1. CETSA beyond Soluble Targets: a Broad Application to Multipass Transmembrane Proteins.
- Author
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Kawatkar A, Schefter M, Hermansson NO, Snijder A, Dekker N, Brown DG, Lundbäck T, Zhang AX, and Castaldi MP
- Subjects
- Aminoquinolines pharmacology, Benzamides pharmacology, Benzimidazoles pharmacology, Benzodiazepinones pharmacology, Benzodioxoles pharmacology, Benzyl Alcohols pharmacology, Biological Assay, Cell Line, Tumor, GABA Antagonists pharmacology, HEK293 Cells, Hot Temperature, Humans, Imidazoles pharmacology, Phase Transition drug effects, Protein Multimerization drug effects, Pyridines pharmacology, Receptor, PAR-2 antagonists & inhibitors, Receptor, PAR-2 chemistry, Receptors, GABA chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases antagonists & inhibitors, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Thapsigargin pharmacology, Receptor, PAR-2 metabolism, Receptors, GABA metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.
- Published
- 2019
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