molecular docking to investigate the interaction between xanthatin and keap1 protein

验证黄素-Keap1 与 Docking 和 CETSA 的相互作用

Proteins are highly important macromolecules in living organisms and possess a diverse range of unique functions within cells, such as membrane composition, cytoskeleton formation, enzyme activity, transportation, cell signaling, and involvement in both intracellular and extracellular mechanisms1,2,3. Proteins manifest their biological functions primarily through specific interactions with a variety of molecules, including other proteins, nucleic acids, small molecule ligands, and metal ions1,4. Ligands are small molecular compounds that specifically bind to proteins in an organism. The interaction between proteins and ligands occurs at specific sites on the protein, called the binding sites, also known as the binding pockets5. In medicinal chemistry research, the focus lies in identifying key proteins that are clearly associated with diseases, which serve as targets for drugs6. Therefore, gaining a deep understanding of the binding sites between proteins and ligands is of utmost importance in promoting drug discovery, design, and research7,8.
Molecular docking is a widely used computational tool for studying protein-ligand binding, which employs the three-dimensional structures of proteins and ligands to explore their primary binding modes and affinities when forming stable complexes9,10,11. The application of molecular docking technology originated in the 1970s. Based on the lock and key pairing principle and utilizing the algorithms of molecular docking software, one can determine the interaction between compounds and molecular targets by analyzing docking results. This approach enables the prediction of active binding sites for both the compound and the target molecule. Consequently, it facilitates the identification of an optimal binding conformation (here called the binding model) for ligand-receptor interactions, which is crucial for understanding the mechanics of these molecular engagements12,13,14,15. While molecular docking provides valuable computer-based predictions of ligand-receptor interactions, it is important to note that these are preliminary findings. Consequently, further experimental verification is essential to confirm these interactions.
The cellular thermal shift assay (CETSA), initially proposed by P&#228;r Nordlund&#39;s research team in 2013, serves as a method for validating drug-target protein interactions. This technique specifically tests the thermal stability of target proteins induced by drug binding, providing a practical approach for confirming molecular interactions16,17,18. This approach is based on the fundamental principle that ligand binding initiates a thermal shift within target proteins and is applicable to a wide array of biological samples, including cell lysates, intact living cells, and tissues19,20. CETSA supports direct target engagement of small molecules in intact cells by detecting thermodynamic stabilization of proteins due to ligand binding and linking the observed phenotypic response to the target compound21,22. Among the various methodologies derived from CETSA, Western Blot-CETSA (WB-CETSA) is considered a classical approach. Following sample preparation using the CETSA method, western blot analysis is utilized to detect alterations in the thermal stability of the target protein. This allows for the precise determination of drug-protein interactions within cellular systems17,23.
Xanthatin is a bioactive compound isolated from the plant Xanthium L. with properties such as anti-inflammatory, which has been used in traditional Chinese medicine to treat diseases like nasal sinusitis and arthritis24,25. The kelch-like ECH-associated protein 1 (Keap1) is a component of the Cullin3-based Cullin-RING E3 ubiquitin ligase multi-subunit protein complex and an important regulator of intracellular redox homeostasis, which influences the intensity and duration of the inflammatory response by modulating the intracellular redox state26. In this study, we first utilized molecular docking to investigate the interaction between xanthatin (small molecule) and Keap1 protein, aiming to predict their binding mode. Subsequently, we employed the CETSA method to validate this interaction by assessing the impact of xanthatin on the thermal stability of the Keap1 protein.

作者聚焦：通过分子对接和 CETSA 简化蛋白质靶标预测和验证

小分子化合物的蛋白质靶点预测与验证

Our research focuses on protein targets, prediction and validation of small molecular compounds. The experiment used here shows a method of molecular docking combined with similar thermal shift assay to predict and validate the interaction between small molecules and protein targets. These steps of CETSA are complicated and any steps errors during the operation can have a great impact on the results.Our protocol combines prediction with verification, effectively reducing both time consumption and economic costs. Furthermore, the experimental instruments and materials are easy to opt in. Finally, the CETSA samples are easy to prepare in test, which ensures the producibility of the samples.

分子对接研究 Xanthatin 和 Keap1 蛋白之间的相互作用

首先，下载 Xanthatin 和 Keap1 的晶体结构。处理完结构后，导航两个任务并选择配体对接选项。从文件中选择受体网格，然后单击“浏览”。然后点击桌面，打开分子对接文件夹，双击滑动网格2FLU文件。选择 glide-grid 2FLU zip 文件，然后单击 Open。现在，单击“从文件中使用配体”。然后单击“浏览”并选择“桌面”。选择分子对接文件夹并打开ligprep_1文件。之后，选择 ligprep_1-out-maegz 文件，然后单击“打开”。访问“设置”，然后选择精度为XP，额外精度选项。将作业名称修改为滑动。xp2flu，然后单击“运行”。要查看停靠结果，请单击“文件”，然后选择“导入结构选项”。导航到桌面并打开分子对接文件夹。然后，选择 glide-dock XP 2FLU 文件。选择滑行码头 XP 1 pv。maegz 文件，然后单击打开。然后双击工作区中的点以选择配体。选择sitemap_1_protein。单击“表”，然后滑动到最右侧以查看对接分数。要可视化 2D 结果，请选择 sitemap_1_protein，如前所述。单击“Ligand Interaction”，选择“View”，然后选中“LID 图例”框。然后单击“文件”，选择“保存屏幕截图”，并输入6， 000作为宽度。取消选中透明背景框，然后单击确定。在桌面上将文件另存为 2D-xanthatin-2FLU。分子对接分析预测了Xanthatin与Keap1蛋白之间的相互作用。

Research

JoVE Journal

Medicine

Molecular Docking to Investigate the Interaction Between Xanthatin and Keap1 Protein

Protein Target Prediction and Validation of Small Molecule Compound

Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of ...

科研

医学

Cell Culture and Cellular Thermal Shift Assay (CETSA) Sample Preparation for Western Blotting

用于蛋白质印迹的细胞培养和细胞热位移测定 （CETSA） 样品制备

Verification of the Interaction Between Xanthatin and Keap1 Protein Using Western Blotting

使用蛋白质印迹法验证 Xanthatin 和 Keap1 蛋白之间的相互作用