As a cellular target engagement assay that determines the level of interaction between a ligand and a target protein of interest in a physiologically relevant setting, CETSA® is perfectly suited for hit confirmation. CETSA® requires no protein or ligand modifications and is much quicker and easier to set up compared to traditional assays, which typically require more time-consuming assay development or molecular biology work. Therefore, CETSA® offers the advantage of investigating compound binding to the protein of interest in its endogenous environment and in its native state. CETSA® can be used to confirm target engagement of hits from a primary screen in a relevant cellular context, which greatly increases the confidence in pursuing these hits further in lead generation. Using CETSA® for hit confirmation allows you to validate faster and progress only the most relevant compounds to the next stages in your drug discovery value chain.
The potential of CETSA® to be used in lead generation for hit identification, hit confirmation as well as for SAR analysis has been demonstrated in the literature by several research teams. In a study from AstraZeneca by Shaw et al., CETSA® Navigate HT was applied for hit confirmation following an HTS campaign based on a biochemical fluorescent polarization (FP) assay for the clinically validated target PARP1. In the study, PARP1 binders were confirmed as hits using a CETSA® Navigate HT assay, with the advantage of simultaneous confirmation of their permeability and target engagement in intact cells.
Almqvist H, Axelssson H, Rozbeh J, Dan C, Mateus A, Haraldsson M, Larsson A, Martinez Molina D, Artursson P, Lundbäck T, Nordlund P. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil. (2016) Nature Communications 7:11040
Shaw J, Dale I, Hemsley P, Leach L, et al. Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1. (2019) SLAS Discov. DOI: 10.1177/2472555218813332