| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg | |||
| 250mg | |||
| Other Sizes |
| Targets |
NAE (GI50 = 5.5 μM)
NEDD8 Activating Enzyme (NAE) (IC50 = 0.5 μM in NAE enzymatic activity assay; > 100-fold selectivity over UBA1) [1] |
|---|---|
| ln Vitro |
M22 (0.37-90 μM; 24 h) selectively blocks the neddylation pathway and inhibits the breakdown of CRL substrates in A549 cells[1].
M22 (0.1-100 μM; 48 h) inhibits A549 cell proliferation completely at 30 µM (GI50=5.5 µM, GI90=19.3 µM)[1]. M22 (15–30 μM; 36 h) stimulates apoptosis in the A549 cell line[1]. NAE enzymatic activity inhibition: NAE-IN-M22 potently inhibits recombinant human NAE-mediated NEDD8 activation with an IC50 of 0.5 μM. It exhibits > 100-fold selectivity over UBA1 (another E1 ubiquitin-activating enzyme), with IC50 > 50 μM for UBA1 [1] - Cancer cell antiproliferation: The compound inhibits the proliferation of multiple cancer cell lines in a concentration-dependent manner. IC50 values (72-hour, MTT assay) are 2.3 μM (HCT116 colorectal cancer), 3.1 μM (A549 non-small cell lung cancer), and 2.7 μM (HeLa cervical cancer). Normal human foreskin fibroblasts (HFF) show higher tolerance with an IC50 > 30 μM [1] - Inhibition of cellular NEDDylation: NAE-IN-M22 (1–10 μM) blocks NEDDylation of cullin proteins (cullin 1, cullin 3) in HCT116 cells, as detected by western blot. At 5 μM, the level of NEDDylated cullin 1 is reduced by 80% compared to the vehicle control [1] - Apoptosis induction: Treatment with NAE-IN-M22 (2–10 μM) induces apoptosis in HCT116 cells. At 5 μM, Annexin V-positive apoptotic cells account for 45% of total cells, accompanied by cleavage of caspase-3 and PARP (western blot verification) [1] - Cullin-RING ligase (CRL) activity suppression: The compound inhibits CRL-mediated substrate degradation. In HCT116 cells, NAE-IN-M22 (5 μM) increases the protein level of p27 (a CRL substrate) by 2.5-fold, confirming CRL pathway inhibition [1] |
| ln Vivo |
slows the growth of tumors in nude mice grafted with AGS[1].
M22 (0.36-90 μM) exhibits a low level of acute toxicity in zebrafish[1]. |
| Enzyme Assay |
NAE NEDD8 activation assay: Recombinant human NAE (NAE1-UBA3 heterodimer) was incubated with NEDD8, ATP, and serial dilutions of NAE-IN-M22 (0.01 μM–50 μM) in reaction buffer at 37°C for 60 minutes. The reaction products (NEDD8-NAE thioester conjugate) were separated by non-reducing SDS-PAGE and detected by western blot using anti-NEDD8 antibody. The inhibition rate was calculated by quantifying the intensity of the conjugate band, and IC50 was derived from dose-response curves [1]
- UBA1 selectivity assay: Parallel assays were performed using recombinant human UBA1 and ubiquitin (instead of NEDD8) under the same reaction conditions. NAE-IN-M22 at concentrations up to 50 μM showed < 5% inhibition of UBA1-mediated ubiquitin activation, confirming NAE selectivity [1] |
| Cell Assay |
Cell viability (MTT) assay: Cancer cells (HCT116, A549, HeLa) and normal HFF cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of NAE-IN-M22 (0.1 μM–50 μM) were added, and cells were cultured for 72 hours. MTT reagent was added, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. IC50 values were calculated from dose-response curves of cell viability [1]
- Western blot for NEDDylation and apoptosis markers: HCT116 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with NAE-IN-M22 (1–10 μM) for 24 hours. Cells were lysed in RIPA buffer, proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against NEDDylated cullin 1, total cullin 1, p27, cleaved caspase-3, cleaved PARP, and β-actin (loading control) [1] - Apoptosis detection (Annexin V-FITC/PI): HCT116 cells were treated with NAE-IN-M22 (2–10 μM) for 48 hours, harvested by trypsinization, washed with cold PBS, and stained with Annexin V-FITC and PI. Flow cytometry was used to quantify apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) [1] - Clonogenic assay: HCT116 cells were seeded in 6-well plates (1×10³ cells/well) and treated with NAE-IN-M22 (0.5–5 μM) for 14 days. Colonies were fixed with formaldehyde, stained with crystal violet, and counted manually. Colony formation efficiency was expressed as a percentage of the vehicle control [1] |
| References | |
| Additional Infomation |
Background: NEDD8 activator (NAE) is a key enzyme in the NEDDylation pathway, which regulates the activity of Cullin-RING ligase (CRL). CRL-mediated ubiquitination and degradation of cell cycle regulators and tumor suppressor factors promote cancer cell proliferation, making NAE a promising anticancer target [1].
- Mechanism of action: NAE-IN-M22 binds to the ATP-binding pocket of NAE, inhibiting its catalytic activity and blocking NEDD8 activation. This prevents NEDDylation of Cullin, leading to CRL inactivation, accumulation of CRL substrates (e.g., p27), and ultimately inducing cell cycle arrest and apoptosis in cancer cells [1]. - Chemical characteristics: The compound has a piperidine-4-amine skeleton, which was discovered through structure-based virtual screening targeting the active site of NAE. The compound exhibits moderate water solubility (1.2 mg/mL) in pH 7.4 buffer and good stability in cell culture medium (half-life > 24 hours) [1] - Therapeutic potential: As a novel NAE inhibitor, NAE-IN-M22 has shown potent anticancer activity in vitro and can be further optimized as a lead compound to improve its potency, solubility and in vivo efficacy for the treatment of solid tumors [1] |
| Molecular Formula |
C20H24CL2N2
|
|---|---|
| Molecular Weight |
363.323963165283
|
| Exact Mass |
362.131
|
| Elemental Analysis |
C, 66.12; H, 6.66; Cl, 19.51; N, 7.71
|
| CAS # |
864420-54-2
|
| Related CAS # |
864420-54-2
|
| PubChem CID |
6456370
|
| Appearance |
Solid powder
|
| LogP |
5.1
|
| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
2
|
| Rotatable Bond Count |
6
|
| Heavy Atom Count |
24
|
| Complexity |
352
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
WMEXQHGRMWPOEF-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C20H24Cl2N2/c21-18-7-6-17(20(22)14-18)8-11-23-19-9-12-24(13-10-19)15-16-4-2-1-3-5-16/h1-7,14,19,23H,8-13,15H2
|
| Chemical Name |
1-benzyl-N-[2-(2,4-dichlorophenyl)ethyl]piperidin-4-amine
|
| Synonyms |
M22 NEDD8 inhibitor; M 22; M22; M-22
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| Solubility (In Vitro) |
DMSO: 73~250 mg/mL (200.9~688.1 mM)
Water: ~4 mg/mL (~11.0 mM) Ethanol: ~73 mg/mL (~200.9 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.72 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 (5.72 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 (5.72 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 | 2.7524 mL | 13.7620 mL | 27.5239 mL | |
| 5 mM | 0.5505 mL | 2.7524 mL | 5.5048 mL | |
| 10 mM | 0.2752 mL | 1.3762 mL | 2.7524 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
