Anton Simeonov Ph.D. National Institute of Health

CETSA Is Now Being Used Routinely to Demonstrate Small Molecule’s Engagement of a Specific Target in the Cell

The cellular thermal shift assay (CETSA) was first described approximately 2 years ago by Par Nordlund's team (Molina et al., Science 2012;341:84), and to date its uptake in the community has been truly widespread. The technique is now being used routinely to demonstrate small molecule's engagement of a specific target in the cell, and improvements have been made to various aspects of the protocol and target quantitation. Two recent reports in this area deserve mention as they provide new ways of using CETSA. In the work by Huber et al., CETSA is being used to discover new target proteins being stabilized by small molecules through the use of quantitative mass spectrometry. After application of the CETSA protocol, the supernatant was subjected to proteolytic digestion and labeling using isobaric tandem mass tags for quantitative mass spectrometry (MS) analysis (Figure 1). 


Figure 1. Schematic representation of the thermal profiling methodology. (a) Binding of a ligand to its target protein (top) increases the enthalpy required for unfolding. As a result, the melting temperature (Tm) is shifted (bottom). This can be exploited with, for example, differential scanning fluorimetry (‘Tm shift assay’). A.U., arbitrary units. (b) Representative workflow. Cells are treated with either small molecule or vehicle, washed and harvested. Equal amounts of cells are then heated to increasing temperatures and lysed, and the supernatant obtained after centrifugation is digested and labeled for quantitative mass spectrometry (MS) analysis. Detection of changes in protein stability in treated versus control samples is enabled by bioinformatic processing and data plotting in the form of differential protein-abundance graphs. (c) The bioinformatics scoring parameters used for data analysis. Continuous lines depict raw data, and dashed lines are curve-fitting results.


The team demonstrated the target discovery approach via a study of the cellular targets of two drugs (methotrexate and (S)-crizotinib) and a metabolite (2′3′-cGAMP), identifying novel molecular targets for these small molecules. In turn, the work by Reinhard et al. extends CETSA to the traditionally difficult-to-study transmembrane proteins. The team performed a careful investigation on the utility of select detergents to extract intact transmembrane proteins after the heat step of the CETSA protocol, while avoiding extraction of the thermally-denatured proteins, and settled on 0.4% Nonidet P-40 (NP-40) as the optimal reagent (Figure 2). The new method was tested in a profiling experiment searching for transmembrane proteins involved in binding adenosine triphosphate. Thus, the use of CETSA in combination with advanced mass spectrometry approaches should allow for a whole new dimension of the technique to be exploited—that of a tool for prospective discovery of molecular targets of drugs and metabolites.


Figure 2. TPP of ATP-binding proteins in cell extracts in the presence or absence of mild detergent. (a) Experiment outline. Cell extracts were generated using PBS with or without 0.4% NP-40, and aliquots were heated to different temperatures, digested, 10-plex tandem mass tag (TMT10) labeled and analyzed by mass spectrometry. Melting curves for vehicle-treated and ATP-treated cell extracts were fitted, and Tm shifts were inferred between the different treatment conditions. (b) Comparison of Tm values of 2,196 proteins in a K562 cell extract in the absence or presence of NP-40. Dashed line represents the identity line. (c) Comparison of Tm shifts induced by the addition of MgATP to cell extracts with or without NP-40. The minimum Tm shift for each protein was calculated from a pair of biological replicates. Dashed line represents the identity line. (d) Density plot of Tm values of membrane and nonmembrane proteins in NP-40 detergent extracts. (e) Comparison of Tm shifts for ATP-binding membrane proteins determined from two biological replicates. (f) Melting and aggregation curves for the inner mitochondrial membrane proteins ABCB10 and BCS1L in the presence (orange symbols) and absence (gray symbols) of ATP. Data from two independent replicate experiments are shown.


* Abstract from Nat Methods 2015;12:1055–1057

Thermal stabilization of proteins after ligand binding provides an efficient means to assess the binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry, the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. We profiled the targets of the drugs methotrexate and (S)-crizotinib and the metabolite 2′3′-cGAMP in intact cells and identified the 2′3′-cGAMP cognate transmembrane receptor STING, involved in immune signaling.


* Abstract from Nat Methods 2015;doi:10.1038/nmeth.3652 AOP.

We extended thermal proteome profiling to detect transmembrane protein–small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced Tcell–receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1and MDR1.





































Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, two papers published in Nature Methods titled "Proteome-wide drug and metabolite interaction mapping by thermal-stability profiling", authors are Huber KVM, Olek KM, Müller AC, Tan CSH, Bennett KL, Colinge J, Superti-Furga G and "Thermal proteome profiling monitors ligand interactions with cellular membrane proteins" authors are Reinhard FBM, Eberhard D, Werner T, Franken H, Childs D, Doce C, Savitski MF, Huber W, Bantscheff M, Savitski1 MM, Drewes G.

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