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| Targets |
NIK (Ki = 0.23 nM)
NIK SMI1 targets NF-κB-inducing kinase (NIK), also known as MAP3K14, a key kinase in the noncanonical NF-κB signaling pathway. NIK activates the noncanonical NF-κB pathway by phosphorylating IKKα, leading to processing of p100 to p52 and nuclear translocation of RelB-containing NF-κB complexes. By inhibiting NIK, NIK SMI1 blocks noncanonical NF-κB activation. This pathway is involved in immune regulation, B cell survival, and inflammation. |
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| ln Vitro |
NIK SMI1 (Compound 4f) has an IC50 of 0.23±0.17 nM and blocks the NIK-catalyzed hydrolysis of ATP to ADP (fluorescence polarization, FP). With an IC50 of 34±6 nM, NIK SMI1 inhibits the expression of the NIK SMI1 response element-regulated firefly luciferase reporter gene in HEK293 cells. The nuclear translocation of p52 (RelB) is shown to be inhibited by NIK SMI1 (IC50=70 nM), as would be expected for a NIK inhibitor. NIK SMI1 has an IC50 of 373±64 nM and inhibits BAFF-induced B cell (mouse) survival in vitro[1].
In vitro, NIK SMI1 potently inhibits NIK-catalyzed ATP hydrolysis with an IC₅₀ of 0.23 ± 0.17 nM. It shows potency against human NIK (IC₅₀ = 230 pM) and mouse NIK (IC₅₀ = 395 pM). The compound is highly selective for NIK over other kinases including KHS1, PKD1, and LRRK2. In cellular assays, it inhibits noncanonical NF-κB signaling, reducing p100 processing and p52 production. |
| ln Vivo |
NIK SMI1 is administered orally to C57BL/6 mice twice daily for 7 days, or as a positive control, three injections of recombinant BAFF receptor fusion protein (Br3- mIgG2a) are given. The saturation of the clearance mechanisms is what causes the nonlinearity of exposure in relation to dose between 100 and 200 mg/kg. With doses of 20, 32, 18, and 7.8 mL/kg per min in the SD rat, CD-1 mouse, beagle, and cynomologous monkey, respectively, the pharmacology of NIK SMI1 is investigated. The respective volumes of distribution (Vd, L/kg) are 1.35, 1.58, 0.778, and 1.39[1].
In vivo, NIK SMI1 shows efficacy in a murine experimental lupus model. It is orally active and selectively disrupts noncanonical NF-κB activation. Pharmacological studies have been conducted in SD rats, CD-1 mice, beagle dogs, and cynomolgus monkeys at infusion rates of 20, 32, 18, and 7.8 mL/kg/min, respectively. The compound is a valuable tool for studying NIK function in autoimmune diseases. |
| Enzyme Assay |
In vitro kinase assays for NIK SMI1 typically use recombinant NIK enzyme and a peptide substrate. The compound is dissolved in DMSO and diluted in assay buffer (50 mM HEPES, pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT). Reactions are initiated by adding ATP (at Km concentration) and incubated at 30°C for 30-60 minutes. Phosphorylated substrate is detected by fluorescence polarization, AlphaScreen, or radiometric methods. IC₅₀ values are calculated from dose-response curves. Selectivity profiling is performed against a panel of kinases.
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| Cell Assay |
For the proliferation assays and the survival assays, human B cells are re-suspended in RPMI containing 2.5% and 10% FBS, respectively. For the survival and proliferation assays, mouse B cells are plated in Co-star 96-well plates at 50,000 or 150,000 cells per well. The cells are given substances (like NIK SMI1) that have been diluted in DMSO (final DMSO assay concentration=0.1%). The cells are incubated with NIK SMI1 for an hour at 37°C. After adding stimulation to the plates, survival or proliferation is assessed after four days. Cells are treated with anti-IgM (20 µg/mL), anti-rhCD40L (10 µg/mL), or anti-mouse CD40 (100 ng/mL) for the proliferation assays. For the BAFF survival assay, cells are treated with human or mouse rBAFF at 10 ng/mL, followed by Cell Titer Glo to assess survival on day four[1].
For cell-based assays, relevant cell lines (e.g., B cells or multiple myeloma cells) are cultured in RPMI-1640 or DMEM with 10% FBS. Cells are seeded in 96-well plates and treated with NIK SMI1 at various concentrations (0.001-10 μM) for 1-24 hours. Noncanonical NF-κB activation is assessed by measuring p100 processing to p52 by Western blotting. Nuclear translocation of RelB is assessed by immunofluorescence. Cell viability is assessed by MTT or CellTiter-Glo. All treatments include vehicle controls and are performed in triplicate. |
| Animal Protocol |
Mice: The C57BL/6 mice used are age-matched. These tests only make use of female mice. NIK SMI1 is administered orally in single doses of 10, 20, 60, 100, and 200 mg/kg. Animals are manually restrained before being dosed orally with a gavage needle of the proper size for PO dosing. Animals are observed for any aspiration or distress symptoms, such as lethargy, pale extremities, and abnormal breathing. For sample collection, three mice per group are tail-pricked eight times with a 27 G needle (lateral tail vein). A pre-filled costar cluster tube containing 40 μL of 1.7 mg/mL EDTA/water and 10 μL of blood is filled with the blood at each timepoint, and the tube is sealed, votexed for five seconds, and then stored on dry ice. Samples are moved to a freezer at -80°C for storage[1].
In vivo, NIK SMI1 is typically administered orally to rodents. For lupus models, compound is dosed at various regimens. Endpoints include autoantibody levels, proteinuria, kidney histopathology, and survival. Pharmacodynamic markers (p52 levels, RelB nuclear translocation) are measured in tissues. Pharmacological studies have been conducted in multiple species. Pharmacokinetic parameters are determined from plasma samples. All procedures follow institutional animal care guidelines. |
| ADME/Pharmacokinetics |
NIK SMI1 is an orally active NIK inhibitor. It shows high potency against human and mouse NIK (IC₅₀ = 230 pM and 395 pM, respectively). Pharmacological studies have been conducted in SD rats, CD-1 mice, beagle dogs, and cynomolgus monkeys. Distribution volume (Vd) has been characterized. Detailed pharmacokinetic parameters are available from preclinical studies. The compound is designed for research applications in immunology.
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| Toxicity/Toxicokinetics |
Toxicology data for NIK SMI1 are limited in publicly available sources. As a kinase inhibitor, potential off-target effects should be considered. The compound has been evaluated in preclinical safety studies in multiple species. The compound is for research use only and not intended for human therapeutic applications. Standard safety pharmacology and toxicology studies would be required for therapeutic development.
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| References | |
| Additional Infomation |
NIK SMI1 is a research-grade NIK inhibitor for studying noncanonical NF-κB signaling. Its primary applications include immunology, autoimmune disease research, and drug discovery. The compound is not approved for clinical use. It is commercially available from chemical suppliers for research purposes only. Its mechanism involves ATP-competitive inhibition of NIK, blocking noncanonical NF-κB activation.
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| Molecular Formula |
C20H19N3O4
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|---|---|
| Molecular Weight |
365.382564783096
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| Exact Mass |
365.137
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| Elemental Analysis |
C, 65.74; H, 5.24; N, 11.50; O, 17.51
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| CAS # |
1660114-31-7
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| Related CAS # |
1660114-31-7;
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| PubChem CID |
117902000
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| Appearance |
Off-white to pink solid powder
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| LogP |
0.9
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
27
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| Complexity |
654
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CN1CC[C@](C1=O)(C#CC2=CC(=CC=C2)C3=NC(=CC(=C3)OC)C(=O)N)O
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| InChi Key |
LQSHXYHWYGKAMX-FQEVSTJZSA-N
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| InChi Code |
InChI=1S/C20H19N3O4/c1-23-9-8-20(26,19(23)25)7-6-13-4-3-5-14(10-13)16-11-15(27-2)12-17(22-16)18(21)24/h3-5,10-12,26H,8-9H2,1-2H3,(H2,21,24)/t20-/m0/s1
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| Chemical Name |
6-[3-[2-[(3R)-3-hydroxy-1-methyl-2-oxopyrrolidin-3-yl]ethynyl]phenyl]-4-methoxypyridine-2-carboxamide
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| Synonyms |
NIK-SMI1; NIK SMI1; NIKSMI1
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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) |
DMSO: 73~125 mg/mL (342.1~199.8 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.84 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (6.84 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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 (5.69 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. Solubility in Formulation 4: ≥ 2.08 mg/mL (5.69 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. Solubility in Formulation 5: ≥ 2.08 mg/mL (5.69 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7369 mL | 13.6844 mL | 27.3688 mL | |
| 5 mM | 0.5474 mL | 2.7369 mL | 5.4738 mL | |
| 10 mM | 0.2737 mL | 1.3684 mL | 2.7369 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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