Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)—A Novel Approach to Characterize Protein–Protein Interactions in Living Cells by Similar Isothermal Dose–Responses

Abstract

Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose–response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein–protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose–responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose–response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named “small molecule arranged thermal proximity coaggregation” (smarTPCA).

Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

“…Furthermore, TMT-DDA detected significant thermal stabilization of myosin light chain kinase (MYLK) (3.6 ± 0.6 °C) following treatment with losmapimod, but the protein was not detected in any DIA datasets (Figure S3A). A recent study reported MYLK to be a potentially new intracellular interaction partner of MAPK14, which may have been stabilized by association due to MAPK14 target engagement. Stability changes in proteins present in a complex have been identified previously with TPP, for example, kinase complexes containing cyclins were stabilized by the kinase inhibitor staurosporine .…”

“…Furthermore, TMT-DDA detected significant thermal stabilization of myosin light chain kinase (MYLK) (3.6 ± 0.6 °C) following treatment with losmapimod, but the protein was not detected in any DIA datasets (Figure S3A). A recent study reported MYLK to be a potentially new intracellular interaction partner of MAPK14, 55 which may have been stabilized by association due to MAPK14 target engagement.…”

A Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling

“…Furthermore, TMT-DDA detected significant thermal stabilisation of myosin light chain kinase (MYLK) (3.6 ± 0.6°C) following treatment with losmapimod, but the protein was not detected in any DIA datasets ( Figure S3A ). A recent study reported MYLK to be a potentially new intracellular interaction partner of MAPK14 52 , which may have been stabilised by association due to MAPK14 target engagement. Stability changes in proteins present in a complex have been identified previously with TPP, for example, kinase complexes containing cyclins were stabilized by the kinase inhibitor staurosporine.…”

Targeting mitochondrial metabolism by the mitotoxin bromoxib as a therapeutic approach for the treatment of leukemia and lymphoma

“…Thermal proteome pro ling with temperature range (TPP-TR) was essentially performed as described [17,34,86,92] employing 40 µM as nal bromoxib concentration and 30 min treatment time on live Ramos cells in three biological replicates (n = 3). In brief, aliquots of compound treated and control cells were shortly incubated side-by-side at ten discrete temperatures, equally distributed in a range between 37°C and 68.5°C, lysed, and the cell extracts containing the fraction of soluble, non-denatured proteins were obtained by centrifugation.…”