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

Sci Rep. 2018 Jun 21;8(1):9472. doi: 10.1038/s41598-018-27834-y.


Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology
  • Cyclin-Dependent Kinase 9 / antagonists & inhibitors
  • Drug Discovery / methods*
  • Enzyme Stability
  • HEK293 Cells
  • HT29 Cells
  • HeLa Cells
  • High-Throughput Screening Assays / methods*
  • Humans
  • L-Lactate Dehydrogenase / antagonists & inhibitors
  • Luciferases / metabolism*
  • Nanotechnology / methods
  • Protein Binding
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology


  • Antineoplastic Agents
  • Protein Kinase Inhibitors
  • L-Lactate Dehydrogenase
  • LDHA protein, human
  • Luciferases
  • Cyclin-Dependent Kinase 9