ActRIIB Enzyme Activity Inhibitor Screening Kit

Supercede's lead 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 patients with obesity, 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 to achieve 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 ActRIIB protein and self-designed suitable substrates, combined with the ADP kinase assay kit, to test the inhibitory effects of compounds on ActRIIB enzyme activity.

Assay System and Procedure

1, Reaction System

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

2, Reagent Preparation

 Please thaw the buffer at room temperature and equilibrate to room temperature before use. Thawed buffers can be stored long-term at2-8 ℃.

 Please thaw other reagents except buffers 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 buffers included in the kit to dilute and prepare relevant reagents to ensure the accuracy and stability of experimental results.

3, Reagent Preparation

3.1 1X Assay buffer D Preparation of Working Solution

 Assay buffer The stock solution is1X, add DTT to a final concentration of50 μM before use to prepare1X Assay

buffer Dworking solution, used for subsequent preparation of compound, protein, substrate, and other working solutions. Prepare fresh for each use.

3.2 Sample Preparation

 Use1X Assay buffer D working solution to prepare test samples. If the sample stock is inDMSO, it is recommended to keep theDMSO concentration consistent in the detection system, with a final concentration below1%in the final5 μL reaction system.

ActRIIB Protein stock is0.3 mg/mL, dilute with1X Assay Buffer Dworking solution to25 μg/mL, to prepare

a2.5X protein working solution. Use2 μLper well, resulting in a final concentration of10 μg/mL. For example, take83.3 μL ActRIIB

stock solution into916.7 μL 1X Assay buffer working solution to obtain1000 μL 2.5X protein working solution.

 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 Stocks are both400X, Substrate stocks are both1000X. Use1X Assay Buffer Dworking solution to diluteSubstrate andATP

to prepare a2.5X substrate mixed working solution. Use2 μLper well. For example, take2.5 μL Substrate stock solution

and6.25 μL ATP stock solution into991.25 μL1X Assay buffer working solution to obtain1000 μL 2.5X

substrate working solution.

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

to avoid repeated freeze-thaw cycles.

4, Operation Steps