用于研究拉帕霉素/mTOR相互作用的半定量药物亲和反应目标稳定性 （DARTS） 测定
Summary
在这项研究中，我们通过监测蛋白质稳定性的变化和估计蛋白质-配体相互作用的亲和力，增强了DARTS实验的数据分析能力。相互作用可以绘制成两条曲线：蛋白质解解曲线和剂量依赖曲线。我们使用 mTOR-拉帕霉素相互作用作为示例案例。
Abstract
药物亲和力响应目标稳定性 （DARTS） 是检测新型小分子蛋白靶点的有力方法。它可用于验证已知的小分子-蛋白质相互作用，并找到天然产物的潜在蛋白质靶点。与其他方法相比，DARTS 使用原生、未经修饰的小分子，操作简单易行。在这项研究中，我们通过监测蛋白质稳定性的变化和估计蛋白质-配体相互作用的亲和力，进一步提高了DARTS实验的数据分析能力。蛋白质-配体相互作用可以绘制成两条曲线：蛋白质解解曲线和剂量依赖曲线。我们使用 mTOR-拉帕霉素相互作用作为建立我们协议的典范案例。从蛋白溶酶曲线中，我们看到，通过蛋白酶抑制mTOR的蛋白解酶被雷帕霉素的存在所抑制。剂量依赖曲线使我们能够估计雷帕霉素和 mTOR 的结合亲和力。该方法可能是准确识别新型靶蛋白和优化药物靶向的一种强大而简单的方法。
Introduction
识别小分子靶蛋白对于机械性理解和开发潜在的治疗药物1，2，3至关重要。亲和色谱作为识别小分子靶蛋白的经典方法，取得了良好的效果4，5。然而，这种方法有局限性，因为小分子的化学修饰往往导致结合特异性或亲和力的减少或改变。为了克服这些限制，最近已经开发并应用了几种新的策略来识别小分子目标，而不需要对小分子进行化学修饰。这些用于无标签小分子目标识别的直接方法包括药物亲和力响应靶稳定（DARTS）6、蛋白质从氧化率（SPROX）7的稳定性、细胞热移位测定（CETSA）8 ，9和热蛋白酶分析 （TPP）10。这些方法是非常有利的，因为它们使用天然的，未经修饰的小分子，并仅依靠直接结合相互作用，以找到目标蛋白质11。
在这些新方法中，DARTS是一种相对简单的方法，大多数实验室都可以轻松采用。DARTS 取决于配体结合蛋白相对于未结合的蛋白质表现出对酶降解的经修饰的易感性的概念。通过液相色谱-质谱法（LC-MS/MS）检查SDS-PAGE凝胶中改变的带状，可以检测出新的靶蛋白。该方法已成功实施，用于识别以前未知的天然产品和药物目标 14、15、16、17、18、19.它作为筛选或验证化合物与特定蛋白质20、21结合的手段也非常强大。在这项研究中，我们通过监测小分子的蛋白质稳定性变化和识别蛋白质配体结合亲和力，对实验进行了改进。我们以mTOR-雷帕霉素相互作用为例来演示我们的方法。
Protocol
1. 收集和酶细胞 使用Dulbeco的改性Eagle培养基（DMEM）与10%胎儿牛血清、2 mM谷氨酰胺和1%抗生素一起培育293T细胞。在 37°C 下孵育培养物，在 5% CO2下孵育。注：细胞的生长状态可能会影响后续实验的稳定性。 扩大培养细胞，直到达到80\u201290%汇合。 将345 μL的细胞溶化试剂（见材料表）与25μL的20x蛋白酶抑制剂鸡尾酒、25μL的1M氟化钠、50μL的100mβ-糖酸磷?…
Representative Results
实验的流程图如图1所示。库马西蓝色染色的结果如图2所示。用小分子孵育可以保护蛋白解。发现三个带，似乎通过孵化与雷帕霉素在车辆控制下孵化。蛋白解曲线实验的预期结果如图3所示。作为原理证明，我们检查了经过充分研究的蛋白质mTOR，这是药物雷帕霉素25的目标。西方印迹说明了mTOR蛋白在低蛋白酶中…
Discussion
DARTS 允许利用蛋白质结合对降解的保护作用来识别小分子靶点。DARTS不需要任何化学修饰或小分子的固定26。这允许使用小分子来确定其直接结合蛋白靶点。经典DARTS方法的标准评估标准包括凝胶染色、质谱和西印12、13。经典方法还提到这些数据可以定量分析，但没有提供这样的示例。在这里，我们使用蜂窝热移变测定（CETSA）的原理?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了NIH研究资助R01NS103931、R01AR062207、R01AR061484和国防部研究补助金W81XWH-16-1-0482的支持。
Materials
| 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 |
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Cite This Article
Zhang, C., Cui, M., Cui, Y., Hettinghouse, A., Liu, C. A Semi-Quantitative Drug Affinity Responsive Target Stability (DARTS) assay for studying Rapamycin/mTOR interaction. J. Vis. Exp. (150), e59656, doi:10.3791/59656 (2019).