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.
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.
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.
1. Collect and lyse cells
2. Incubate protein lysates with the small molecule
3. Perform proteolysis
NOTE: For proteolysis, steps are carried out at room temperature unless otherwise noted
4. Quantification and analysis
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 mTOR, which is the target for the drug rapamycin25. Western blotting illustrates the presence of mTOR protein at low pronase:protein ratios and its reduction and loss with increasing ratios (Figure 3A). Proteolysis of mTOR by pronase is clearly inhibited by the presence of rapamycin and the addition of rapamycin generates an obvious shift in the proteolytic curve (Figure 3B). To investigate effects of drug concentration, we maintained a constant pronase:protein ratio while varying concentrations of rapamycin. As ligand concentration nears target binding saturation, an increased presence of target protein is observed. Rapamycin dose-dependently enhanced the level of mTOR, suggesting the rising stability of mTOR with rapamycin treatment (Figure 4A). Quantification of the target protein band intensities allows representation of target stability as a function of ligand concentration as exemplified by the curve in Figure 4B. These results strongly suggest that mTOR is the target protein of rapamycin.
Figure 1: Schematic of the DARTS approach for drug target semi-quantitative analysis. Cell lysate is incubated in the presence or absence of a small molecule, followed by proteolysis and protein electrophoresis. Protected protein bands are excised and subjected to mass spectroscopy. Protein targets are identified as those proteins that display increased protease resistance in the presence of the small molecule. Then western-blot is used to identify and semi-quantitatively analyze the target proteins. Data are expressed as means ± standard deviation (SD). Please click here to view a larger version of this figure.
Figure 2: Example of Coomassie blue staining visualization of DARTS with the small molecule rapamycin. Red dots flank the protected bands. Please click here to view a larger version of this figure.
Figure 3: Illustration of the remaining amount of mTOR accessible for detection as a function of the pronase:protein ratio used for treatment of 293T cell lysates. (A) Protection of mTOR from proteolysis by rapamycin was evaluated by western blot analysis. (B) The intensity of the mTOR bands were quantified using the statistical analysis and drawing software. The line was fitted with a four-parameter logistic curve. Data were obtained from the three independent experiments and were expressed as mean ± SD. Please click here to view a larger version of this figure.
Figure 4: Illustration of the amount of stabilized mTOR accessible for detection in the presence of increasing concentrations of rapamycin. 293T cell lysates were incubated with rapamycin (0, 1, 10, 100, 1000, 10000 nM) for 1 h, then the cell lysates were subjected to digestion at the pronase:protein ratio of 1:400. (A) The stabilization effect of rapamycin on mTOR was evaluated by western blot. (B) The intensity of the mTOR bands were quantified using the statistical analysis and drawing software. The line was fitted with a four-parameter logistic curve. Data were obtained from the three independent experiments and were expressed as mean ± SD. Please click here to view a larger version of this figure.
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. The classic methodology also mentions that these data can be quantitatively analyzed, but there is no such example provided. Here, we use principles of the cellular thermal shift assay (CETSA) to semi-quantitatively analyze the data, and obtain parameters similar to those supplied by CESTA (Tm and EC50) which increases the utility of DARTS analysis8. The ligand–target interaction can be plotted against pronase:protein ratio to display obvious shifts in proteolytic curves. Carrying out proteolysis using different pronase:protein ratios can help in narrowing down the concentration of pronase that should be used in downstream experiments. Further, using the optimized concentration of pronase, the proteolysis carried out in the presence of different concentrations of the small molecule may provide an indirect measure of the affinity of the small molecule with its target protein. In addition, generation of a dose-dependence curve allows for approximation of effects on target proteins dependent upon ligand concentration. Inclusion of analytic capacity for dose-dependence is a powerful expansion of DARTS methodology; providing a straightforward and quick approach to probing the therapeutic mechanism of small molecules. This gel-based approach is the easiest to implement. It can be used for high-throughput screening for compounds that bind a specific protein20,27,28. Additionally, DARTS can be utilized for analyzing true interactions with low affinity, because washing is not included as an experimental step12,26. Moreover, compared with CETSA, DARTS has advantages in identifying the targets of membrane proteins as DARTS allows a better assessment of membrane proteins through use of mild, stabilizing detergents11.
The experiment also has some limitations. First, when the cell lysate has a low abundance of target protein, the DARTS method cannot be used to easily visualize alterations in proteolysis of the target protein. Additional steps to concentrate these proteins are required in order to apply this methodology. Second, we only test rapamycin/mTOR interaction. The interaction is known to be potent and stable. However, some small molecules may bind to their targets less selectively, or transiently, and it is not clear if such small molecules can be analyzed with this assay. Third, some target proteins may be extremely sensitive or resistant to the proteases used.
DARTS assay analysis allows for identification of potential protein interactions through assessment of proteolytic curves generated across a range of pronase:protein ratios in the presence or absence of a small molecule ligand. Excitingly, our modifications to the standard procedural outline of the DARTS assay highlight the capacity of this method to be used in generation of concentration-response curve. These outputs are of special utility in drug development, allowing identification of mechanistically relevant drug concentrations. Moreover, comparison of concentration-response curves generated using disparate ligands offers insight to the comparative binding affinities for several ligands of the same target; a capacity potentially useful in prediction of small molecule efficacy and refinement of dosing. We hope that this demonstration of expanded analytic power of DARTS will be useful in the development, implementation, and understanding of small molecule drugs, particularly for ligands and targets difficult to analyze using alternative approaches.
The authors have nothing to disclose.
This work was supported partly by NIH research grants R01NS103931, R01AR062207, R01AR061484, and a DOD research grant W81XWH-16-1-0482.
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 |