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| Targets |
Eukaryotic initiation factor 4A-3 (eIF4A3). eIF4A3 is a member of the DEAD-box RNA helicase family and is a core component of the exon junction complex (EJC), which is deposited on mRNAs after splicing. eIF4A3 plays critical roles in mRNA splicing, export, translation, and nonsense-mediated decay (NMD). The active enantiomer (eIF4A3-IN-2) binds noncompetitively and selectively to eIF4A3, inhibiting its ATPase and helicase activities. (R)-eIF4A3-IN-2 is the inactive enantiomer used as a negative control.
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| ln Vitro |
Asp-Glu-Ala-Asp (DEAD) box family adenosine triphosphate (ATP)-dependent RNA helicase is known as eukaryotic initiation factor 4A-3 (eIF4A3) [1].
In cell-free biochemical assays, eIF4A3-IN-2 inhibits eIF4A3 ATPase and helicase activities with an IC50 of 110 nM. The inhibition is noncompetitive with respect to ATP, meaning the inhibitor binds to a site distinct from the ATP-binding site. Specificity is high, with eIF4A3-IN-2 showing minimal activity against other DEAD-box helicases. The (R)-enantiomer has substantially lower inhibitory activity; precise IC50 values for the (R)-enantiomer are not publicly disclosed. The compound impacts RNA binding and unwinding by eIF4A3. |
| ln Vivo |
Cell-based assays for (R)-eIF4A3-IN-2 are not extensively reported, as this compound is the less active enantiomer used as a control. The active enantiomer eIF4A3-IN-2 inhibits eIF4A3 function in cells, impacting mRNA splicing and nonsense-mediated decay (NMD) processes. Inhibition of eIF4A3 reduces the expression of NMD-sensitive transcripts and alters alternative splicing patterns. The (R)-enantiomer is expected to have minimal cellular activity, making it an appropriate negative control for distinguishing specific target engagement from off-target effects. At concentrations used for the active enantiomer (e.g., 0.1-10 uM), the (R)-enantiomer shows no significant effects.
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| Enzyme Assay |
The ATPase activity of eIF4A3 is measured in a cell-free format using a coupled luminescent assay. Recombinant eIF4A3 protein (typically 10-100 nM) is incubated with varying concentrations of (R)-eIF4A3-IN-2 or the active enantiomer (0.001-100 uM) in assay buffer containing ATP (0.1-1 mM), RNA (poly(U) or other RNA substrate), Mg2+, and KCl. ATP hydrolysis is quantitated using a Kinase-Glo® or ATPase-Glo™ reagent that measures remaining ATP or ADP production. Luminescence is measured on a plate reader, and IC50 values are calculated from dose-response curves. Alternatively, the malachite green assay can be used to measure released phosphate. For helicase activity, a FRET-based or gel-based unwinding assay is used with fluorescently labeled duplex RNA.
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| Cell Assay |
Cell-based assays for eIF4A3 inhibition are not standard for the (R)-enantiomer, which is used as a control. For the active enantiomer, cells (e.g., HeLa, HEK293) are treated with eIF4A3-IN-2 (0.1-10 uM) for 24-72 hours. NMD efficiency is assessed using reporter constructs (e.g., beta-globin or TCR-beta reporters containing premature termination codons) by measuring mRNA and protein levels. Alternative splicing is analyzed by RT-PCR of eIF4A3 target genes. The (R)-enantiomer is added at the same concentrations as the control, and any minor effects are noted to account for off-target or non-specific activities. Cell viability is typically not affected by eIF4A3 inhibitors unless long-term NMD inhibition disrupts cellular homeostasis.
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| Animal Protocol |
In vivo animal studies for (R)-eIF4A3-IN-2 have not been reported, as this compound is the less active control. The active enantiomer eIF4A3-IN-2 has been used in cancer research. In mouse xenograft models of eIF4A3-dependent cancers (e.g., certain leukemias, gliomas, or solid tumors), treatment with the active enantiomer (e.g., 10-50 mg/kg IP or oral) reduces tumor growth and alters NMD-dependent gene expression. The (R)-enantiomer would be used as a negative control to confirm that in vivo effects are due to specific eIF4A3 inhibition rather than non-target activities. Typical study designs include daily dosing for 2-4 weeks, tumor volume measurement, and endpoint analysis of NMD-sensitive transcripts.
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| ADME/Pharmacokinetics |
Dedicated pharmacokinetic studies for (R)-eIF4A3-IN-2 have not been published. As the less active enantiomer, it is presumed to have similar physicochemical properties (MW 602.71, C25H19Br2ClN4O2) to the active enantiomer. Standard PK parameters such as half-life, oral bioavailability, and tissue distribution would need to be evaluated for the racemate or individual enantiomers. For in vivo studies using the active enantiomer, typical formulations use DMSO-based vehicles diluted in PEG300 or saline. The (R)-enantiomer is expected to have similar absorption, distribution, metabolism, and excretion (ADME) properties due to its structural identity, though enantioselective metabolism cannot be ruled out.
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| Toxicity/Toxicokinetics |
Preclinical toxicology data for (R)-eIF4A3-IN-2 are not available. As the less active enantiomer, it is not the focus of drug development and is used only as a research control tool. The active enantiomer eIF4A3-IN-2, when used at therapeutic doses, is expected to be well-tolerated in animal models. Common toxicity concerns for RNA helicase inhibitors may include effects on cellular RNA metabolism and splicing, but these are generally not associated with acute toxicity. Standard laboratory safety precautions should be followed. No human clinical data are available.
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| References | |
| Additional Infomation |
(R)-eIF4A3-IN-2 is a research-grade compound used as a negative control for eIF4A3-IN-2. The active enantiomer (not the (R)-form) is a highly selective and noncompetitive inhibitor of eIF4A3, an RNA helicase involved in mRNA splicing and nonsense-mediated decay (NMD). The compound is useful for studying cancer biology, novel therapeutic pathways, and the regulation of gene expression. eIF4A3 is overexpressed in several cancers, and its inhibition represents a potential therapeutic strategy. The compound's molecular formula is C25H19Br2ClN4O2, molecular weight 602.71. (R)-eIF4A3-IN-2 is not approved for clinical use. The active enantiomer's selectivity stems from binding to a noncompetitive site unique to eIF4A3, minimizing off-target effects on other helicases.
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| Molecular Formula |
C25H19BR2CLN4O2
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| Molecular Weight |
602.7050
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| Exact Mass |
601.954
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| CAS # |
2095484-82-3
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| Related CAS # |
eIF4A3-IN-2;2095677-20-4
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| PubChem CID |
131953886
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| Appearance |
White to light yellow solid powder
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| LogP |
5
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
34
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| Complexity |
744
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| Defined Atom Stereocenter Count |
1
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| SMILES |
BrC1C([H])=C([H])C2=C(C([H])=NN2C=1[H])C(N1C([H])([H])C([H])([H])N(C(C2C([H])=C([H])C(=C([H])C=2[H])Br)=O)[C@]([H])(C2C([H])=C([H])C(=C([H])C=2[H])Cl)C1([H])[H])=O
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| InChi Key |
WKKAVTNXNVPCCN-QHCPKHFHSA-N
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| InChi Code |
InChI=1S/C25H19Br2ClN4O2/c26-18-5-1-17(2-6-18)24(33)31-12-11-30(15-23(31)16-3-8-20(28)9-4-16)25(34)21-13-29-32-14-19(27)7-10-22(21)32/h1-10,13-14,23H,11-12,15H2/t23-/m0/s1
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| Chemical Name |
(4-bromophenyl)-[(2R)-4-(6-bromopyrazolo[1,5-a]pyridine-3-carbonyl)-2-(4-chlorophenyl)piperazin-1-yl]methanone
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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 : ~50 mg/mL (~82.96 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.15 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 25.0 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: ≥ 2.5 mg/mL (4.15 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6592 mL | 8.2959 mL | 16.5917 mL | |
| 5 mM | 0.3318 mL | 1.6592 mL | 3.3183 mL | |
| 10 mM | 0.1659 mL | 0.8296 mL | 1.6592 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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