| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| Other Sizes |
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| Targets |
IC50: 0.2 μM (eIF4A3)
Kd: 0.043 μM (eIF4A3) |
|---|---|
| ln Vitro |
Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor 53a exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of 53a and its analog 52a to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds 52a and 53a represent novel molecular probes for further study of eIF4A3, the EJC, and NMD [1].
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| Enzyme Assay |
RNA-Dependent ATPase Assay
The RNA-dependent ATPase assay was performed using the ADP-Glo assay system (Promega). Single-stranded RNA poly(U) was purchased from MP Biomedicals. The assay buffer comprised 20 mM Tris-HCl (pH7.5), 2.5 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol (DTT), and 0.01% (v/v) Tween20. To enhance ATPase activity for eIF4A, the equivalent molar concentration of MLN51 for 150 nM eIF4A3 or eIF4B and eIF4G for 100 nM eIF4A1 or eIF4A2 were added. Regarding the ATPase assays for DHX29 or BRR2, the optimal concentrations were 6.4 nM and 6.25 nM, respectively. Concentrations of ATP or RNA were set at the Km value of each substrate for each enzyme as follows: 35 μM ATP and 1.5 μg/mL poly(U) for eIF4A1 and eIF4A3; 20 μM ATP and 3.0 μg/mL poly(U) for eIF4A2; 30 μM ATP and 1.8 μg/mL poly(U) for DHX29; and 20 μM ATP and 2.5 μg/mL poly(U) for BRR2. After the addition of the substrates and test compounds, the ATPase reactions were started by the addition of the enzymes. They were incubated at room temperature for 30 min for eIF4A3, DHX29, and BRR2 or 40 min for eIF4A1 and eIF4A2. The enzymatic reactions were terminated by ADP-Glo reagent, and then ADP-Glo detection reagent was added to detect ADP. Luminescent signals were measured using an EnVision 2102 multilabel plate reader (PerkinElmer). We defined the luminescent signals of the reaction without enzyme as 100% inhibitory activity and those of the complete reaction mixture as 0% inhibitory activity. Curve fittings and calculations of IC50 values were performed using the program XLfit version 5 (ID Business Solutions) with the maximum and minimum of the curve constrained to 100 and 0, respectively. |
| Cell Assay |
HEK293T cells were transfected with the pGL4.12 SV40-Luc2CP-BGG vector and a pRL-TK vector as an internal control using Lipofectamine LTX (Thermo Fisher Scientific). The compounds were treated for 6 h after transfection of the two vectors for 24 h. Relative firefly to renilla luciferase activity was determined using the dual luciferase kit (Promega). Luminescent signals were detected by EnVision 2102 multilabel plate reader.[1]
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| References |
| Molecular Formula |
C29H23BRCLN5O2
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|---|---|
| Molecular Weight |
588.882224321365
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| Exact Mass |
587.072
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| CAS # |
2095486-67-0
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| PubChem CID |
137640621
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| Appearance |
White to off-white solid powder
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| LogP |
5.2
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
38
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| Complexity |
897
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC1=C(C=NN1C2=CC=CC(=C2)C#N)C(=O)N3CCN([C@H](C3)C4=CC=C(C=C4)Cl)C(=O)C5=CC=C(C=C5)Br
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| InChi Key |
BDGKKHWJYBQRIE-HHHXNRCGSA-N
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| InChi Code |
InChI=1S/C29H23BrClN5O2/c1-19-26(17-33-36(19)25-4-2-3-20(15-25)16-32)29(38)34-13-14-35(28(37)22-5-9-23(30)10-6-22)27(18-34)21-7-11-24(31)12-8-21/h2-12,15,17,27H,13-14,18H2,1H3/t27-/m1/s1
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| Chemical Name |
3-[4-[(3S)-4-(4-bromobenzoyl)-3-(4-chlorophenyl)piperazine-1-carbonyl]-5-methylpyrazol-1-yl]benzonitrile
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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 : ~200 mg/mL (~339.63 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (8.49 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: Solubility in Formulation 1: ≥ 5 mg/mL (8.5 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 take 100 μL of 50 mg/mL DMSO stock solution and add to 900 μL of corn oil, mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6981 mL | 8.4907 mL | 16.9814 mL | |
| 5 mM | 0.3396 mL | 1.6981 mL | 3.3963 mL | |
| 10 mM | 0.1698 mL | 0.8491 mL | 1.6981 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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