molecular docking to investigate the interaction between xanthatin and keap1 protein

Validating Xanthatin-Keap1 Interaction with Docking and CETSA

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.

Author Spotlight: Streamlining Protein Target Prediction and Validation via Molecular Docking and CETSA

Prédiction et validation de cibles protéiques de composés à petites molécules

Amarrage moléculaire pour étudier l’interaction entre la xanthatine et la protéine Keap1

Pour commencer, téléchargez les structures cristallines de Xanthatin et Keap1. Après avoir traité les structures, naviguez dans deux tâches et sélectionnez l’option d’amarrage des ligands. Choisissez la grille réceptrice à partir du fichier et cliquez sur Parcourir.Cliquez ensuite sur Bureau, ouvrez le dossier d’amarrage moléculaire et double-cliquez sur le fichier 2FLU de glide-grid. Sélectionnez le fichier zip 2FLU et cliquez sur Ouvrir. Maintenant, cliquez sur utiliser les ligands des fichiers.Cliquez ensuite sur Parcourir et sélectionnez Bureau. Sélectionnez le dossier d’ancrage moléculaire et ouvrez le fichier ligprep_1. Après cela, sélectionnez le fichier ligprep_1-out-maegz et cliquez sur Ouvrir.Accédez aux paramètres et choisissez la précision comme XP, précision supplémentaire, option. Modifiez le nom de la tâche pour qu’elle glisse. xp2flu, puis cliquez sur Exécuter.Pour afficher les résultats de l’ancrage, cliquez sur Fichier et sélectionnez Options d’importation des structures. Accédez au bureau et ouvrez le dossier d’amarrage moléculaire. Ensuite, sélectionnez le fichier glide-dock XP 2FLU.Choisissez le dock de glissement XP 1 pv. maegz et cliquez sur Ouvrir. Double-cliquez ensuite sur le point dans l’espace de travail pour sélectionner le ligand.Choisissez sitemap_1_protein. Cliquez sur Tableau et faites glisser vers l’extrême droite pour afficher le score d’ancrage. Pour visualiser les résultats 2D, sélectionnez-sitemap_1_protein comme indiqué précédemment.Cliquez sur Interaction avec le ligand, sélectionnez Afficher et cochez la case de légende LID. Cliquez ensuite sur Fichier, choisissez Enregistrer la capture d’écran et saisissez 6 000 pour la largeur. Décochez la case Arrière-plan transparent et cliquez sur OK. Enregistrez le fichier sur le bureau sous le nom 2D-xanthatin-2FLU.L’analyse de l’amarrage moléculaire a prédit l’interaction entre la xanthatine et la protéine Keap1.

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 ...

Recherche

Médecine

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

Préparation d’échantillons pour le transfert thermique occidental (CETSA)

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

Vérification de l’interaction entre la xanthatine et la protéine Keap1 à l’aide du Western Blot