molecular docking to investigate the interaction between xanthatin and keap1 protein

التحقق من صحة تفاعل Xanthatin-Keap1 مع الإرساء و 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.

تسليط الضوء على المؤلف: تبسيط التنبؤ بالهدف البروتيني والتحقق من صحته عبر الالتحام الجزيئي و CETSA

التنبؤ بالبروتين المستهدف والتحقق من صحة مركب الجزيئات الصغيرة

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.

الالتحام الجزيئي للتحقيق في التفاعل بين الزانثاتين وبروتين Keap1

للبدء ، قم بتنزيل الهياكل البلورية ل Xanthatin و Keap1. بعد معالجة الهياكل ، انتقل إلى مهمتين وحدد خيار إرساء الرباط. اختر شبكة المستقبل من الملف وانقر فوق تصفح.ثم انقر فوق سطح المكتب ، وافتح مجلد الإرساء الجزيئي وانقر نقرا مزدوجا فوق ملف 2FLU للشبكة المنزلقة. حدد الملف المضغوط 2FLU للشبكة المنزلقة وانقر فوق فتح. الآن ، انقر فوق استخدام الروابط من الملفات.ثم انقر فوق استعراض وحدد سطح المكتب. حدد مجلد الإرساء الجزيئي وافتح ملف ligprep_1. بعد ذلك ، حدد ملف ligprep_1-out-maegz وانقر فوق فتح.الوصول إلى الإعدادات واختيار الدقة مثل XP ، دقة إضافية ، خيار. قم بتعديل اسم الوظيفة للانزلاق. xp2flu ومن ثم انقر فوق تشغيل.لعرض نتائج الإرساء ، انقر فوق ملف وحدد خيارات استيراد الهياكل. انتقل إلى سطح المكتب وافتح مجلد الإرساء الجزيئي. بعد ذلك ، حدد ملف XP 2FLU المنزلق.اختر الانزلاق رصيف XP 1 الكهروضوئية. maegz وانقر فوق فتح. ثم انقر نقرا مزدوجا فوق النقطة في مساحة العمل لتحديد الرابطة.اختر sitemap_1_protein. انقر فوق جدول ومرر إلى أقصى اليمين لعرض درجة الإرساء. لتصور نتائج 2D ، حدد sitemap_1_protein كما هو موضح سابقا.انقر فوق تفاعل Ligand ، وحدد عرض وحدد مربع أسطورة LID. ثم اضغط قم بتقديم ، اختر حفظ لقطة الشاشة، وأدخل 6،000 للعرض. قم بإلغاء تحديد مربع الخلفية الشفافة وانقر فوق موافق. احفظ الملف على سطح المكتب ك 2D-زانثاتين-2FLU.توقع تحليل الالتحام الجزيئي التفاعل بين بروتين Xanthatin و 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 ...

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

زراعة الخلايا ومقايسة التحول الحراري الخلوي (CETSA) تحضير العينة للنشاف الغربي

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

التحقق من التفاعل بين الزانثاتين وبروتين Keap1 باستخدام النشاف الغربي