This work was supported partly by NIH research grants R01NS103931, R01AR062207, R01AR061484, and a DOD research grant W81XWH-16-1-0482.

1. Collect and lyse cells
<ol>
	<li>Grow 293T cells using Dulbecco&#39;s modified Eagle medium (DMEM) with 10% fetal bovine serum, 2 mM glutamine and 1% antibiotics. Incubate cultures at 37 &#176;C under 5% CO2. 
	NOTE: The growth state of the cells may affect the stability of subsequent experiments.</li>
	<li>Expand cells in culture until reaching 80&#8210;90% confluence.</li>
	<li>Mix 345 &#956;L of cell lysis reagent (see the Table of Materials) with 25 &#956;L of 20x protease inhibi...

<ol>
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L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coomassie+blue+staining.">Coomassie blue staining.</a> Methods in Enzymology. 541, 161-167 (2014).</li><li>Domon, B., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+and+protein+analysis.">Mass spectrometry and protein analysis.</a> Science. 312 (5771), 212-217 (2006).</li><li>Hnasko, T. S., Hnasko, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> Methods in Molecular Biology. 1318, 87-96 (2015).</li><li>Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. 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The authors have nothing to disclose.

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 blotting12,13. ...

Identifying small molecule target proteins is essential to the mechanistic understanding and development of potential therapeutic drugs1,2,3. Affinity chromatography, as a classical method for identifying the target proteins of small molecules, has yielded good results4,5. 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 applied to identify the small molecule targets without chemical modification of the small molecules. These direct methods for target identification of label-free small molecules include drug affinity responsive target stability (DARTS)6, stability of proteins from rates of oxidation (SPROX)7, cellular thermal shift assay (CETSA)8,9, and thermal proteome profiling (TPP)10. These methods are highly advantageous because they use natural, unmodified small molecules and rely only on direct binding interactions to find target proteins11.
Among these new methods, DARTS is a comparatively simple methodology that can easily be adopted by most labs12,13. DARTS depends on the concept that ligand-bound proteins demonstrate modified susceptibility to enzymatic degradation relative to unbound proteins. The new target protein can be detected by examination of the altered band in SDS-PAGE gel through liquid chromatography-mass spectrometry (LC-MS/MS). This approach has been successfully implemented for identification of previously unknown targets of natural products and drugs14,15,16,17,18,19. It is also powerful as a means to screen or validate binding of compounds to a specific protein20,21. In this study, we present an improvement to the experiment by monitoring the changes in protein stability with small molecules and identifying protein-ligand binding affinities. We use mTOR- rapamycin interaction as an example to demonstrate our approach.

<table>
	<tbody>
		<tr>
			<td>100X Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
			<td>Dilute to 20X with ultrapure water</td>
		</tr>
		<tr>
			<td>293T cell line</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>DMEM medium with 10% FBS</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Fisher</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Thermo Fisher</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250 Staining Solution</td>
			<td>Bio-Rad</td>
			<td>1610436</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide(DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software</td>
			<td>Version 6.0</td>
			<td>statistical analysis and drawing software</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>Version 1.52</td>
			<td>image processing and analysis software</td>
		</tr>
		<tr>
			<td>M-PER Cell Lysis Reagent</td>
			<td>Thermo Fisher</td>
			<td>78501</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Corning</td>
			<td>R21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Roche</td>
			<td>PRON-RO</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>S7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium orthovanadate</td>
			<td>Sigma-Aldrich</td>
			<td>450243</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>221368</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td>adjust to pH 8.0</td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

a semi-quantitative drug affinity responsive target stability (darts) assay for studying rapamycin/mtor interaction

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.

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...

Watch this Scientific Journal Video about A Semi-Quantitative Drug Affinity Responsive Target Stability DARTS assay for studying Rapamycin/mTOR interaction at JoVE.com

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

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.

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

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 ...

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. In this study, we further enhance 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 against two curves, a proteolytic curve and a dose-dependence curve. We've used the mTOR-rapamycin interaction as an exemplary case for the establishment of our protocol. Grow 293T cells using DMEM with 10%fetal bovine serum, two-millimolar glutamine, and 1%antibiotics.Incubate the cultures at 37 degrees Celsius under 5%carbon dioxide. When the culture reaches 80 to 90%confluence, wash the cells twice with cold PBS. Use a cell scraper to collect the cells into an appropriate amount of cold cell lysis buffer and transfer the lysing cells into a 1.5-millimeter tube.Invert to mix the lysis buffer and lysing cells well and incubate the tube on ice for 10 minutes. Centrifuge the tube at 18, 000 times g for 10 minutes at four degrees Celsius. Transfer the supernatant into a new 1.5-milliliter tube and keep chilled on ice.Perform BCA assay to approximate the protein concentration of lysates and calculate the dilution of the pronase based on the protein concentration. First, transfer the lysate into two 1.5-milliliter tubes, 99 microliters each. Add one microliter of small-molecule stock solution to each aliquot of lysate and incubate the two tubes for 30 to 60 minutes at room temperature with shaking.On ice, establish serial dilutions of freshly thawed pronase solution in 1x TNC. Following the incubation with the small molecule, divide each aliquot into 20 microliters of samples in five tubes. To ensure the same digestion time, at specific intervals of every 30 seconds, add two microliters of the prepared pronase solutions to the samples accordingly.For the one control group, add two microliters of TNC buffer. After five to 20 minutes, halt the digestion of the six tubes via the addition of two microliters of cold 20x protease inhibitor cocktail at 30-second intervals. Mix well and incubate on ice for 10 minutes.Dilute each sample with six microliters of 5x SDS-PAGE loading buffer and boil the samples in a water bath at 70 degrees Celsius for 10 minutes. Now, to perform Western blot, load equal amounts of protein into the wells of an 8%SDS-PAGE gel, along with an appropriate molecular weight marker. Run the gel for 30 minutes at 80 volts, then adjust the voltage to 120 volts and continue running for one to two hours.Verify that the small molecule can bind directly to the potential target proteins. In this protocol, incubation with the small molecule confirmed the protection against proteolysis. Three bands were found to be protected by incubation with rapamycin over vehicle control.Western blotting illustrated the presence of mTOR protein at low pronase to protein ratios and its reduction and loss with increasing ratios. Proteolysis of mTOR by pronase was clearly inhibited by the presence of rapamycin and the addition of rapamycin generated an obvious shift in the proteolytic curve. Rapamycin dose-dependently enhanced the level of mTOR, suggesting the rising stability of mTOR with rapamycin treatment.Quantification of the target protein band intensities suggested that mTOR is the target protein of rapamycin. In order to get the best stepwise effect, this experiment may have to be repeated several times to obtain a suitable range of pronase over protein ratios.

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Research

JoVE Journal

Biochemistry

15.6K visualizações.  New York University Medical Center. A estabilidade do alvo responsivo da afinidade medicamentosa, DARTS, é um método robusto para detecção de novos alvos proteicos de pequenas moléculas. Pode ser usado para verificar interações conhecidas de proteínas de pequenas moléculas e para encontrar potenciais alvos proteicos para produtos naturais. Neste estudo, melhoramos ainda mais as capacidades de análise de dados do experimento DARTS, monitorando as mudanças na estabilidade proteica e estimando a afinidade das interaçõ...