A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

ABSTRACT: Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophos

SUBMITTER: Massey AJ 

PROVIDER: S-EPMC5884524 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature