UA-Glo®ActRIIB Enzyme Activity Inhibitor Screening Kit

Activin receptor type II (ActR IIA and IIB) is a transmembrane serine/threonine kinase receptor belonging to the TGF-β superfamily of type II receptors. ActR IIA/IIB serves as the primary receptor for activin and GDF11. Upon ligand binding, it recruits type I receptors, which phosphorylate and activate downstream SMAD2/3 signaling. Additionally, this receptor mediates the MAPK and PI3K/AKT signaling pathways.

In Phase II clinical studies for obesity treatment, inhibiting the ActRII signaling pathway has been shown to significantly reduce fat while stimulating an increase in lean body mass (muscle). For obese patients, achieving substantial fat reduction while maintaining or increasing lean body mass is a critical need. Whether as a monotherapy or in combination with incretin (GLP-1)-based therapies, ActRII inhibition is clinically the only mechanism proven to achieve this beneficial overall body composition change.

The Kinase ADP Assay Kit quantitatively measures kinase activity in enzymatic experiments. This kit determines kinase activity by quantifying the amount of ADP, a product of the kinase reaction: the amount of ADP is positively correlated with kinase activity. The ATP concentration in the kinase reaction can be up to 1 mM when using this kit. It can detect most kinases, with substrates including peptides, proteins, lipids, or sugars. The kit is suitable for high-throughput homogeneous screening of kinase inhibitors and can distinguish between ATP-competitive and non-competitive inhibitors by adjusting ATP concentration. It can also be used to measure the activity of any enzyme that produces ADP, such as ATPase.

The ActR IIB Inhibitor Screening Kit includes ActR IIB enzyme protein, substrate, kinase reaction buffer (1x Assay buffer), and Kinase ADP Assay Reagent. It can be used to assess the effect of compounds on ActR IIB enzyme activity and is particularly suitable for high-throughput screening of ActR IIB inhibitors.

The components and specifications of the ActR IIB inhibitor screening kit are as follows. The detectable number of reactions is calculated based on a 384-well plate with 5μL kinase reaction per well, under the conditions specified in the manual.

Species: Human

Molecular alias: ACVR 2B, Activin receptor type IIB, ACTR IIB

Components and specifications

The optimized reaction system for the ActR IIB kinase reaction includes 1x Assay buffer, ActR IIB enzyme at a final concentration of 15 µg/mL, substrate (Substrate) at a final concentration of 2 µM, and ATP at a final concentration of 10 µM. Customers may further optimize these conditions as needed.

The ActR IIB kinase reaction is performed in 96-well or 384-well white opaque assay plates. A reaction volume of 25 µL is recommended for 96-well plates, and 5 µL for 384-well plates. Gradient concentrations of test compounds may be added to the kinase reaction. The reaction is typically conducted at room temperature (23°C–25°C) for 1 hour, or under conditions optimized by the customer. No additional reagents are required to terminate the kinase reaction. If termination is necessary for specific experimental requirements, avoid using magnesium ion chelators such as EDTA, as the kinase ADP detection reaction requires magnesium ions.

Some commercially available ATP contains residual ADP. Due to the high sensitivity of the kinase ADP detection reagents, residual ADP in ATP can lead to high background signals. Therefore, high-purity ATP should be used for the kinase reaction, such as the ATP provided with the kit or other high-purity ATP sources (e.g., Sigma-Aldrich ATP, Cat# A2383, purity ≥99%).

The following example demonstrates the measurement of IC₅₀ for an ActR IIB inhibitor. The kinase reaction volume was 5 µL, with final concentrations of ActR IIB enzyme at 15 µg/mL, substrate (Substrate) at 2 µM, and ATP at 10 µM. The final concentration of DMSO should not exceed 1%. If the user has liquid handling equipment capable of dispensing very small volumes, it is recommended to prepare gradient-diluted test compounds directly in 100% DMSO (e.g., 25 nL of a 200x test concentration) into the 384-well assay plate. The volume and concentration of the subsequent kinase reaction working solution and substrate working solution should be adjusted accordingly (e.g., adding 4 µL of 1.25x kinase reaction solution and 1 µL of 5x substrate working solution), as long as the final concentrations match the above conditions.

ActR IIB Kinase Reaction

1)Prepare 1x Assay buffer D working solution: Add DTT to the required volume of 1x Assay buffer to a final concentration of 50 µM. This is the 1x Assay buffer D working solution, used for preparing subsequent compound, protein, and substrate working solutions. Prepare fresh for each experiment.

2)Compound preparation: Use the 1x Assay buffer D working solution to prepare 5x final concentration of 3x gradient-diluted test compounds. Note that the concentration of organic solvents (e.g., DMSO) should not exceed 3% at this step, ensuring that the final DMSO concentration does not exceed 1% after adding the ActR IIB kinase reaction solution in step 5.

3)Add 1 µL/well of gradient-diluted test compounds to a 384-well white (opaque bottom) assay plate. Include solvent control wells, positive compound control wells, and no-enzyme control wells.

4)Prepare 2.5x kinase reaction solution: Add ActR IIB to the required volume of 1x Assay buffer D to a final concentration of 37.5 µg/mL (or customer-optimized enzyme concentration). Mix gently by pipetting. This is the 2.5x kinase reaction solution.

5)Add 2 µL/well of the 2.5x ActR IIB kinase reaction solution to the 384-well plate prepared in step 3 (containing 1 µL of 5x gradient-diluted test compounds). For no-enzyme control wells, add 2 µL/well of 1x Assay buffer D working solution.

6)Incubate at room temperature for 15 minutes to allow binding of the test compounds to the ActR IIB enzyme.

7)Prepare 2.5x substrate working solution: Add Substrate to 5 µM and ATP to 25 µM in the required volume of 1x Assay buffer D working solution. Mix by pipetting or inversion. This is the 2.5x substrate working solution.

8)Add 2 µL/well of the 2.5x substrate working solution to the 384-well plate from step 6 to initiate the kinase reaction.

9)Mix by plate shaking and incubate at room temperature (23°C–25°C) for 1 hour.

10)Proceed with ADP detection as follows.

ADP Detection After ActR IIB Kinase Reaction

1.ATP Removal After Kinase Reaction

1)Thaw the kinase ATP removal reagent and equilibrate to room temperature. Mix by inversion. The luciferase reaction in the ADP detection reagent is sensitive to temperature changes. Reagents and test samples/assay plates must be equilibrated to room temperature (23°C–25°C), and the temperature should remain stable (±1°C) during testing.

2)Add 5 µL/well of ATP removal reagent to the 384-well plate containing the 5 µL/well kinase reaction. Mix by shaking. The volume ratio of kinase reaction, ATP removal reagent, and ADP detection reagent should be 1:1:2.

3)Incubate at room temperature for 40 minutes.

2.ADP Detection

1)Thaw the kinase ADP detection reagent and equilibrate to room temperature. Mix by inversion. If precipitation occurs after thawing, use the supernatant directly or centrifuge to remove the precipitate.

2)Add 10 µL/well of ADP detection reagent to the 384-well plate after ATP removal. Mix by shaking and incubate in the dark at room temperature for 40 minutes.

3)Measure luminescence signals using a luminometer or multifunctional plate reader. The signal can be read as early as 30 minutes after adding the ADP detection reagent. Due to signal stability, readings can be taken up to 3 hours or longer after adding the detection reagent if needed.

1) Different batches are not recommended for mixed use 2) Without strict validation, it is not recommended to alter the dosage of detection reagents 3) For research use only