1 research outputs found
<i>In Situ</i> Target Engagement Studies in Adherent Cells
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 <i>in situ</i>, 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