ACVR2A,Activin receptor type IIA,ACTRIIA

Supercede's leading program, STX-001, is a novel oral small-molecule Activin receptor type II (ACTRII) kinase inhibitor currently in preclinical development for the treatment of obesity. Phase II clinical studies have demonstrated that inhibiting the ACTRII signaling pathway significantly reduces 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 monotherapy or in combination with glucagon-like peptide-1 (GLP-1)-based therapies, ACTRII inhibition is the only clinically proven mechanism capable of achieving this beneficial overall body composition change. Based on this, we have developed ActRIIA and ActRIIB enzyme activity inhibitor screening kits for research and development use.

This kit includes our self-expressed ActRIIA protein, as well as a self-designed suitable substrate, combined with the ADP kinase assay kit, which can be used to test the inhibitory effect of compounds on ActRIIA enzyme activity.

Assay System and Procedure

1. Reaction System

20 μL In the reaction system, the volume and concentration reference information for each component are shown in the table below.

2. Reagent Preparation

 Thaw the buffer at room temperature and equilibrate to room temperature before use. The thawed buffer can be stored long-term at 2-8 ℃.

 Thaw all other reagents except the buffer on ice, and equilibrate to room temperature before use. These reagents should avoid repeated freeze-thaw cycles. It is recommended to aliquot them into small tubes as needed and store properly at the recommended temperature.

 Please use the buffer provided in the kit to dilute and prepare related reagents to ensure the accuracy and stability of experimental results.

3. Reagent Preparation

3.1 1X Assay buffer D Working Solution Preparation

 Assay buffer The stock solution is 1X. Add DTT to a final concentration of 50 μM before use to prepare 1X Assay

buffer D working solution, which is used for subsequent preparation of compound, protein, substrate, and other working solutions. Prepare fresh each time.

3.2 Sample Preparation

 Use 1X Assay buffer D working solution to prepare the test samples. If the sample stock is in DMSO, it is recommended to keep the DMSO concentration consistent across the assay system, with a final concentration below 1% in the final 5 μL reaction system.

ActRIIA The protein stock solution is 0.5 mg/mL. Dilute with 1X Assay Buffer D working solution to 25 μg/mL to obtain

prepare the 2.5X protein working solution. Use 2 μL per well, with a final concentration of 10 μg/mL. For example, take 50μL ActRIIB

stock solution into 950 μL 1X Assay buffer working solution to obtain 1000 μL 2.5X protein working solution.

 The protein working solution must be prepared fresh. After initial use, it is recommended to aliquot the protein on ice as needed and store at -80 ℃ to avoid ,

Substrate +ATP solution preparation (2.5x): ATP stock solutions are all 400X, Substrate stock solutions are all 1000X. Use 1X Assay Buffer D working solution to dilute Substrate and ATP

to prepare the 2.5X substrate mixed working solution, using 2 μL per well. For example, take 2.5 μL Substrate stock solution

and 6.25 μL ATP stock solution into 991.25 μL1X Assay buffer working solution to obtain 1000 μL 2.5X

substrate working solution.

 The substrate working solution must be prepared fresh. After initial use, it is recommended to aliquot Substrate and ATP separately as needed and store at -20 ℃

to avoid repeated freeze-thaw cycles.

4. Procedure