A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction

Area of Science:

• Biochemistry
• Pharmacology
• Drug Discovery

Background:

• Drug Affinity Responsive Target Stability (DARTS) is a validated technique for identifying small molecule protein targets.
• Existing methods for target identification can be complex and require modified small molecules.
• DARTS offers a simpler approach using native small molecules for target detection.

Purpose of the Study:

• To enhance the data analysis capabilities of DARTS experiments.
• To enable the monitoring of protein stability changes.
• To estimate the affinity of protein-ligand interactions.

Main Methods:

• Developed enhanced data analysis for DARTS experiments.
• Monitored protein stability and quantified protein-ligand binding affinity.
• Utilized proteolytic and dose-dependence curves for analysis.

Main Results:

• Demonstrated the protocol using the mTOR-rapamycin interaction.
• Observed inhibition of mTOR proteolysis by rapamycin.
• Estimated the binding affinity between rapamycin and mTOR.

Conclusions:

• The enhanced DARTS method provides powerful and simple tools for target identification.
• This approach facilitates accurate identification of novel protein targets.
• The method aids in optimizing drug target engagement for drug development.