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.
Highlights
To evaluate multiplex proximity extension assay (PEA) readout for cellular thermal shift assay (CETSA) analysis, we applied two exploratory, noncommercial PEA panels developed in collaboration with Olink Proteomics and targeting a total of 67 distinct proteins (Table S6 and Supporting File 1)
mass spectrometry (MS) provides a powerful means to investigate target engagement by drugs using the CETSA and for off-target profiling, both in the context of repurposing established drugs and to avoid adverse events by new entities.[14,16−22] We reasoned that this ability of MS to investigate whole proteomes might for some applications be balanced against PEA’s superior sensitivity and convenience by targeting specific sets of proteins of interest in small sample aliquots.[29−33] We selected as a cell model system the human K-562 lymphoblast cell line treated with three ATP-competitive kinase inhibitors: the clinical cancer drugs dasatinib and gefitinib, both having narrow target specificity, and the preclinical pan-inhibitor staurosporine
Assays that quantitatively measure engagement by candidate drugs with their targets can help focus drug discovery programs on those compounds that reach their targets in a cellular context with minimal off-target effects
Summary
Drug development programs often fail for reasons of safety or efficacy during clinical phases I−III, and half of these failures have been reported to be due to lack of efficacy.[1,2] The efficacy of drugs depends on how well the compounds can modulate the primary target molecule, a process referred to as target engagement (TE).[3,4] It is of great value for successful drug development if TE can be ascertained in physiologically relevant tissues.[5,6] Analysis of purified proteins by thermal shift assays (TSAs) takes advantage of the biophysical principle that binding of ligands can stabilize or sometimes destabilize a target protein subjected to a heat challenge.[7,8] By incubating proteins at variable temperatures, it is often possible to observe that the addition of a drug that binds a given protein causes a shift for that protein in the melting (denaturing) temperature, that is, the temperature that reduces the relative abundance by one half (Tm),[7−10] with a low rate of false positives. The cellular thermal shift assay (CETSA) is the first broadly applicable technique for TE studies directly in cells and tissues.[11,12] The CETSA technology builds on the observation that proteins in cells precipitate after heat-induced unfolding, and melting curves can be generated by quantifying the remaining soluble proteins after treatment at increasing temperatures or drug concentrations. Article used with a smaller number of cells but are limited in practice by the requirement that the proteins give very high-contrast melting curves.[19,20] MS on the other hand enables studies of intracellular drug binding and downstream effects in the context of proteome-wide measurement for assessing drug safety and efficacy.[18,21−23] typical MS data sets often miss ensembles of proteins of specific interest, and none of the current approaches allows high-throughput, multiplex analysis of a specific targeted set of proteins in samples with limited numbers of cells such as clinical sample materials.[13,24−26]. See the Supporting Information for the LC− MS/MS analysis and NPARC workflow
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