Proteome thermal stability reflects organ physiology and identifies drug-target engagement in vivo

Abstract

Studying biological processes at a molecular level and monitoring drug-target interactions is established for simple cell systems but challenging in vivo. We introduce and apply a methodology for proteome-wide thermal stability measurements to characterize organ physiology and activity of many fundamental biological processes across tissues, such as energy metabolism and protein homeostasis. This method, termed tissue thermal proteome profiling (tissue-TPP), also enabled target and off-target identification and occupancy measurements in tissues derived from animals dosed with the non-covalent histone deacetylase inhibitor, panobinostat. Finally, we devised blood-CETSA, a thermal stability-based method to monitor target engagement in whole blood. Our study generates the first proteome-wide map of protein thermal stability in tissue and provides tools that will be of great impact for translational research. Main Text:Thermal proteome profiling (TPP) determines thermal stability across the proteome by measuring the soluble fractions of proteins upon heating cells and lysates to a range of temperatures. It also enables the proteome-wide assessment of drug-protein interactions by combining quantitative mass spectrometry (1) with the cellular thermal shift assay, (CETSA)(2)(3)(4).Beyond the identification of direct drug targets (4) TPP detects drug treatment induced perturbations of metabolic and signaling pathways (5)(3)(6) and modulation of protein complexes regulating complex biological processes (7)(8)(9) in in vitro cellular systems. The CETSA methodology has recently been applied to tissues for detecting thermal stability changes of known drug targets upon in vivo dosing (2)(10), however, a strategy for the proteome-wide assessment of thermal stability and for target profiling in vivo has been lacking. Here we report a TPP strategy to assess thermal stability across tissue proteomes, henceforth referred to as tissue-TPP, and to study engagement of targets and off-targets in organs of animals dosed with the marketed HDAC inhibitor panobinostat.

Thermal proteome profiling for interrogating protein interactions

“…In theory, any cell type can be used, provided that the lysis method does not resolubilize the heat-induced insoluble protein fraction. To date, the method has been used to profile bacteria ( Intact tissues can also be used to preserve the in vivo context of cells (Martinez Molina et al, 2013;Ishii et al, 2017;Perrin et al, 2020). These can either be collected and treated with a perturbation, or be collected after the perturbation is performed in the whole organism and systematically analyzed (Perrin et al, 2020).…”

“…To date, the method has been used to profile bacteria ( Intact tissues can also be used to preserve the in vivo context of cells (Martinez Molina et al, 2013;Ishii et al, 2017;Perrin et al, 2020). These can either be collected and treated with a perturbation, or be collected after the perturbation is performed in the whole organism and systematically analyzed (Perrin et al, 2020). This allows the collection of multiple tissues from a single animal, which provides a holistic view of the perturbation in the organism.…”

“…This allows the collection of multiple tissues from a single animal, which provides a holistic view of the perturbation in the organism. Biological fluids, such as blood, can also be collected (Perrin et al, 2020). In the future, these might offer new therapeutic monitoring strategies or disease biomarkers.…”

Perspective on CETSA Literature: Toward More Quantitative Data Interpretation

“…To fully exploit these opportunities, the analysis should incorporate also the CETSA amenability (i.e., the extent to which primary pharmacologies and downstream events respond to different heat pulses). It will be interesting to follow how such developments are integrated with, for example, blood–CETSA 51 and other innovative approaches that can bring us closer to measuring individual patient responses.…”

“…3A ), there are only a few published examples of ex vivo and in vivo experiments. 8,9,48–51 Particularly noteworthy examples are the studies on RIPK1 inhibitors 50 and panobinostat, 52 respectively, because they include demonstration of cellular target engagement in blood cells, as well as multiple tissues from treated animals. These pivotal translational studies pave the way for further development toward the potential use of CETSA as a biomarker of pharmacologic response.…”