Large-scale characterization of drug mechanism of action using proteome-wide thermal shift assays

Abstract

In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to characterize compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified 1.498 million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.

Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays

“…In summary, our data show that live-cell PISA can be useful to study pathway interactions but falls short for the unambiguous assignment of direct rocaglate targets. We therefore opted to measure thermal stabilization in cell lysate to allow for improved detection of direct ligand-protein engagement while muting network effects (47).…”

“…apparent stabilization) through multiple indirect mechanisms ( e.g. protein expression, cellular localization) (47). This is particularly salient for rocaglates, which acutely alter protein abundance through cap-dependent translation inhibition, making it unclear whether fold-change differences arise from ligand-induced stabilization or modulation of protein synthesis.…”

“…One way to mitigate sensitivity tradeoffs is through informed selection of heating temperatures. Others have demonstrated that higher temperature ranges can magnify stabilization readouts, provided that the linear portion of the stability curve is captured for proteins of interest (47, 62). It was a matter of serendipity that most DDXs have half-maximal melting points between those of DDX3X and eIF4A2 ( Figure 4A ) – this helped us infer that the temperatures we tested were representative of the target class.…”

“…Live cell-based formats report on the downstream effects of compound treatment ( e.g. changes in protein abundance) in addition to compound-induced thermal stabilization, which makes identification of direct protein targets more challenging (47). Lysate-based formats are useful in identifying direct targets but are performed under non-physiological conditions in which co-factors or co-substrates necessary for target engagement may become diluted or disrupted (43, 48).…”

Defining the heterogeneous molecular landscape of lung cancer cell responses to epigenetic inhibition

“…For example, we compared total proteomics and thermal stability analyses after belinostat treatment to integrate understanding of early HDACi action (thermal stability) and downstream effects (total proteomics) (Supplementary Figure 8A, B, D). Owing to the potential importance of cellular and chromatin context in affecting HDACi engagement, we have previously found that both cell- and lysate-based analyses were complementary for deciphering the molecular effects of chemical perturbations in cancer cells (Van Vranken et al, 2024). From the thermal stability analysis, we observed clear engagement of belinostat and vorinostat with HDAC1, HDAC2, HDAC6 (Figure 8B).…”