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

Summary

In this study, we enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used mTOR-rapamycin interaction as an exemplary case.

Abstract

Drug Affinity Responsive Target Stability (DARTS) is a robust method for detection of novel small molecule protein targets. It can be used to verify known small molecule-protein interactions and to find potential protein targets for natural products. Compared with other methods, DARTS uses native, unmodified, small molecules and is simple and easy to operate. In this study, we further enhanced the data analysis capabilities of the DARTS experiment by monitoring the changes in protein stability and estimating the affinity of protein-ligand interactions. The protein-ligand interactions can be plotted into two curves: a proteolytic curve and a dose-dependence curve. We have used the mTOR-rapamycin interaction as an exemplary case for establishment of our protocol. From the proteolytic curve we saw that the proteolysis of mTOR by pronase was inhibited by the presence of rapamycin. The dose-dependency curve allowed us to estimate the binding affinity of rapamycin and mTOR. This method is likely to be a powerful and simple method for accurately identifying novel target proteins and for the optimization of drug target engagement.

Introduction

Identifying small molecule target proteins is essential to the mechanistic understanding and development of potential therapeutic drugs^1,2,3. Affinity chromatography, as a classical method for identifying the target proteins of small molecules, has yielded good results^4,5. However, this method has limitations, in that chemical modification of small molecules often results in reduced or altered binding specificity or affinity. To overcome these limitations, several new strategies have recently been developed and applie....

Protocol

1. Collect and lyse cells

1. Grow 293T cells using Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum, 2 mM glutamine and 1% antibiotics. Incubate cultures at 37 °C under 5% CO₂.
   NOTE: The growth state of the cells may affect the stability of subsequent experiments.
2. Expand cells in culture until reaching 80‒90% confluence.
3. Mix 345 μL of cell lysis reagent (see the Table of Materials) with 25 μL of 20x protease inhibitor cocktail, 25 μL of 1 M sodium fluoride, 50 μL of 100 mM β-glycerophosphate, 50 μL of 50 mM sodium pyrophosphate, and 5 μL....

Results

The flow chart of the experiment is outlined in Figure 1. The result of Coomassie blue staining is shown in Figure 2. Incubation with the small molecule confers protection against proteolysis. Three bands that appear to be protected by incubation with rapamycin over vehicle control are found. The expected results from proteolytic curve experiment are shown in Figure 3. As a proof-of-principle, we examined the well-studied protein mT.......

Discussion

DARTS allows for identification of small molecule targets by exploiting the protective effect of protein binding against degradation. DARTS does not require any chemical modification or immobilization of the small molecule²⁶. This allows small molecules to be used to determine their direct binding protein targets. Standard assessment criteria for the classical DARTS method include gel staining, mass spectrometry and western blotting^12,13. .......

Materials

Name	Company	Catalog Number	Comments
100X Protease inhibitor cocktail	Sigma-Aldrich	P8340	Dilute to 20X with ultrapure water
293T cell line	ATCC	CRL-3216	DMEM medium with 10% FBS
Acetic acid	Sigma-Aldrich	A6283
BCA Protein Assay Kit	Thermo Fisher	23225
Calcium chloride	Sigma-Aldrich	C1016
Cell scraper	Thermo Fisher	179693
Coomassie Brilliant Blue R-250 Staining Solution	Bio-Rad	1610436
Dimethyl sulfoxide(DMSO)	Sigma-Aldrich	D2650
GraphPad Prism	GraphPad Software	Version 6.0	statistical analysis and drawing software
Hydrochloric acid	Sigma-Aldrich	H1758
ImageJ	National Institutes of Health	Version 1.52	image processing and analysis software
M-PER Cell Lysis Reagent	Thermo Fisher	78501
Phosphate-buffered saline (PBS)	Corning	R21-040-CV
Pronase	Roche	PRON-RO	10 mg/ml
Sodium chloride	Sigma-Aldrich	S7653
Sodium fluoride	Sigma-Aldrich	S7920
Sodium orthovanadate	Sigma-Aldrich	450243
Sodium pyrophosphate	Sigma-Aldrich	221368
Trizma base	Sigma-Aldrich	T1503	adjust to pH 8.0
β-glycerophosphate	Sigma-Aldrich	G9422