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Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays
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Author Spotlight: Advancing Structural and Biochemical Studies of Proteins Through Thermal Shift Assays

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A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase

Natalia J Martinez1, Rosita R Asawa1, Matthew G Cyr1

  • 1National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, 20850, USA.

Scientific Reports
|June 23, 2018
PubMed

Insights

A new Split Nano Luciferase Cellular Thermal Shift Assay (SplitLuc CETSA) platform enables high-throughput drug target engagement studies in intact cells. This standardized assay accelerates pre-clinical drug discovery for diverse targets, even when other methods fail.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Assessing drug-target interactions in cells is crucial for pre-clinical drug discovery.
  • The Cellular Thermal Shift Assay (CETSA) measures protein thermal stability changes upon ligand binding.
  • Existing methods face challenges in throughput and applicability.

Purpose of the Study:

  • To develop and validate a high-throughput, standardized, and target-independent CETSA platform.
  • To utilize a split Nano Luciferase approach (SplitLuc CETSA) for enhanced cellular thermal shift assays.
  • To demonstrate the platform's utility for drug target engagement studies and high-throughput screening.

Main Methods:

  • Development of a homogeneous, high-throughput (384- and 1536-well) SplitLuc CETSA.
  • Validation using diverse protein targets and well-characterized inhibitors.
  • Comparison with independent biochemical and cell-based assays.

Main Results:

  • Demonstrated broad applicability of SplitLuc CETSA across various targets.
  • Showcased comparable performance to existing biochemical and cell-based assays.
  • Validated the platform for medium and high-throughput screening applications.

Conclusions:

  • SplitLuc CETSA offers a robust solution for cellular target engagement studies.
  • The platform facilitates rapid assay development and screening, especially for challenging targets.
  • Enables efficient pre-clinical drug discovery by overcoming limitations of traditional assays.

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