molecular docking to investigate the interaction between xanthatin and keap1 protein

Validación de la interacción de Xanthatin-Keap1 con el acoplamiento y 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.

Autor destacado: Optimización de la predicción y validación de dianas proteicas mediante acoplamiento molecular y CETSA

Predicción y validación de dianas proteicas de compuestos de moléculas pequeñas

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.

Acoplamiento molecular para investigar la interacción entre la xanthatina y la proteína Keap1

Para empezar, descarga las estructuras cristalinas de Xanthatin y Keap1. Después de procesar las estructuras, navegue por dos tareas y seleccione la opción de acoplamiento de ligandos. Elija la cuadrícula del receptor como en el archivo y haga clic en Examinar.A continuación, haga clic en Escritorio, abra la carpeta de acoplamiento molecular y haga doble clic en el archivo 2FLU de glide-grid. Seleccione el archivo zip glide-grid 2FLU y haga clic en Abrir. Ahora, haga clic en usar ligandos de archivos.A continuación, haga clic en Examinar y seleccione Escritorio. Seleccione la carpeta de acoplamiento molecular y abra el archivo ligprep_1. Después de eso, seleccione el archivo ligprep_1-out-maegz y haga clic en Abrir.Acceda a Configuración y elija la opción precisión como XP, precisión extra. Modifique el nombre del trabajo para deslizarlo. xp2flu y, a continuación, haga clic en Ejecutar.Para ver los resultados del acoplamiento, haga clic en Archivo y seleccione Opciones de importación de estructuras. Navegue hasta el escritorio y abra la carpeta de acoplamiento molecular. A continuación, seleccione el archivo glide-dock XP 2FLU.Elija el muelle deslizante XP 1 pv. maegz y haga clic en Abrir. A continuación, haga doble clic en el punto del espacio de trabajo para seleccionar el ligando.Elija sitemap_1_protein. Haga clic en Tabla y deslice hacia el extremo derecho para ver la puntuación de acoplamiento. Para visualizar los resultados 2D, seleccione sitemap_1_protein como se ha demostrado anteriormente.Haga clic en Interacción del ligando, seleccione Ver y marque la casilla de leyenda LID. A continuación, haga clic en Archivo, elija Guardar captura de pantalla e introduzca 6.000 para el ancho. Desmarque la casilla de fondo transparente y haga clic en Aceptar. Guarde el archivo en el escritorio como 2D-xanthatin-2FLU.El análisis de acoplamiento molecular predijo la interacción entre la xanthatina y la proteína 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 ...

Investigación

Medicina

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

Cultivo celular y ensayo de desplazamiento térmico celular (CETSA) Preparación de muestras para Western Blotting

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

Verificación de la interacción entre xanthatin y la proteína keap1 mediante Western blotting