Sensitive Measurement of Drug-Target Engagement by a Cellular Thermal Shift Assay with Multiplex Proximity Extension Readout.
Rasel A Al-AminCaroline J GallantPhathutshedzo M MutheloUlf LandegrenPublished in: Analytical chemistry (2021)
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.
Keyphrases
- high throughput
- mass spectrometry
- social media
- multiple sclerosis
- drug discovery
- real time pcr
- ms ms
- healthcare
- gene expression
- electronic health record
- adverse drug
- clinical practice
- emergency department
- liquid chromatography
- single molecule
- high performance liquid chromatography
- cell proliferation
- health information
- signaling pathway
- transcription factor
- pi k akt
- label free
- capillary electrophoresis
- simultaneous determination