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

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

Scientific reports (2018-06-23)
Natalia J Martinez, Rosita R Asawa, Matthew G Cyr, Alexey Zakharov, Daniel J Urban, Jacob S Roth, Eric Wallgren, Carleen Klumpp-Thomas, Nathan P Coussens, Ganesha Rai, Shyh-Ming Yang, Matthew D Hall, Juan J Marugan, Anton Simeonov, Mark J Henderson
ABSTRACT

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 LuciferaseĀ approach (SplitLucĀ 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 SplitLucĀ 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Isocitrate Dehydrogenase 1 (IDH1)-R132H Antibody, clone HMab-1, clone HMab-1, from mouse
Sigma-Aldrich
LY2857785 free base, ≥98% (HPLC)