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 inhibitor cocktail, 25 &#956;L of 1 M sodium fluoride, 50 &#956;L of 100 mM &#946;-glycerophosphate, 50 &#956;L of 50 mM sodium pyrophosphate, and 5 &#956;L of 200 mM sodium orthovanadate. Keep the lysis buffer chilled on ice. 
	NOTE: Other lysis buffers with various detergents (e.g., Triton X-100 or NP-40) can be used with DARTS as long as they are non-denaturing. Membrane proteins or nuclear proteins can be extracted by adding 0.4% Triton X-100 or 0.4% NP-40 to the cell lysate.</li>
	<li>Wash the cells twice with cold phosphate-buffered saline (PBS).</li>
	<li>Use a cell scraper to collect the cells in an appropriate amount of cell lysis buffer and transfer the lysing cells into a 1.5 mL tube. 
	NOTE: The number of cells needed for each DARTS experiment will vary based on how much protein can be extracted from various cell lines. In general, the protein concentration of the lysate used is between 4&#8210;6 &#956;g/&#956;L. One 10 cm plate of 293T cells at 85&#8210;90% confluency, lysed with 300 &#956;L of lysis buffer typically results in a lysate with a protein concentration of ~5 &#956;g/&#956;L.</li>
	<li>Mix the lysis buffer/lysing cells well and incubate the tube on ice for 10 min.</li>
	<li>Centrifuge the tube at 18,000 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Transfer the supernatant into a new 1.5 mL tube and keep chilled on ice.</li>
	<li>Perform a BCA assay to approximate the protein concentration of lysates.</li>
</ol>
2. Incubate protein lysates with the small molecule
<ol>
	<li>Split 99 &#956;L of the lysates into two 1.5 mL tubes.</li>
	<li>Make a starting stock concentration of 10 mM small molecule. When performing DARTS, one may begin with a higher concentration of the small molecule (5&#8210;10x the EC50 value) to ensure optimal binding.
	<ol>
		<li>Add either 1 &#956;L of solvent that the small molecule is soluble in or 1 &#181;L of small molecule stock solutions to each aliquot of lysate. Incubate cell lysate with the solutions for 30&#8210;60 min at room temperature with shaking.</li>
	</ol>
	</li>
	<li>On ice, establish serial dilutions (1:200, 1:400, 1:800 and 1:1600) of freshly thawed pronase solution in 1x TNC (For 1 mL of 10x TNC buffer, mix 300 &#956;L of ultrapure water with 100 &#956;L of 5 M sodium chloride, 100 &#956;L of 1 M calcium chloride, and 500 &#956;L of 1 M Tris-HCl, pH 8.0).</li>
	<li>Examine a wide-breadth of pronase:protein ratios (e.g., spanning from 1:100 to 1:2000) to guarantee observability of the step-wise effect of pronase on target(s). 
	NOTE: To calculate pronase concentrations (example): 5 &#956;g/&#956;L protein concentration x 20 &#956;L sample = 100 &#956;g protein. For a pronase:protein ratio of 1:100 we need 0.5 &#956;g/&#956;L (100 &#956;g &#247; 100 &#247; 2 &#956;L) pronase solution. This experiment may have to be repeated several times to obtain a suitable range of pronase:protein ratios.</li>
</ol>
3. Perform proteolysis
NOTE: For proteolysis, steps are carried out at room temperature unless otherwise noted
<ol>
	<li>Following incubation with the small molecule, divide each aliquot into 20 &#956;L samples.</li>
	<li>Add 2 &#956;L of the range of pronase solutions in each sample at specific intervals (every 30 s). Use an equal volume of 1x TNC buffer to establish an undigested control sample.</li>
	<li>After 5&#8210;20 min, halt digestion via addition of 2 &#956;L of cold 20x protease inhibitor cocktail every 30 s. Mix well and incubate on ice for 10 min.</li>
	<li>Dilute samples with the appropriate volume of 5x SDS-PAGE loading buffer and boil at 95 &#176;C for 5 min.</li>
	<li>Carry out the next portion of the experiment or store the protein sample at &#8722;80 &#176;C.</li>
</ol>
4. Quantification and analysis
<ol>
	<li>After DARTS, perform Coomassie blue staining according to the previously published protocol22.</li>
	<li>The stained protein bands should be very clear after destaining. Pour off the used destain solution and add fresh 1% acetic acid solution to cover the gel. Put the gel under the light to observe the bands with significant differences between groups with (sample group) or without (control group) the small molecule.</li>
	<li>Cut the two corresponding bands from the gel with a sterile instrument and perform LC-MS/MS immediately. 
	NOTE: LC-MS/MS needs to be done as soon as possible because the proteins in the gel will degrade continuously.</li>
	<li>Digest the bands, extract peptides and perform LC-MS/MS analysis23.
	<ol>
		<li>To analyze LC-MS/MS data, first identify all the proteins in the individual band. Second, use peptide spectrum match (PSM) to represent the abundance of each protein. 
		NOTE: PSM provides the total number of identified peptide sequences for the protein, including those redundantly identified. In general, how often a peptide is identified/sequenced can be used as a rough estimate of how abundant the protein is in the sample. Proteins enriched in the sample group over the control group are proteins of interest.</li>
	</ol>
	</li>
	<li>Procure the primary antibodies of the selected proteins. Perform western blot to verify that the small molecule can bind directly to the potential target proteins24.
	<ol>
		<li>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 min at 80 V, then adjust the voltage to 120 V and continue running for 1&#8210;2 h.</li>
	</ol>
	</li>
	<li>Quantify the different target protein bands using image processing and analysis software (see the Table of Materials).</li>
	<li>Analyze the data and draw the curves.
	<ol>
		<li>Determination of the proteolytic curve for a target protein
		<ol>
			<li>The pronase:protein ratio is varied when performing proteolysis. Normalize data from image analysis by attributing the band intensity values corresponding to the undigested bands to 100%.</li>
			<li>Use nonlinear regression analysis of the statistical analysis and drawing software to plot a curve of normalized data for relative band intensity and pronase:protein ratios.</li>
			<li>First, open the software, select the type of table and graph as XY and allow for 3 replicate values in side-by-side subcolumns. Then enter the corresponding data in the space below x and y. Under x, input the numbers 0, 1, 2, 3, 4, 5 (as place holders corresponding pronase:protein ratios).</li>
			<li>In the y column, enter the normalized data for relative band intensities. Perform &#8220;Nonlinear regression&#8221; and implement a &#8220;Dose-response-Stimulation&#8221; using the &#8220;Log (agonist) versus response&#8212;Variable slope (four parameters)&#8221; equation.</li>
			<li>Convert the annotations of the X-axis in the curve to the corresponding pronase:protein ratios.</li>
		</ol>
		</li>
		<li>Determination of the dose-dependence curve for a target protein 
		NOTE: For dose-dependence analysis, the molarity of the small molecule is differed across samples. The stable pronase:protein ratio should be informed by analysis of proteolysis data. The pronase:protein ratio that showed maximal difference in target protein intensity during the proteolytic curve should be used for the dose-dependence experiment.
		<ol>
			<li>Quantify the different target protein bands using an image analysis software. As in generation of the dose-dependence curve, normalize data from image analysis by attributing the band intensity values corresponding to the least digested band to 100%.</li>
			<li>Apply the nonlinear regression analysis within the statistical analysis and drawing software to the normalized data. First, open the software, select the type of table and graph as XY, and enter 3 replicate values in side-by-side subcolumns.</li>
			<li>Then, enter the corresponding data in the space below x and y. For the &#8216;x&#8217; variable, input different concentrations of the small molecule; &#8216;y&#8217; includes the corresponding normalized data for relative band intensity.</li>
			<li>Transform x values using X = Log(X). Finally, employ nonlinear regression, and implement a dose-response stimulation using the &#8220;Log (agonist) versus response&#8212;Variable slope (four parameters)&#8221; equation.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Rask-Andersen, M., Masuram, S., Schioth, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+druggable+genome%3A+Evaluation+of+drug+targets+in+clinical+trials+suggests+major+shifts+in+molecular+class+and+indication%5BTitle%5D)+AND+%22Annual+Review+of+Pharmacology+and+Toxicology%22%5BJournal%5D)">The druggable genome: Evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication.</a> Annual Review of Pharmacology and Toxicology. 54, 9-26 (2014).</li>
<li>O'Connor, C. J., Laraia, L., Spring, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+genetics%5BTitle%5D)+AND+%22Chemical+Society+Reviews%22%5BJournal%5D)">Chemical genetics.</a> Chemical Society Reviews. 40 (8), 4332-4345 (2011).</li>
<li>McFedries, A., Schwaid, A., Saghatelian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+the+elucidation+of+protein-small+molecule+interactions%5BTitle%5D)+AND+%22Chemistry+%26+Biology%22%5BJournal%5D)">Methods for the elucidation of protein-small molecule interactions.</a> Chemistry & Biology. 20 (5), 667-673 (2013).</li>
<li>Sato, S., Murata, A., Shirakawa, T., Uesugi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+target+isolation+for+novices%3A+affinity-based+strategies%5BTitle%5D)+AND+%22Chemistry+%26+Biology%22%5BJournal%5D)">Biochemical target isolation for novices: affinity-based strategies.</a> Chemistry & Biology. 17 (6), 616-623 (2010).</li>
<li>Sleno, L., Emili, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+methods+for+drug+target+discovery%5BTitle%5D)+AND+%22Current+Opinion+in+Chemical+Biology%22%5BJournal%5D)">Proteomic methods for drug target discovery.</a> Current Opinion in Chemical Biology. 12 (1), 46-54 (2008).</li>
<li>Lomenick, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target+identification+using+drug+affinity+responsive+target+stability+%28DARTS%29%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Target identification using drug affinity responsive target stability (DARTS).</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (51), 21984-21989 (2009).</li>
<li>Strickland, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermodynamic+analysis+of+protein-ligand+binding+interactions+in+complex+biological+mixtures+using+the+stability+of+proteins+from+rates+of+oxidation%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.</a> Nature Protocols. 8 (1), 148-161 (2013).</li>
<li>Jafari, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cellular+thermal+shift+assay+for+evaluating+drug+target+interactions+in+cells%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">The cellular thermal shift assay for evaluating drug target interactions in cells.</a> Nature Protocols. 9 (9), 2100-2122 (2014).</li>
<li>Martinez Molina, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monitoring+drug+target+engagement+in+cells+and+tissues+using+the+cellular+thermal+shift+assay%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay.</a> Science. 341 (6141), 84-87 (2013).</li>
<li>Savitski, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tracking+cancer+drugs+in+living+cells+by+thermal+profiling+of+the+proteome%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Tracking cancer drugs in living cells by thermal profiling of the proteome.</a> Science. 346 (6205), 1255784(2014).</li>
<li>Chang, J., Kim, Y., Kwon, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+identification+and+validation+of+protein+targets+of+natural+products+without+chemical+modification%5BTitle%5D)+AND+%22Natural+Product+Reports%22%5BJournal%5D)">Advances in identification and validation of protein targets of natural products without chemical modification.</a> Natural Product Reports. 33 (5), 719-730 (2016).</li>
<li>Pai, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+affinity+responsive+target+stability+%28DARTS%29+for+small-molecule+target+identification%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Drug affinity responsive target stability (DARTS) for small-molecule target identification.</a> Methods in Molecular Biology. 1263, 287-298 (2015).</li>
<li>Lomenick, B., Jung, G., Wohlschlegel, J. A., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Target+identification+using+drug+affinity+responsive+target+stability+%28DARTS%29%5BTitle%5D)+AND+%22Current+Protocols+in+Chemical+Biology%22%5BJournal%5D)">Target identification using drug affinity responsive target stability (DARTS).</a> Current Protocols in Chemical Biology. 3 (4), 163-180 (2011).</li>
<li>Xu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision+therapeutic+targeting+of+human+cancer+cell+motility%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Precision therapeutic targeting of human cancer cell motility.</a> Nature Communications. 9 (1), 2454(2018).</li>
<li>Lim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+autophagy+enhancer+as+a+therapeutic+agent+against+metabolic+syndrome+and+diabetes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">A novel autophagy enhancer as a therapeutic agent against metabolic syndrome and diabetes.</a> Nature Communications. 9 (1), 1438(2018).</li>
<li>Schulte, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+blockade+of+ASCT2-dependent+glutamine+transport+leads+to+antitumor+efficacy+in+preclinical+models%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacy in preclinical models.</a> Nature Medicine. 24 (2), 194-202 (2018).</li>
<li>Skrott, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alcohol-abuse+drug+disulfiram+targets+cancer+via+p97+segregase+adaptor+NPL4%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.</a> Nature. 552 (7684), 194-199 (2017).</li>
<li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endosidin2+targets+conserved+exocyst+complex+subunit+EXO70+to+inhibit+exocytosis%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (1), 41-50 (2016).</li>
<li>Chin, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+metabolite+alpha-ketoglutarate+extends+lifespan+by+inhibiting+ATP+synthase+and+TOR%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The metabolite alpha-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.</a> Nature. 510 (7505), 397-401 (2014).</li>
<li>Robinson, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+screen+identifies+disulfiram+as+a+potential+therapeutic+for+triple-negative+breast+cancer+cells%3A+interaction+with+IQ+motif-containing+factors%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: interaction with IQ motif-containing factors.</a> Cell Cycle. 12 (18), 3013-3024 (2013).</li>
<li>Aghajan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+genetics+screen+for+enhancers+of+rapamycin+identifies+a+specific+inhibitor+of+an+SCF+family+E3+ubiquitin+ligase%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase.</a> Nature Biotechnology. 28 (7), 738-742 (2010).</li>
<li>Brunelle, J. L., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coomassie+blue+staining%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">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/pubmed?term=((Mass+spectrometry+and+protein+analysis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+Western+Blot%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">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. L., Clardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atomic+structures+of+the+human+immunophilin+FKBP-12+complexes+with+FK506+and+rapamycin%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin.</a> Journal of Molecular Biology. 229 (1), 105-124 (1993).</li>
<li>Lomenick, B., Olsen, R. W., Huang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+direct+protein+targets+of+small+molecules%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">Identification of direct protein targets of small molecules.</a> ACS Chemical Biology. 6 (1), 34-46 (2011).</li>
<li>Park, Y. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+Multiple+Cryptococcal+Fungicidal+Drug+Targets+by+Combined+Gene+Dosing+and+Drug+Affinity+Responsive+Target+Stability+Screening%5BTitle%5D)+AND+%22MBio%22%5BJournal%5D)">Identification of Multiple Cryptococcal Fungicidal Drug Targets by Combined Gene Dosing and Drug Affinity Responsive Target Stability Screening.</a> MBio. 7 (4), (2016).</li>
<li>Qu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecule+promotes+beta-catenin+citrullination+and+inhibits+Wnt+signaling+in+cancer%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Small molecule promotes beta-catenin citrullination and inhibits Wnt signaling in cancer.</a> Nature Chemical Biology. 14 (1), 94-101 (2018).</li>
</ol>

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>&amp;beta;-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 ...

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JoVE Journal

Biochemistry

16.2K Views.  New York University Medical Center. 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.

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

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular functions and events1, 2. Their spatiotemporally dynamic nature has been revealed by visualization of their activity and localization in real time3. In order to gain deeper understanding of their roles in diverse cellular functions at the molecular level, the next step should be perturbation of protein activities at a precise subcellular location and timing. 
To achieve this goal, we have developed a method for light-induced, spatio-temporally controlled activation of small GTPases by combining two techniques: (1) rapamycin-induced FKBP-FRB heterodimerization and (2) a photo-caging method of rapamycin. With the use of rapamycin-mediated FKBP-FRB heterodimerization, we have developed a method for rapidly inducible activation or inactivation of small GTPases including Rac4, Cdc424, RhoA4 and Ras5, in which rapamycin induces translocation of FKBP-fused GTPases, or their activators, to the plasma membrane where FRB is anchored. For coupling with this heterodimerization system, we have also developed a photo-caging system of rapamycin analogs. A photo-caged compound is a small molecule whose activity is suppressed with a photocleavable protecting group known as a caging group. To suppress heterodimerization activity completely, we designed a caged rapamycin that is tethered to a macromolecule such that the resulting large complex cannot cross the plasma membrane, leading to virtually no background activity as a chemical dimerizer inside cells6. Figure 1 illustrates a scheme of our system. With the combination of these two systems, we locally recruited a Rac activator to the plasma membrane on a timescale of seconds and achieved light-induced Rac activation at the subcellular level6.

A method for spatio-temporal control of small GTPase activity by light is described. This method is based on rapamycin-induced FKBP-FRB heterodimerization and photo-caging systems. Optimization of light-irradiation enables the spatio-temporally controlled activation of small GTPases at the subcellular level.

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

Bioengineering

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Cancer Research

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Quantitative Assessment of Macropinocytosis in mTORC1-Hyperactive Cells using Flow Cytometry

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically.
In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.

This protocol provides experimental tools to evaluate macropinocytic uptake of nutrients (carbohydrate and protein) by mTORC1-hyperactive cells. Detailed steps to quantify the uptake of fluorescently labeled dextran and bovine serum albumin (BSA) are described.

Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. ...

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

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