High-Throughput Cellular Thermal Shift Assays in Research and Drug Discovery

Abstract

Thermal shift assays (TSAs) can reveal changes in protein structure, due to a resultant change in protein thermal stability. Since proteins are often stabilized upon binding of ligand molecules, these assays can provide a readout for protein target engagement. TSA has traditionally been applied using purified proteins and more recently has been extended to study target engagement in cellular environments with the emergence of cellular thermal shift assays (CETSAs). The utility of CETSA in confirming molecular interaction with targets in a more native context, and the desire to apply this technique more broadly, has fueled the emergence of higher-throughput techniques for CETSA (HT-CETSA). Recent studies have demonstrated that HT-CETSA can be performed in standard 96-, 384-, and 1536-well microtiter plate formats using methods such as beta-galactosidase and NanoLuciferase reporters and AlphaLISA assays. HT-CETSA methods can be used to select and characterize compounds from high-throughput screens and to prioritize compounds in lead optimization by facilitating dose–response experiments. In conjunction with cellular and biochemical activity assays for targets, HT-CETSA can be a valuable addition to the suite of assays available to characterize molecules of interest. Despite the successes in implementing HT-CETSA for a diverse set of targets, caveats and challenges must also be recognized to avoid overinterpretation of results. Here, we review the current landscape of HT-CETSA and discuss the methodologies, practical considerations, challenges, and applications of this approach in research and drug discovery. Additionally, a perspective on potential future directions for the technology is presented.

Applied Biophysical Methods in Fragment-Based Drug Discovery

“…Cocktailing can be taken to its extreme when screening fluorine libraries. The enormous 19 F chemical shift range of >100 ppm means that cocktail size is no longer dictated by spectral overlap. Factors such as the maximally acceptable total organic load, expected hit rate, and NMR spectrometer hardware limitations, such as the maximal chemical shift range that can be covered reliably with radiofrequency pulses, 69 put an upper limit on the cocktail size.…”

“…Screening campaigns using up to 36 fragments per cocktail have been reported in the literature. 70 Together with the advent of cryogenically cooled 19 F detection coils, 71 this allows a library of several thousand fragments to be screened in a matter of days. We are not going to discuss the pros and cons of the 19 F nucleus compared with 1 H. Instead, we refer the reader to a series of excellent papers discussing this in great detail.…”

“…We are not going to discuss the pros and cons of the 19 F nucleus compared with 1 H. Instead, we refer the reader to a series of excellent papers discussing this in great detail. 70,72,73 As far as this review is concerned, 19 F-NMR will be treated as just another flavor of NMR that can be applied to the same tasks as 1 H-NMR (e.g., for screening and for reporter assay development; see below).…”

“…15 More recently, intrinsic tryptophan fluorescence has made a return to Tm shift buffer screens, 16 and the thermal shift principle has also been adapted to a cell-based readout and the issue of target engagement. [17][18][19][20][21] The following section discusses Tm shift fragment screening monitored by Sypro Orange binding, but similar principles should apply to any fluorescent method that is used to monitor the thermal denaturation process.…”

Advances in Cellular Target Engagement and Target Deconvolution

“…Henderson et al 9 provide an analysis of high-throughput (HT) CETSA methods, which utilize conventional plate-based assay readouts to detect soluble, folded protein, and therefore allow application in a higher-throughput setting, for example, analysis of hits from a high-throughput screen where it has been possible to rank hits according to the EC 50 value of protein stabilization. Several formats of HT CETSA have been described, including antibody-based (measuring endogenous protein) and reporter-based, where a tag is added to the target protein.…”