Objective

Thermal shift assays (TSA) reveal changes in protein structure upon binding to small molecules due to a resultant change in the thermal melting temperature of the protein. Originally, thermal shift experiments were performed with purified proteins, but over the last few years, the TSA was reported in a cellular context, and the cellular thermal shift assay (CETSA) was born. We have combined CETSA with a high throughput protein detection method, to increase the throughput of the assay, since traditional protein detection methods such as western blots are low throughput. To develop high throughput 384-well and 1536-well protocols, we used a protein reporter system in a homogeneous (additions only, no wash) assay format. Some 384-well work was published and showed the utility of the assay in dose response curve format to generate small molecule compound-target engagement potency values [McNulty et al, SLAS Discovery 2018, 23(1)34-46)]. These studies were extended to include lysate formats, and 1536-well methods. Results have shown the feasibility of applying this technique to high throughput screening (HTS) for hit identification. Assay parameters tested during optimization included target expression levels, and titration of detection reagents. Innovation in equipment and methods used for thermal melting contributed to our ability to successfully translate this technology into an assay format that can be used for HTS.