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HiBiT Cellular Thermal Shift Assay (HiBiT CETSA).

Sarath Ramachandran1, Magdalena Szewczyk2, Samir H Barghout2,3

  • 1Centre for Targeted Protein Degradation, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, James Black Centre, Dundee, UK.

Methods in Molecular Biology (Clifton, N.J.)
|August 9, 2023
PubMed
Summary
This summary is machine-generated.

The HiBiT CETSA method quantifies compound target engagement by measuring protein stability changes. This high-throughput assay tracks protein stabilization in cells, offering a significant advancement over traditional methods.

Keywords:
CETSADrug discoveryHiBiTSplit nanoluciferaseTarget engagementThermal shift

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Area of Science:

  • Biochemistry
  • Pharmacology
  • Molecular Biology

Background:

  • Cellular Thermal Shift Assay (CETSA) measures compound-target interactions by assessing protein thermal stabilization.
  • Traditional Western-based CETSA is effective but lacks high throughput and quantitative capabilities.
  • Compound binding can alter protein stability, providing a basis for target engagement assays.

Purpose of the Study:

  • To describe a protocol for the quantitative and high-throughput HiBiT CETSA.
  • To enable tracking of protein stability changes in response to compounds using a novel tagging system.
  • To facilitate screening of target engagement in cellular models.

Main Methods:

  • Utilized a HiBiT tag derived from NanoLuciferase (NanoLuc) for protein labeling.
  • Employed LgBiT NanoLuc tag for complementation, generating a quantifiable luminescent signal.
  • Applied increasing temperatures to induce protein denaturation and measured signal changes.
  • Assessed protein stability in the presence and absence of various compounds in living or permeabilized cells.

Main Results:

  • The HiBiT CETSA method provides a quantitative measure of protein stabilization.
  • The assay demonstrates higher throughput compared to traditional Western-based CETSA.
  • The NanoLuc signal accurately reflects changes in protein stability due to compound binding.
  • The method successfully tracks the effects of temperature on protein stability and compound interactions.

Conclusions:

  • HiBiT CETSA is a robust, quantitative, and high-throughput method for assessing cellular target engagement.
  • The protocol enables precise measurement of compound-induced protein stabilization.
  • This assay is valuable for drug discovery and chemical biology research, allowing for screening in cellular contexts.