molecular docking to investigate the interaction between xanthatin and keap1 protein

डॉकिंग और CETSA के साथ Xanthatin-Keap1 इंटरैक्शन को मान्य करना

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.

लेखक स्पॉटलाइट: आणविक डॉकिंग और सीईटीएसए के माध्यम से प्रोटीन लक्ष्य भविष्यवाणी और सत्यापन को सुव्यवस्थित करना

प्रोटीन लक्ष्य भविष्यवाणी और छोटे अणु यौगिक का सत्यापन

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.

Xanthatin और Keap1 प्रोटीन के बीच बातचीत की जांच करने के लिए आणविक डॉकिंग

शुरू करने के लिए, Xanthatin और Keap1 की क्रिस्टल संरचनाओं को डाउनलोड करें। संरचनाओं को संसाधित करने के बाद, दो कार्यों को नेविगेट करें और लिगैंड डॉकिंग विकल्प चुनें। फ़ाइल से रिसेप्टर ग्रिड चुनें और ब्राउज़ पर क्लिक करें।फिर डेस्कटॉप पर क्लिक करें, आणविक डॉकिंग फ़ोल्डर खोलें और ग्लाइड-ग्रिड 2FLU फ़ाइल पर डबल क्लिक करें। ग्लाइड-ग्रिड 2FLU ज़िप फ़ाइल का चयन करें और क्लिक करें खुला हुआ. अब, Use ligands from files पर क्लिक करें।फिर ब्राउज़ करें पर क्लिक करें और डेस्कटॉप चुनें। आणविक डॉकिंग फ़ोल्डर का चयन करें और ligprep_1 फ़ाइल खोलें। उसके बाद, ligprep_1-आउट-मेगज़ फ़ाइल का चयन करें और क्लिक करें खुला हुआ.सेटिंग्स तक पहुंचें और एक्सपी, अतिरिक्त परिशुद्धता, विकल्प के रूप में सटीकता चुनें। कार्य नाम को ग्लाइड करने के लिए संशोधित करें। xp2flu और उसके बाद चलाएँक्लिक करें।डॉकिंग परिणाम देखने के लिए, फ़ाइल पर क्लिक करें और आयात संरचनाएं विकल्प चुनें। डेस्कटॉप पर नेविगेट करें और आणविक डॉकिंग फ़ोल्डर खोलें। बाद में, ग्लाइड-डॉक XP 2FLU फ़ाइल का चयन करें।ग्लाइड-डॉक XP 1 पीवी चुनें। maegz फ़ाइल खोलें और खोलें पर क्लिक करें। फिर लिगैंड का चयन करने के लिए कार्यक्षेत्र में डॉट पर डबल क्लिक करें।sitemap_1_protein चुनें. टेबल पर क्लिक करें और डॉकिंग स्कोर देखने के लिए सबसे दाईं ओर स्लाइड करें। 2D परिणामों की कल्पना करने के लिए, पहले दिखाए गए अनुसार sitemap_1_protein का चयन करें।लिगैंड इंटरैक्शन पर क्लिक करें, देखें चुनें और एलआईडी लीजेंड बॉक्स को चेक करें। फिर क्लिक करें फ़ाइल, चुनें स्क्रीनशॉट सहेजें, और चौड़ाई के लिए 6, 000 इनपुट करें। पारदर्शी पृष्ठभूमि बॉक्स को अनचेक करें और ठीक क्लिक करें. डेस्कटॉप पर फ़ाइल को 2D-xanthatin-2FLU के रूप में सहेजें।आणविक डॉकिंग विश्लेषण ने ज़ैंथाटिन और केएपी 1 प्रोटीन के बीच बातचीत की भविष्यवाणी की।

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

सेल संस्कृति और सेलुलर थर्मल शिफ्ट परख (CETSA) पश्चिमी सोख्ता के लिए नमूना तैयार

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

पश्चिमी सोख्ता का उपयोग Xanthatin और Keap1 प्रोटीन के बीच बातचीत का सत्यापन