molecular docking to investigate the interaction between xanthatin and keap1 protein

Validering af Xanthatin-Keap1-interaktion med Docking og 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.

Forfatter Spotlight: Strømlining af forudsigelse og validering af proteinmål via molekylær docking og CETSA

Proteinmålforudsigelse og validering af småmolekyleforbindelse

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.

Molekylær docking for at undersøge interaktionen mellem Xanthatin og Keap1 protein

For at begynde skal du downloade krystalstrukturerne af Xanthatin og Keap1. Efter behandling af strukturerne skal du navigere i to opgaver og vælge liganddockingindstillingen. Vælg receptorgitteret fra filen, og klik på Gennemse.Klik derefter på Desktop, åbn molekylær dockingmappe og dobbeltklik på glide-grid 2FLU-filen. Vælg zip-filen glide-grid 2FLU, og klik på Åbn. Klik nu på brug ligander fra filer.Klik derefter på Gennemse, og vælg Skrivebord. Vælg den molekylære dockingmappe, og åbn ligprep_1 filen. Vælg derefter filen ligprep_1-out-maegz og klik på Åbn.Få adgang til Indstillinger, og vælg præcisionen som XP, ekstra præcision, mulighed. Rediger jobnavnet til at glide. xp2flu og derefter klikke på Kør.For at se dockingresultater skal du klikke på Filer og vælge Indstillinger for importstrukturer. Naviger til skrivebordet, og åbn den molekylære dockingmappe. Vælg derefter glide-dock XP 2FLU-filen.Vælg glide-dock XP 1 pv. maegz-fil, og klik på Åbn. Dobbeltklik derefter på prikken i arbejdsområdet for at vælge liganden.Vælg sitemap_1_protein. Klik på Tabel og skub helt til højre for at se dockingscoren. Hvis du vil visualisere 2D-resultaterne, skal du vælge sitemap_1_protein som vist tidligere.Klik på Ligand Interaction, vælg Vis og marker LID legend boks. Klik derefter på Filer, vælg Gem skærmbillede, og indtast 6.000 for bredde. Fjern markeringen i afkrydsningsfeltet gennemsigtig baggrund, og klik på OK. Gem filen på skrivebordet som 2D-xanthatin-2FLU.Molekylær dockinganalyse forudsagde interaktionen mellem Xanthatin og Keap1-protein.

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 ...

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

Cellekultur og cellulær termisk forskydningsanalyse (CETSA) prøveforberedelse til Western Blotting

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

Verifikation af interaktionen mellem xanthatin og Keap1-protein ved anvendelse af Western blotting