In Situ Target Engagement Studies in Adherent Cells.

Axelsson H, Almqvist H, Otrocka M, Vallin M, Lundqvist S, Hansson P, Karlsson U, Lundbäck T, Seashore-Ludlow B

ACS Chem. Biol. 13 (4) 942-950 [2018-04-20; online 2018-02-21]

A prerequisite for successful drugs is effective binding of the desired target protein in the complex environment of a living system. Drug-target engagement has typically been difficult to monitor in physiologically relevant models, and with current methods, especially, while maintaining spatial information. One recent technique for quantifying drug-target engagement is the cellular thermal shift assay (CETSA), in which ligand-induced protein stabilization is measured after a heat challenge. Here, we describe a CETSA protocol in live A431 cells for p38α (MAPK14), where remaining soluble protein is detected in situ, using high-content imaging in 384-well, microtiter plates. We validate this assay concept using a number of known p38α inhibitors and further demonstrate the potential of this technology for chemical probe and drug discovery purposes by performing a small pilot screen for novel p38α binders. Importantly, this protocol creates a workflow that is amenable to adherent cells in their native state and yields spatially resolved target engagement information measurable at the single-cell level.

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PubMed 29433316

DOI 10.1021/acschembio.7b01079

Crossref 10.1021/acschembio.7b01079