<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

Discovery of AZD2716: A Novel Secreted Phospholipase A₂ (sPLA₂) Inhibitor for the Treatment of Coronary Artery Disease

Expedited structure-based optimization of the initial fragment hit <b>1</b> led to the design of (<i>R</i>)-<b>7</b> (AZD2716) a novel, potent secreted phospholipase A₂ (sPLA₂) inhibitor with excellent preclinical pharmacokinetic properties across species, clear <i>in vivo</i> efficacy, and minimized safety risk. Based on accumulated profiling data, (<i>R</i>)-<b>7</b> was selected as a clinical candidate for the treatment of coronary artery disease