molecular docking to investigate the interaction between xanthatin and keap1 protein

ドッキングおよびCETSAによるキサンタチン-Keap1相互作用の検証

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.

著者スポットライト:Molecular DockingおよびCETSAによるタンパク質標的の予測と検証の効率化

低分子化合物のタンパク質標的予測と検証

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.

キサンタチンとKeap1タンパク質の相互作用を調べるための分子ドッキング

まず、キサンタチンとKeap1の結晶構造をダウンロードしてください。構造を処理した後、2つのタスクをナビゲートし、リガンドドッキングオプションを選択します。ファイルからレセプターグリッドを選択し、[参照]をクリックします。次に、デスクトップをクリックし、分子ドッキングフォルダーを開き、glide-grid2FLUファイルをダブルクリックします。glide-grid 2FLU zipファイルを選択して、Openをクリックします。次に、ファイルからのリガンドを使用をクリックします。次に、[参照]をクリックして[デスクトップ]を選択します。分子ドッキングフォルダーを選択し、ligprep_1ファイルを開きます。その後、ligprep_1-out-maegzファイルを選択し、[開く]をクリックします。[設定]にアクセスし、精度をXP、追加精度、オプションを選択します。ジョブ名を glide に変更します。xp2fluと入力し、[実行]をクリックします。ドッキング結果を表示するには、ファイルをクリックし、構造のインポートオプションを選択します。デスクトップに移動し、分子ドッキングフォルダーを開きます。その後、グライドドックXP2FLUファイルを選択します。グライドドックXP1pvを選択します。maegz ファイルを開き、[開く] をクリックします。次に、ワークスペース内のドットをダブルクリックして、リガンドを選択します。[sitemap_1_protein] を選択します。表をクリックし、右端にスライドしてドッキングスコアを表示します。2D 結果を視覚化するには、前に示したように [sitemap_1_protein] を選択します。Ligand Interaction をクリックし、View を選択して LID legend ボックスにチェックを入れます。次に、をクリックします フィレット、選択 スクリーンショットを保存、幅に6, 000を入力します。透明な背景ボックスのチェックを外して、[OK]をクリックします。ファイルを 2D-xanthatin-2FLU という名前でデスクトップに保存します。分子ドッキング解析により、キサンタチンとKeap1タンパク質の相互作用が予測されました。

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 ...

医学

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

ウェスタンブロッティング用の細胞培養および細胞熱シフトアッセイ(CETSA)サンプル調製

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

ウェスタンブロッティングを用いたキサンタチンとKeap1タンパク質の相互作用の検証