molecular docking to investigate the interaction between xanthatin and keap1 protein

Validatie van Xanthatin-Keap1-interactie met docking en 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.

Auteur in de schijnwerpers: Stroomlijning van de voorspelling en validatie van eiwitdoelen via moleculaire docking en CETSA

Voorspelling en validatie van eiwitdoelen van een verbinding met kleine moleculen

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.

Moleculair docken om de interactie tussen xanthatin en Keap1-eiwit te onderzoeken

Download om te beginnen de kristalstructuren van Xanthatin en Keap1. Nadat u de structuren hebt verwerkt, navigeert u door twee taken en selecteert u de optie ligandkoppeling. Kies het receptorraster uit het bestand en klik op Bladeren.Klik vervolgens op Bureaublad, open de moleculaire dockingmap en dubbelklik op het glide-grid 2FLU-bestand. Selecteer het zip-bestand van het glijraster 2FLU en klik op Openen. Klik nu op liganden uit bestanden gebruiken.Klik vervolgens op Bladeren en selecteer Bureaublad. Selecteer de moleculaire dockingmap en open het ligprep_1 bestand. Selecteer daarna het ligprep_1-out-maegz-bestand en klik op Openen.Ga naar Instellingen en kies de precisie als XP, extra precisie, optie. Wijzig de taaknaam om te glijden. xp2flu en klik vervolgens op Uitvoeren.Als u de dockingresultaten wilt bekijken, klikt u op Bestand en selecteert u Opties voor importstructuren. Navigeer naar het bureaublad en open de moleculaire dockingmap. Selecteer daarna het XP 2FLU-bestand met glide-dock.Kies dan voor de glide-dock XP 1 pv. maegz bestand en klik op Openen. Dubbelklik vervolgens op de stip in de werkruimte om de ligand te selecteren.Kies sitemap_1_protein. Klik op Tafel en schuif helemaal naar rechts om de dockingscore te bekijken. Als u de 2D-resultaten wilt visualiseren, selecteert u sitemap_1_protein zoals eerder gedemonstreerd.Klik op Ligand Interactie, selecteer Bekijken en vink het vakje LID-legenda aan. Klik vervolgens op Bestand, kies Screenshot opslaan en voer 6.000 in voor breedte. Schakel het vakje voor transparante achtergrond uit en klik op OK. Sla het bestand op het bureaublad op als 2D-xanthatin-2FLU.Moleculaire docking-analyse voorspelde de interactie tussen Xanthatin en Keap1-eiwit.

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

Celcultuur en cellulaire thermische verschuivingstest (CETSA) monstervoorbereiding voor western bletting

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

Verificatie van de interactie tussen xanthanyine en Keap1-eiwit met behulp van western blotting