A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells

PLOS ONE, Nov 2019

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

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A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells

April A high content, high throughput cellular thermal stability assay for measuring drug- target engagement in living cells Andrew J. Massey 0 1 0 Vernalis Research , Granta Park, Cambridge , United Kingdom 1 Editor: Bing Xu, Brandeis University , UNITED STATES Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells. - Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by Vernalis Research who agreed to the publication of this manuscript. The funder provided support in the form of salaries for author AJM, but did not have any additional role in the study design, data collection and analysis, or preparation of the manuscript. The specific role of the author are articulated in the `author contributions' section. Introduction A critical component of small molecule drug discovery is determining and understanding ligand-protein interactions (target engagement) at the site of drug action in the cell. For a large number of potential drug targets, ªclassicalº approaches (e.g. monitoring changes to substrate or product generation) are not amenable. The cellular thermal shift assay (CETSA) first described by Martinez Molina et al [ 1 ] has become frequently used in target engagement studies. The assay relies on the principle that ligand binding results in thermal stabilization (or sometimes destabilization) of the bound protein. Practically, the CETSA method measures the amount of soluble protein remaining in cells following heating at various temperatures in the absence or presence of a ligand. The classic method [ 1,2 ] relies on treating cells with ligand and then heating in suspension at relatively high densities (of the order of 1-3x107/mL) in a thermocycler. Following cell lysis, cell debris as well as aggregated and precipitated proteins are removed and the remaining soluble protein detected by, for Competing interests: AJM is an employee of Vernalis. This does not alter the author's adherence to PLOS ONE policies on sharing data and materials. example, western blotting or homogenous detection methods (e.g. AlphaScreen, ELISA, referred to as HT-CETSA etc.) [ 2,3 ]. As this method does not rely on modification of either the target or an interacting ligand, it can in theory, be applied to any target in any cellular system. A recent advance has seen the application of high-resolution mass spectrometry to the whole proteome enabling not only the measurement of desired on-target effects but also the identification of potential off-target liabilities [4±7]. For adherent cells, the requirement to heat the cells in suspension at high density is an obvious drawback and the process of trypsinization and resuspension may alter cellular physiology and target pharmacology. Additionally, having to treat cells at high cell densities may result in an underestimation of target engagement potency and make comparisons to downstream pharmacology assays more difficult. As the CETSA method determines the amount of soluble, folded protein remaining, we hypothesized that heating cells growing attached to a 96 well plate (96WP) and determining changes in the amount of target protein still correctly folded by high content immunofluorescent imaging might be a useful adaption of the CETSA method for adherent cells. We have therefore developed a novel cellular target engagement assay in adherent live cells using the principle of ligand-induced changes to protein thermal stability coupled with high-content single cell immunofluorescent imaging in an attempt to mitigate some of these potential liabilities. Materials and methods Cell lines and cell culture HT29 and U2OS cells were purchased from the ATCC and grown in DMEM or McCoys 5a media supplemented with 10% fetal bovine ser (...truncated)


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Andrew J. Massey. A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells, PLOS ONE, 2018, Volume 13, Issue 4, DOI: 10.1371/journal.pone.0195050