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| Targets |
The primary molecular target of TAS4464 hydrochloride is NEDD8-activating enzyme (NAE), a heterodimeric enzyme composed of NAE1 and UBA3 subunits. NAE catalyzes the first step in the neddylation pathway, activating the ubiquitin-like protein NEDD8 via an ATP-dependent adenylation reaction. TAS4464 hydrochloride binds to the active site of NAE with high affinity, competitively inhibiting the enzyme with an IC50 of 0.955 nM. By inhibiting NAE, the compound prevents the conjugation of NEDD8 to cullin proteins, which are essential components of cullin-RING ubiquitin ligases (CRLs). CRLs regulate the ubiquitination and proteasomal degradation of numerous proteins involved in cell cycle progression, DNA replication, and signal transduction. The compound shows high selectivity for NAE over other ubiquitin-like activating enzymes (E1s).
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| ln Vitro |
In cell-free enzymatic assays, TAS4464 hydrochloride demonstrates potent inhibition of NEDD8-activating enzyme with an IC50 of 0.955 nM. The compound shows high selectivity for NAE over other E1 enzymes, including ubiquitin-activating enzyme (UBA1) and SUMO-activating enzyme (SAE), ensuring specificity for the neddylation pathway. In biochemical assays, TAS4464 hydrochloride inhibits the ATP-dependent formation of the NEDD8-NAE thioester intermediate, preventing the downstream transfer of NEDD8 to its conjugating enzyme (Ubc12) and subsequently to cullin substrates. The compound exhibits a prolonged duration of target inhibition, with sustained suppression of cullin neddylation even after compound washout. Enzyme kinetic studies may be performed to determine the mode of inhibition (competitive vs. non-competitive) and to calculate Ki values. In cellular studies, TAS4464 hydrochloride effectively blocks the neddylation of cullin proteins, leading to the accumulation of CRL substrates such as p21, p27, and Wee1. This results in cell cycle arrest, induction of apoptosis, and inhibition of cancer cell proliferation. The compound shows activity in both hematologic and solid tumor cell lines. In various cancer cell lines, TAS4464 hydrochloride reduces cell viability with IC50 values in the low nanomolar to sub-micromolar range. The compound induces DNA damage and replication stress due to the dysregulation of cell cycle checkpoint proteins. TAS4464 hydrochloride has been shown to sensitize cancer cells to other therapeutic agents, including DNA-damaging agents and targeted therapies. The compound's prolonged target inhibition contributes to its sustained anti-proliferative effects, with activity observed even after compound removal.
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| ln Vivo |
In vivo studies of TAS4464 hydrochloride have demonstrated superior antitumor activity in mouse xenograft models of both hematologic and solid tumors. The compound shows prolonged target inhibition, with sustained suppression of cullin neddylation in tumor tissues after dosing. TAS4464 hydrochloride is typically administered orally or via intraperitoneal injection at doses determined from pharmacokinetic and toxicology studies. In xenograft models, the compound causes significant tumor growth inhibition and, in some cases, tumor regression. The anti-tumor activity is accompanied by pharmacodynamic evidence of target engagement, including reduced cullin neddylation and accumulation of CRL substrates (e.g., p21, p27) in tumor tissues. The compound is well-tolerated at efficacious doses, with minimal body weight loss observed. TAS4464 hydrochloride is considered a promising agent for the treatment of a variety of tumors.
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| Enzyme Assay |
For NAE inhibition assays, the enzymatic activity of recombinant human NAE is measured using a fluorescence-based or radiometric assay. Purified NAE (NAE1/UBA3 heterodimer) is incubated with varying concentrations of TAS4464 hydrochloride (typically 0.001-10 µM) in reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM ATP, and 0.1% BSA at 37°C for 15-30 minutes. The reaction is initiated by the addition of NEDD8 and the NEDD8-conjugating enzyme Ubc12, and incubated for 30-60 minutes. Product formation (NEDD8-Ubc12 thioester or neddylated cullin) is detected by Western blot using anti-NEDD8 or anti-cullin antibodies. Alternatively, fluorescence polarization or time-resolved FRET assays using labeled NEDD8 can be used for high-throughput screening. IC50 values are calculated by non-linear regression analysis of dose-response curves. Selectivity assays against other E1 enzymes (e.g., UBA1, SAE) are performed using similar protocols with their respective substrates.
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| Cell Assay |
For cellular neddylation inhibition studies, cancer cells are cultured in appropriate medium (RPMI-1640 or DMEM) with 10% FBS and antibiotics at 37°C in 5% CO₂. Cells are seeded in 6-well or 12-well plates and allowed to reach 70-80% confluence. TAS4464 hydrochloride is dissolved in DMSO and diluted in culture medium to final concentrations (typically 0.001-10 µM). Cells are treated for 4-24 hours, then lysed in RIPA buffer with protease and phosphatase inhibitors. Lysates are analyzed by Western blot for cullin neddylation status (neddylated vs. unneddylated cullins), accumulation of CRL substrates (p21, p27, Wee1, NRF2), and apoptosis markers (cleaved PARP, caspase-3). For cell viability assays, cells are seeded in 96-well plates (5,000-10,000 cells/well) and treated with serial dilutions of TAS4464 hydrochloride (0.001-100 µM) for 48-96 hours. Viability is assessed by MTT, CCK-8, or CellTiter-Glo assay. IC50 values are calculated. For cell cycle analysis, treated cells are fixed, stained with propidium iodide, and analyzed by flow cytometry. Apoptosis is assessed by Annexin V/PI staining or caspase-3/7 activity assays.
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| Animal Protocol |
For in vivo efficacy studies, 6-8 week old female immunodeficient mice (e.g., nude or SCID) are used. Mice are subcutaneously implanted with 5 × 10⁶ cancer cells (hematologic or solid tumor lines) in the flank. When tumors reach approximately 100-200 mm³, animals are randomized into treatment groups (n = 6-10 per group). TAS4464 hydrochloride is formulated in vehicle (e.g., 0.5% methylcellulose or 10% DMSO in PEG400) and administered orally or intraperitoneally at doses of 1-100 mg/kg, typically on a daily or q.d. schedule for 2-4 weeks. Tumor volumes are measured twice weekly using calipers (volume = length × width² / 2). Body weights are monitored for toxicity. At study termination, tumors are excised, weighed, and processed for Western blot analysis of cullin neddylation, CRL substrate accumulation, and apoptosis markers. Blood samples may be collected for pharmacokinetic analysis. For combination studies, TAS4464 hydrochloride may be combined with other agents (e.g., chemotherapy, targeted therapy) to assess synergistic effects.
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| ADME/Pharmacokinetics |
Pharmacokinetic data for TAS4464 hydrochloride indicate favorable oral bioavailability and prolonged target inhibition. As a small-molecule inhibitor, the compound is expected to have moderate to high oral bioavailability, with a half-life sufficient for once- or twice-daily dosing. TAS4464 hydrochloride shows superior antitumor activity with prolonged target inhibition, as evidenced by sustained suppression of cullin neddylation in tumor tissues after dosing. The compound is likely metabolized by hepatic CYP450 enzymes and cleared via biliary and renal excretion. Its pharmacokinetic profile supports its use in preclinical efficacy studies. Detailed pharmacokinetic parameters, including Cmax, Tmax, AUC, half-life, and clearance, are available from published studies but may vary depending on formulation and route of administration.
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| Toxicity/Toxicokinetics |
Toxicological data for TAS4464 hydrochloride are primarily derived from preclinical toxicology studies in animal models. As a potent NAE inhibitor, the compound is expected to have on-target toxicities related to disruption of the neddylation pathway, which is essential for normal cell cycle regulation and protein homeostasis. In preclinical studies, TAS4464 hydrochloride is well-tolerated at efficacious doses with minimal body weight loss observed. The compound shows a favorable therapeutic window, with significant antitumor activity at doses that do not cause overt toxicity. Potential toxicities may include effects on rapidly dividing tissues (bone marrow, gastrointestinal tract) due to the role of the neddylation pathway in cell cycle regulation. However, no detailed toxicology data (e.g., LD50, organ-specific toxicity) have been reported in the public domain. As with all research compounds, appropriate safety precautions should be taken when handling TAS4464 hydrochloride.
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| References | |
| Additional Infomation |
TAS4464 hydrochloride is a highly potent and selective NEDD8-activating enzyme (NAE) inhibitor with an IC50 of 0.955 nM. It is a valuable research tool for investigating the neddylation pathway in oncology research, demonstrating activity in both cellular and in vivo models of cancer. NAE inhibition represents a promising therapeutic strategy for targeting cancers that are dependent on CRL-mediated protein degradation for survival. By blocking neddylation, TAS4464 hydrochloride causes accumulation of CRL substrates, including cell cycle inhibitors (p21, p27) and tumor suppressors (e.g., NRF2), leading to cell cycle arrest and apoptosis. The compound shows activity in both hematologic and solid tumors. TAS4464 hydrochloride contains an alkyne group, enabling click chemistry applications. It has not entered clinical trials and is strictly for research use only.
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| Molecular Formula |
C21H24CLFN6O6S
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| Molecular Weight |
542.968265533447
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| Exact Mass |
542.115
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| CAS # |
1848959-11-4
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| Related CAS # |
TAS4464;1848959-10-3
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| PubChem CID |
124121823
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| Appearance |
Light yellow to khaki solid powder
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
36
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| Complexity |
911
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| Defined Atom Stereocenter Count |
4
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| SMILES |
O[C@@H]1[C@@H]([C@@H](CNS(=O)(=O)N)O[C@H]1N1C=C(C#CC2C(=CC=CC=2OCC)F)C2=C(N=CN=C12)N)O.Cl
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| InChi Key |
CDCFJYCKNAWPTL-WVAMHNCPSA-N
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| InChi Code |
InChI=1S/C21H23FN6O6S.ClH/c1-2-33-14-5-3-4-13(22)12(14)7-6-11-9-28(20-16(11)19(23)25-10-26-20)21-18(30)17(29)15(34-21)8-27-35(24,31)32/h3-5,9-10,15,17-18,21,27,29-30H,2,8H2,1H3,(H2,23,25,26)(H2,24,31,32)1H/t15-,17-,18-,21-/m1./s1
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| Chemical Name |
A name could not be generated for this structure.
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| Synonyms |
TAS4464 HCl TAS 4464 hydrochloride TAS-4464 hydrochloride
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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 : ~100 mg/mL (~184.17 mM)
H2O : ~5 mg/mL (~9.21 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.83 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 (3.83 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 (3.83 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 | 1.8417 mL | 9.2086 mL | 18.4172 mL | |
| 5 mM | 0.3683 mL | 1.8417 mL | 3.6834 mL | |
| 10 mM | 0.1842 mL | 0.9209 mL | 1.8417 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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