No abstract

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance, and reaching “undruggable” proteins compared with traditional small-molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation; instead, one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs), understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The cellular thermal shift assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS), CETSA can simultaneously evaluate compound-induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g., immunomodulatory drugs) and PROTACs, to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment, we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e., binding to the PoI and ligases) and functional readout (i.e., degrader induced protein degradation).

Cellular thermal shift assay (CETSA) coupled with high-resolution mass-spectrometry (MS) has proven to be indispensable tool to track thermal stability changes in cellular proteins caused by various external or internal perturbations. One of the major applications of CETSA MS is still binding profile characterization of small molecule drugs. Before applying the method for characterization of novel compound it is crucially important to understand the limitations, sensitivity and ways to improve the throughput of the different method implementations. Here we present deep comprehensive profiling of Staurosporine induced proteome thermal stability alteration utilizing different experiment layouts. By applying unbiased straightforward sample preparation and data analysis approaches we were able to compare and benchmark different experiment layouts for detecting compound-induced protein stability changes in CETSA MS experiments.

The reuse of preexisting small molecules for a novel emerging disease threat is a rapid measure to discover unknown applications for previously validated therapies. A pertinent and recent example where such a strategy could be employed is in the fight against coronavirus disease 2019 (COVID-19). Therapies designed or discovered to target viral proteins also have off-target effects on the host proteome when employed in a complex physiological environment. This study aims to assess these host cell targets for a panel of FDA-approved antiviral compounds including remdesivir, using the cellular thermal shift assay (CETSA) coupled with mass spectrometry (CETSA MS) in noninfected cells. CETSA MS is a powerful method to delineate direct and indirect interactions between small molecules and protein targets in intact cells. Biologically active compounds can induce changes in thermal stability, in their primary binding partners, and in proteins that in turn interact with the direct targets. Such engagement of host targets by antiviral drugs may contribute to the clinical effect against the virus but can also constitute a liability. We present here a comparative study of CETSA molecular target engagement fingerprints of antiviral drugs to better understand the link between off-targets and efficacy.

Cyclin-dependent-kinases (CDKs) are members of the serine/threonine kinase family and are highly regulated by cyclins, a family of regulatory subunits that bind to CDKs. CDK9 represents one of the most studied examples of these transcriptional CDKs. CDK9 forms a heterodimeric complex with its regulatory subunit cyclins T1, T2 and K to form the positive transcription elongation factor b (P-TEFb). This complex regulates transcription via the phosphorylation of RNA polymerase II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription elongation and thus remains an attractive therapeutic target. Herein, we have utilized classical affinity purification chemical proteomics, kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS and Limited Proteolysis (LiP) to study the selectivity, target engagement and downstream mechanistic insights of a CDK9 tool compound. The above experiments highlight the value of quantitative mass spectrometry approaches to drug discovery, specifically proteome wide target identification and selectivity profiling. The approaches utilized in this study unanimously indicated that the CDK family of kinases are the main target of the compound of interest, with CDK9, showing the highest target affinity with remarkable consistency across approaches. We aim to provide guidance to the scientific community on the available chemical biology/proteomic tools to study advanced lead molecules and to highlight pros and cons of each technology while describing our findings in the context of the CDKs biology.