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The experiment used here shows a method of molecular docking combined with cellular thermal shift assay to predict and validate the interaction between small molecules and protein targets.
Proteins are fundamental to human physiology, with their targets being crucial in research and drug development. The identification and validation of crucial protein targets have become integral to drug development. Molecular docking is a computational tool widely utilized to investigate protein-ligand binding, especially in the context of drug and protein target interactions. For the experimental verification of the binding and to access the binding of the drug and its target directly, the cellular thermal shift assay (CETSA) method is used. This study aimed to integrate molecular docking with CETSA to predict and validate interactions between drugs and vital protein targets. Specifically, we predicted the interaction between xanthatin and Keap1 protein as well as its binding mode through molecular docking analysis, followed by verification of the interaction using the CETSA assay. Our results demonstrated that xanthatin could establish hydrogen bonds with specific amino acid residues of Keap1 protein and reduce the thermostability of Keap1 protein, indicating that xanthatin could directly interact with Keap1 protein.
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 mole....
1. Downloading the structures of xanthatin and Keap1
Molecular docking analysis predicted the interaction between xanthatin and Keap1 protein. Figure 2 demonstrates the formation of hydrogen bonds between xanthatin and amino acid residues Gly-367 and Val-606 of Keap1 protein, with a hydrogen bond length of 2.17 Ã… for Gly-367 and 2.13 Ã… for Val-606. In addition, the calculated docking score of -5.69 kcal/mol signifies a good binding affinity between xanthatin and Keap1 protein.
The CETSA method showed that .......
The identification of disease targets and the discovery and development of drugs are closely interconnected27. By precisely targeting specific targets, drug candidates can be developed to treat particular diseases more effectively while concurrently minimizing the side effects associated with the drugs28,29. The most commonly used targets are protein targets30. However, the identification of special protein targets .......
This work was supported by National Natural Science Foundation of China (82004031) and Sichuan Science and Technology Program (2022NSFSC1303). We express our great appreciation to Jiayi Sun at Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, for the assistance with western blot.
....Name | Company | Catalog Number | Comments |
0.45 μm Polyvinylidene fluoride membrane | Millipore | PR05509 | |
Anhydrous ethanol | Chron chemicals | 64-17-5 | |
Bovine serum albumin | BioFroxx | 4240GR100 | |
Broad-spectrum protease inhibitor mixtures | Boster Biological Technology Co., Ltd | AR1193 | |
DMSO | Boster Biological Technology Co., Ltd | PYG0040 | |
Enhanced chemiluminescence reagent | Beyotime Biotechnology Co., Ltd | P0018S | |
GAPDH antibody | ProteinTech Group Co., Ltd | 10494-1-AP | |
Gel Imaging Instrument | E-BLOT | Touch Imager Pro | |
Gradient PCR instrument | Biometra TADVANCED | Biometra Tadvanced 96SG | |
High-speed freezing centrifuge | Beckman Coulter | Allegra X-30R | |
Horseradish peroxidase-conjugated affiniPure goat antibody | ProteinTech Group Co., Ltd | SA00001-2 | |
Isopropyl alcohol | Chron chemicals | 67-63-0 | |
Keap1 antibody | Zen BioScience Co., Ltd | R26935 | |
Metal bath | Analytik Jena | TSC | |
Methanol | Chron chemicals | 67-56-1 | |
Ncmblot rapid transfer buffer (20×) | NCM Biotech Co., Ltd | WB4600 | |
Omni-Easy OneStep PAGE gel fast preparation kie | Epizyme Biotech Co., Ltd | PG212 | |
Phosphate buffer saline | Boster Biological Technology Co., Ltd | PYG0021 | |
Prestained Color Protein Marker | Biosharp | BL741A | |
Protein Blotting Electrophoresis System | Bio-Rad | MiniPROTEANÃ’Tetra Cell | |
RAW264.7 cell | Beyotime Biotechnology Co., Ltd | C7505 | |
RAW264.7 cell-specific medium | Procell Life Science&Technology Co., Ltd | CM-0597 | |
SDS-PAGE protein loading buffer | Boster Biological Technology Co., Ltd | AR1112-10 | |
SDS-PAGE running buffer powder | Servicebio | G2018 | |
Tris buffered saline powder | Servicebio | G0001 | |
Tween 20 | BioFroxx | 1247ML100 | |
Water bath | Memmert | WNE10 | |
Water purifier | Millipore | Milli- IQ 7005 | |
Xanthatin | ChemConst Biotechnology Co., Ltd | CONST210706 |
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