Poster
186 |
Cellular Thermal Shift Assay: Case studies of membrane proteins |
Target engagement is an important parameter for successful drug
discovery, hence it is applied as an integral component of most screening cascades. The
cellular thermal shift assay (CETSA) is a powerful new method for assessing
target engagement in the physiological environment of living cells. A unique
advantage is the possibility to run this assay without labels on either
compound or proteins, allowing studies also in patient-derived cells.
Originally, CETSA was based on extraction of soluble target proteins in a
detergent free setting, thus it was not applicable to membrane-bound targets.
This isn’t sufficient as a significant portion of drug targets are integrated
with cellular membranes. Later
protocols included detergents to allow analysis also of membrane-bound
proteins. Here we further extend these results by showcasing CETSA data
for multi pass membrane spanning proteins, with particular focus on the unique
challenges associated with the establishment of thermal aggregation curves and
interpretation of observed shifts thereof.
Our case studies for various
transmembrane proteins showcases unique challenges in establishing protocols
for successful melt curves. Different membrane proteins have different
CETSA behaviors and some are not amenable to the approach despite optimized
conditions. Sample preparation strongly influences the melting behavior. Our
Work provides the drug discovery community with a broad picture of what to
expect when establishing target engagement using CETSA for complex
membrane-bound proteins.