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

Abstract

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.

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

“…The PEA method has shown high sensitivity and specificity and has thus been able to detect femtomolar levels of interleukin-8 (IL-8) and glial-derived neurotrophic factor (GDNF) proteins, even in human plasma. Recently, PEA strategies have also been utilized for measuring the levels of single-cell proteins [34], detecting extracellular vesicle (EV) proteins [35,36], C-reactive protein, cardiac troponin I [37], or plasma proteins [38], monitoring drug-target interactions, [39] or profiling proteins in plasma samples of COVID-19 patients [31,40]. In addition to PLAs and PEAs, relatively simpler designs have been adopted for biosensors that afford signals arising directly from proximity-induced hybridization.…”

Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies

“… 1 In addition, molecular protein detection assays with improved proofreading are needed for efficient detection of target molecules at high efficiency and with a minimal nonspecific background to ensure highly specific and sensitive detection. Oligo-assisted proximity-based immunoassays 2 have been adapted for different proteomic applications, such as for high-throughput plasma proteomics, 3 visualization of protein and their complexes in situ , 4 detection of drug–target interactions and extracellular vesicles, 5 , 6 infectious diagnostics, 7 flow cytometry, 6 and western blotting. 8 Variants of proximity-based assays have been developed where each target molecule must be recognized by three different Abs for enhanced detection in solution-phase assays 9 or on solid supports, 10 and prostate-derived microvesicles called prostasomes have been detected at elevated levels in plasma from prostate cancer patients using sets of five different Abs.…”

Sensitive protein detection using site-specifically oligonucleotide-conjugated nanobodies

“…1 In addition, molecular protein detection assays with improved proofreading are needed for efficient detection of target molecules at high efficiency and with minimal nonspecific background to ensure highly specific and sensitive detection. Oligo-assisted proximity-based immunoassays 2 have been adapted for different proteomic applications, such as for high-throughput plasma proteomics, 3 visualization of protein and their complexes in situ, 4 detection of drug-target engagement and of extracellular vesicles, [5][6] infectious diagnostics, 7 flow cytometry 6 and western blotting. 8 Variants of proximity-based assays have been developed where each target molecule must be recognized by three different Abs, for enhanced detection in solution-phase assays 9 or on solid supports 10 and prostate derived microvesicles called prostasomes have been detected at elevated levels in plasma from prostate cancer patients using sets of five different Abs.…”