Fig. 1.

Principles and Workflow of the InCELL Pulse STEP₄₆ Cellular Target Engagement Assay. The miniaturized STEP₄₆ target engagement assay is a form of reporter-based cellular thermal shift assay that can be reliably integrated into a drug discovery campaign. (a) An assay plate is prepared using an Echo Liquid Handler (or similar) to spot compounds of interest in the wells to be probed. HEK293T cells are transiently transfected with a pICP-ePL-N-STEP46 plasmid that expresses STEP₄₆ with an enhanced ProLabel (ePL, 42 amino acids) fusion tag. After 1 d, cells are detached, resuspended in fresh growth media, and are transferred to the assay plate. The cells are incubated with candidate compounds for 1 h. (b) For a thermal profile, the assay plate is subjected to a temperature gradient pulse for 3 min. Increasing temperatures cause proteins to denature and form insoluble, inaccessible aggregates. Specific target engagement of a small molecule can stabilize STEP₄₆ against aggregation. A mixture of enzyme acceptor (EA) complementation reagent and lysis buffer enables the quantification of soluble ePL-tagged STEP₄₆ via the reporter enzyme chemiluminescence system. The signal for each well is recorded and the data are analyzed