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In Situ Target Engagement Studies in Adherent Cells.
Axelsson, Hanna; Almqvist, Helena; Otrocka, Magdalena; Vallin, Michaela; Lundqvist, Sara; Hansson, Pia; Karlsson, Ulla; Lundbäck, Thomas; Seashore-Ludlow, Brinton.
Affiliation
  • Axelsson H; Chemical Biology Consortium Sweden, Science for Life Laboratory , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Almqvist H; Department of Medical Biochemistry and Biophysics , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Otrocka M; Chemical Biology Consortium Sweden, Science for Life Laboratory , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Vallin M; Department of Medical Biochemistry and Biophysics , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Lundqvist S; Chemical Biology Consortium Sweden, Science for Life Laboratory , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Hansson P; Department of Medical Biochemistry and Biophysics , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Karlsson U; Chemical Biology Consortium Sweden, Science for Life Laboratory , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Lundbäck T; Department of Medical Biochemistry and Biophysics , Karolinska Institutet , SE-171 65 Solna , Sweden.
  • Seashore-Ludlow B; Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg , Sweden.
ACS Chem Biol ; 13(4): 942-950, 2018 04 20.
Article in En | MEDLINE | ID: mdl-29433316
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.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Drug Design / Protein Array Analysis / Enzyme Inhibitors / Protein Stability / Hot Temperature Limits: Humans Language: En Journal: ACS Chem Biol Year: 2018 Document type: Article

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Drug Design / Protein Array Analysis / Enzyme Inhibitors / Protein Stability / Hot Temperature Limits: Humans Language: En Journal: ACS Chem Biol Year: 2018 Document type: Article