Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout

The ability to monitor
target engagement in cellular contexts is
a key for successful drug discovery and also valuable in clinical
routine. A cellular thermal shift assay (CETSA) provides realistic
information about drug binding in cells and tissues, revealing drug-target
engagement in clinically relevant samples. The CETSA combined with
mass spectrometry (MS) detection can be applied in the early hit identification
phase to generate target engagement data for large sets of proteins.
However, the analysis is slow, requires substantial amounts of the
sample material, and often misses proteins of specific interest. Here,
we combined the CETSA and the multiplex proximity extension assay
(PEA) for analysis of target engagement of a set of 67 proteins from
small amounts of the sample material treated with kinase inhibitors.
The results were concordant with the corresponding analyses read out
via MS. Our approach allows analyses of large numbers of specific
target proteins at high sensitivity in limited sample aliquots. Highly
sensitive multiplex CETSA-PEA assays are therefore promising for monitoring
drug-target engagement in small sample aliquots in the course of drug
development and potentially in clinical settings.