6 (human AML) cells are seeded in 6-well plates and treated with various concentrations of WD6305 TFA (20, 50, 100, 250, 500, 1000, 2000, and 5000 nM) for 24 hours. After treatment, cells are lysed, and the lysates are subjected to SDS-PAGE and immunoblotted with antibodies against METTL3, METTL14, and a loading control (e.g., GAPDH or beta-actin). The band intensities are quantified. The DC50 is calculated by plotting the percentage of remaining target protein against the log of the compound concentration. Apoptosis induction is assessed by treating Mono-Mac-6 cells with 1 and 2.5 uM of WD6305 for 48 hours, then measuring the levels of cleaved caspase-3 and cleaved PARP by Western blot.
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| Animal Protocol |
A general in vivo protocol for evaluating efficacy involves a mouse xenograft model using AML cells. Subcutaneously implant Mono-Mac-6 cells into immunocompromised mice (e.g., NOD/SCID). When tumors are established (e.g., ~150 mm3), mice are treated with WD6305 TFA via intraperitoneal (IP) injection at a dose of, for example, 20 mg/kg every other day for 2-3 weeks. Tumor volume and body weight are measured twice weekly. At the end of the study, tumors are harvested for Western blot analysis to confirm METTL3 and METTL14 degradation, and for m6A quantification by ELISA or LC-MS/MS. TUNEL or Caspase-3 staining could be performed to assess apoptosis.
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| ADME/Pharmacokinetics |
A general PK protocol for WD6305 TFA would involve administering a single dose (e.g., 10 mg/kg) to male CD-1 mice via intravenous (IV) and oral (PO) routes. Blood samples are collected at various time points (0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 hours). Plasma concentrations are quantified by a validated LC-MS/MS method. Parameters like Cmax, Tmax, half-life, clearance, volume of distribution, and oral bioavailability (%F) are calculated. PROTACs often have higher molecular weights (~1226.4 for WD6305 TFA) [49L4-L5] and may exhibit suboptimal oral bioavailability, but the IV route can be used for in vivo efficacy studies.
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| Toxicity/Toxicokinetics |
General toxicity for PROTAC molecules such as WD6305 TFA would be evaluated in a 14-day repeated-dose study in rats. Animals would receive IV or IP doses (e.g., 5, 15, and 40 mg/kg) once daily. Parameters would include body weight, food consumption, and clinical observations. Standard panels for hematology, serum chemistry (liver enzymes, kidney function), and coagulation would be performed. A full histopathology assessment of major organs (liver, kidney, spleen, adrenal glands, etc.) would be conducted at the end of the study. Off-target degradation is a general safety concern for PROTACs, so proteomics analysis may also be performed to assess selectivity.
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| References | |
| Additional Infomation |
WD6305 TFA is a heterobifunctional PROTAC molecule consisting of a METTL3-targeting ligand linked to a ligand for the Von Hippel-Lindau (VHL) E3 ubiquitin ligase. By bringing the target protein into close proximity to the E3 ligase, it leads to ubiquitination and subsequent proteasomal degradation of METTL3 and the METTL14 complex. The TFA salt form improves the compound's solubility. The molecular weight of WD6305 TFA is 1226.4, and its molecular formula is C₆3H₇₆F₉N11O₇S.
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| Molecular Formula |
C63H76F5N11O7S
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| Molecular Weight |
1226.40
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| Appearance |
Solid powder
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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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 : 95 mg/mL (77.46 mM; with sonication)
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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 | 0.8154 mL | 4.0770 mL | 8.1539 mL | |
| 5 mM | 0.1631 mL | 0.8154 mL | 1.6308 mL | |
| 10 mM | 0.0815 mL | 0.4077 mL | 0.8154 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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