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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Downloading the structures of xanthatin and Keap1

  1. Open the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), input xanthatin (small molecule), then press Search and click on The First Result. Click on Download and click Save under 2D structure to save the compound in .sdf format.
  2. Download the crystal structure of the protein.
    1. Open the UniProt database (https://www.uniprot.org/), input Keap1 a.......

Representative Results

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 .......

Discussion

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 .......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
0.45 μm Polyvinylidene fluoride membraneMilliporePR05509
Anhydrous ethanolChron chemicals64-17-5
Bovine serum albuminBioFroxx4240GR100
Broad-spectrum protease inhibitor mixturesBoster Biological Technology Co., LtdAR1193
DMSOBoster Biological Technology Co., LtdPYG0040
Enhanced chemiluminescence reagentBeyotime Biotechnology Co., LtdP0018S
GAPDH antibodyProteinTech Group Co., Ltd10494-1-AP
Gel Imaging InstrumentE-BLOTTouch Imager Pro
Gradient PCR instrumentBiometra TADVANCEDBiometra Tadvanced 96SG
High-speed freezing centrifugeBeckman CoulterAllegra X-30R
Horseradish peroxidase-conjugated affiniPure goat antibodyProteinTech Group Co., LtdSA00001-2
Isopropyl alcohol Chron chemicals67-63-0
Keap1 antibodyZen BioScience Co., LtdR26935
Metal bathAnalytik JenaTSC
MethanolChron chemicals67-56-1
Ncmblot rapid transfer buffer (20×)NCM Biotech Co., LtdWB4600
Omni-Easy OneStep PAGE gel fast preparation kieEpizyme Biotech Co., LtdPG212
Phosphate buffer salineBoster Biological Technology Co., LtdPYG0021
Prestained Color Protein MarkerBiosharp BL741A
Protein Blotting Electrophoresis SystemBio-RadMiniPROTEANÃ’Tetra Cell
RAW264.7 cellBeyotime Biotechnology Co., LtdC7505
RAW264.7 cell-specific mediumProcell Life Science&Technology Co., LtdCM-0597
SDS-PAGE protein loading bufferBoster Biological Technology Co., LtdAR1112-10
SDS-PAGE running buffer powderServicebioG2018
Tris buffered saline powderServicebioG0001
Tween 20BioFroxx1247ML100
Water bathMemmertWNE10
Water purifierMilliporeMilli- IQ 7005
XanthatinChemConst Biotechnology Co., LtdCONST210706

References

  1. Soleymani, F., Paquet, E., Viktor, H., Michalowski, W., Spinello, D. Protein-protein interaction prediction with deep learning: A comprehensive review. Computat Struct Biotechnol J. 20, 5316-5341 (2022).
  2. Du, X., et al.

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Protein Target PredictionSmall Molecule CompoundsMolecular DockingThermal Shift AssayCETSAKeap1 ProteinXanthatinProtein ligand BindingDrug Development

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