Assay Development and Screening
Development of Cellular Assays to Understand Protein Homeostasis
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. Experimentally, this change in melt temperature can be measured by exposure of the protein to a temperature gradient, followed by quantification of the protein level or activity at each temperature. Originally, protein thermal shift experiments were performed with purified protein samples, but recently 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 1536-well CETSA, we used a protein reporter system in a homogeneous (additions only, no wash) assay format. We have successfully utilized this assay to characterize compounds in dose response curves for drug discovery programs at GSK. This method can also be applied to identify hits in high throughput screening. Assay parameters optimized included target expression level, the amount of detection reagents added after thermal melting, plate type, and thermal melt methodologies. Uses and applications in drug discovery will be presented.