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Determining the In Vitro Ligand-Target Interaction by Cellular Thermal Shift Assay and Isothermal Dose-Response

Danyu Du1,2,3, Shengtao Yuan2, Jing Xiong1

  • 1Department of Pharmacology, School of Pharmacy, China Pharmaceutical University, Nanjing, China.

Bio-Protocol
|August 12, 2024
PubMed
Summary

This study adapts cellular thermal shift assays (CETSA) for RNA-binding proteins (RBPs), successfully determining enasidenib

Keywords:
Cellular thermal shift assay (CETSA)Isothermal dose-response fingerprint assay (ITDRFCETSA)Ligand–target interactionRNA binding motif protein 45

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint assay (ITDRFCETSA) are established for target engagement studies.
  • These methods typically measure ligand-induced stabilization of cellular target proteins.
  • Their application to RNA-binding proteins (RBPs) remains underexplored.

Purpose of the Study:

  • To adapt CETSA and ITDRFCETSA for assessing ligand interactions with RNA-binding proteins (RBPs).
  • To determine the interaction between enasidenib and the RBM45 protein using this adapted methodology.
  • To establish a sensitive and time-efficient protocol for RBP-ligand interaction studies.

Main Methods:

  • Utilized modified cellular thermal shift assay (CETSA) and isothermal dose-response fingerprint assay (ITDRFCETSA).
  • Applied the assay to investigate the interaction between the drug enasidenib and the RNA-binding protein RBM45.
  • Adapted established CETSA protocols for a new class of proteins (RBPs).

Main Results:

  • Successfully determined the interaction between enasidenib and RBM45 using the adapted CETSA and ITDRFCETSA.
  • Demonstrated the sensitivity and time-efficiency of the developed protocol.
  • Validated the applicability of CETSA-based methods to RNA-binding proteins.

Conclusions:

  • The adapted CETSA and ITDRFCETSA provide a robust method for studying RBP-ligand interactions.
  • This protocol can be extended to investigate RBM45 interactions with other potential drug candidates.
  • The study expands the utility of thermal shift assays to RNA-binding proteins.