IC50: 0.2 μM (eIF4A3)
Kd: 0.043 μM (eIF4A3)
The primary target of eIF4A3-IN-1 is eukaryotic initiation factor 4A3 (eIF4A3), an ATP-dependent RNA helicase that is a core component of the exon junction complex (EJC). eIF4A3 plays critical roles in RNA splicing, translation, and nonsense-mediated mRNA decay (NMD). eIF4A3-IN-1 acts as a selective inhibitor that binds to a non-ATP binding site of eIF4A3 with an IC₅0 of 0.26 microM and a Kd of 0.043 microM. By binding to this allosteric site, the compound inhibits eIF4A3 function without competing with ATP, providing specificity. eIF4A3 is a validated target for cancer research and other diseases driven by aberrant RNA metabolism.

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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In vitro studies demonstrate that eIF4A3-IN-1 is a selective inhibitor of eIF4A3 with an IC₅0 of 0.26 microM and a Kd of 0.043 microM. The compound binds to a non-ATP binding site of eIF4A3 and shows significant cellular nonsense-mediated RNA decay (NMD) inhibition at 10 microM and 3 microM. These properties make eIF4A3-IN-1 a valuable probe for studying eIF4A3, the exon junction complex (EJC), and NMD. The compound is also a valuable research tool for exploring RNA-binding protein function and post-transcriptional control mechanisms.

In vivo studies of eIF4A3-IN-1 are limited, as it is primarily used as a research tool in cellular assays. However, given its potent and selective inhibition of eIF4A3 with an IC₅0 of 0.26 microM and a Kd of 0.043 microM, the compound may have potential for in vivo efficacy studies in animal models of diseases where eIF4A3 plays a role, such as cancer. eIF4A3 is involved in RNA splicing and translation, processes that are often dysregulated in cancer. Further studies are needed to evaluate its pharmacokinetic properties, bioavailability, and efficacy in vivo.

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.

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For in vitro enzyme/receptor binding assays, eIF4A3-IN-1 is evaluated using helicase activity assays that measure the ATP-dependent RNA unwinding by eIF4A3. The compound is incubated with recombinant eIF4A3 protein, RNA substrate, and ATP at various concentrations. eIF4A3-mediated RNA unwinding is quantified using fluorescence-based or gel-based methods. IC₅0 values and Kd values are determined from dose-response curves and binding studies. Binding affinity to the non-ATP binding site can be assessed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). Selectivity profiling against other helicases may be performed to confirm specificity.

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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For in vitro cellular experiments, eIF4A3-IN-1 is tested in cell lines to evaluate its effects on eIF4A3 function and downstream RNA processing. Cells are cultured in appropriate media and treated with various concentrations of the compound (typically ranging from nanomolar to micromolar). eIF4A3 inhibition is assessed by measuring its effects on nonsense-mediated mRNA decay (NMD) using reporter assays or by measuring the levels of NMD target transcripts by qPCR. Gene expression changes are analyzed by RNA sequencing. Cell viability and proliferation are monitored using standard assays. The compound's effects on EJC formation can be assessed using RNA immunoprecipitation.

For in vivo animal experiments, eIF4A3-IN-1 can be administered to animals via various routes including oral gavage, intravenous injection, or intraperitoneal injection, depending on its solubility and pharmacokinetic properties. The compound's efficacy can be evaluated in tumor models or other disease models where eIF4A3 plays a role. Typical dosing regimens may range from 1 to 50 mg/kg administered daily or intermittently. Pharmacodynamic markers such as NMD inhibition or RNA splicing changes are measured in tissues. Tumor volume, body weight, and overall health are monitored.

Pharmacokinetic properties of eIF4A3-IN-1 are not extensively detailed in the public literature. As a small molecule with a molecular weight of approximately 602.7 g/mol, it may have reasonable oral bioavailability and tissue distribution. The presence of bromine and chlorine atoms may influence its metabolic stability and clearance. Detailed parameters such as Cₘₐₓ, Tₘₐₓ, AUC, half-life, and clearance would need to be determined through comprehensive PK studies. The compound's protein binding, metabolism, and excretion pathways remain to be fully characterized. Formulation development may be necessary for optimal in vivo administration.

Toxicological data for eIF4A3-IN-1 are limited, as it is primarily a research tool. As an eIF4A3 inhibitor, its toxicity would depend on the importance of eIF4A3 for normal cellular function. eIF4A3 is a core component of the exon junction complex and plays critical roles in RNA splicing and translation. Its inhibition could have significant effects on gene expression and cell function. Comprehensive toxicology studies would be needed for further development. Appropriate safety precautions should be taken when handling this compound, including the use of personal protective equipment and adherence to institutional safety guidelines.

[1]. Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors. J Med Chem. 2017 Apr 27;60(8):3335-3351.

eIF4A3-IN-1 is a research compound used to study eIF4A3 biology and RNA processing. No clinical trials or regulatory approvals have been reported for this compound as a therapeutic agent. It is available from various chemical suppliers for research purposes only. The compound is a selective eIF4A3 inhibitor with an IC₅0 of 0.26 microM and a Kd of 0.043 microM that binds to a non-ATP binding site. It shows significant cellular NMD inhibition at 10 microM and 3 microM and can be used as a probe for studying eIF4A3, EJC, and NMD.

C29H23BRCLN5O2

588.882224321365

587.072

2095486-67-0

137640621

White to off-white solid powder

5.2

0

4

4

38

897

1

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

BDGKKHWJYBQRIE-HHHXNRCGSA-N

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

3-[4-[(3S)-4-(4-bromobenzoyl)-3-(4-chlorophenyl)piperazine-1-carbonyl]-5-methylpyrazol-1-yl]benzonitrile

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~200 mg/mL (~339.63 mM)

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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)