Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Mortison, Jonathan D.; Cornella-Taracido, Ivan; Venkatchalam, Gireedhar; Partridge, Anthony W.; Siriwardana, Nirodhini; Bushell, Simon M.

doi:10.1021/acsmedchemlett.1c00276

Cited by 22 publications

(11 citation statements)

References 29 publications

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“…The absence of any significant activity of compound 11 in the cellular NF-κB reporter assay prompted us to establish a cellular thermal shift assay (CETSA) to investigate cell permeability and cellular target engagement (Figure A). In particular, we utilized a HiBiT CETSA assay format , conducted in intact HEK293T cells. Cells were transfected with USP21 fused to a small proluminescent NanoLuc HiBiT tag, which, when complemented with the Large BiT NanoLuc fragment, produces a quantifiable luminescence signal.…”

Section: Resultsmentioning

confidence: 99%

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21

Göricke

Smith

et al. 2023

J. Med. Chem.

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USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

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Section: Resultsmentioning

confidence: 99%

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21

Göricke

Smith

et al. 2023

J. Med. Chem.

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“…To verify these data, we established a cellular thermal shift assay (HiBIT-CETSA) using the sensitive HiBIT-tagged WDR5 (HEK293T HiBIT-WDR5 ) as a detection system. 22 Surprisingly, a relatively high cellular melting point of WDR5 was determined (71.4 °C) while PROTAC addition further stabilized WDR5 towards temperature induced denaturation. We obtained relative shifts in melting temperature up to 13.8 °C for MS67 increasing the WDR5 melting point to 85.2 °C (Table 1).…”

Section: Detection Of Binary Complexesmentioning

confidence: 93%

Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization

Schwalm

Krämer

Dölle

et al. 2023

Preprint

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The multistep PROTAC (PROteolysis TArgeting Chimeras) induced degradation process poses challenges on the rational development of these chimeric molecules, as the rate limiting steps determining PROTAC efficiency are largely unknown. Moreover, the slow throughput and the limited dynamic range of currently used endpoint assays such as Western blotting, does not allow the comprehensive analysis of PROTAC SAR series. To this end, we developed fluorescent based assays using NanoLuciferase and HaloTag based technologies, that allow measuring PROTAC induced ternary complex formation kinetics and stability between the target protein and the chosen E3 ligase. Using available PROTACs developed for degradation of WDR5, the comprehensive characterization of the mode of action of these PROTACs in the early degradation cascade revealed a key role of ternary complex formation and stability over the corresponding binary PROTAC-WDR5 and PROTAC-E3 complexes. Comparing a series of ternary complex crystal structures highlighted the importance of an efficient E3-target interface for ternary complex stability. The developed assays outline a strategy for the rational optimization of PROTACs using a series of live cell assays monitoring key steps of the early PROTAC induced degradation pathway.

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“…The ligand binding to the target protein changes the protein conformation which decreases the luciferase activity ( Dale et al, 2019 ). Scientists from Merck have developed a nano luciferase detection system for CETSA called HiBiT thermal shift assay (BiTSA) ( Mortison et al, 2021 ). BiTSA combines the CETSA and parts of NanoBRET to create an antibody-free high-throughput detection system.…”

Section: Different Label-free Cell-based Methods For Target Engagementmentioning

confidence: 99%

“…When in complex together HiBiT-LgBiT produces bioluminescence, but when the target protein is denatured, LgBiT cannot bind to HiBiT. The luciferase signal attenuates linearly together with the amount of ligand bound to the target protein ( Dale et al, 2019 ; Mortison et al, 2021 ). NanoBRET and BiTSA both require an engineered cell system to produce a tagged target protein but can be used for screening molecular libraries against pre-determined targets and for lead optimization.…”

Section: Different Label-free Cell-based Methods For Target Engagementmentioning

confidence: 99%

“…In recent years, many new drug leads have been found from phenotypic screenings, creating a demand for target deconvolution and confirmation of lead molecules' target engagement ( Kaur et al, 2018 ; Friman, 2020 ; Mateus et al, 2021 ). The most used label-free techniques using intact cells or cell lysates are stability of proteins from rates of oxidation (SPROX) ( West et al, 2008 ; Strickland et al, 2013 ), drug affinity responsive target stability (DARTS) ( Lomenick et al, 2009 ), limited proteolysis (LiP) ( Feng et al, 2014 ), and cellular thermal shift assay (CETSA ® ) ( Martinez Molina et al, 2013 ) with all its variations.…”

Section: Introductionmentioning

confidence: 99%

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Current Advances in CETSA

Tolvanen

2022

Front. Mol. Biosci.

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Knowing that the drug candidate binds to its intended target is a vital part of drug discovery. Thus, several labeled and label-free methods have been developed to study target engagement. In recent years, the cellular thermal shift assay (CETSA) with its variations has been widely adapted to drug discovery workflows. Western blot–based CETSA is used primarily to validate the target binding of a molecule to its target protein whereas CETSA based on bead chemistry detection methods (CETSA HT) has been used to screen molecular libraries to find novel molecules binding to a pre-determined target. Mass spectrometry–based CETSA also known as thermal proteome profiling (TPP) has emerged as a powerful tool for target deconvolution and finding novel binding partners for old and novel molecules. With this technology, it is possible to probe thermal shifts among over 7,000 proteins from one sample and to identify the wanted target binding but also binding to unwanted off-targets known to cause adverse effects. In addition, this proteome-wide method can provide information on the biological process initiated by the ligand binding. The continued development of mass spectrometry labeling reagents, such as isobaric tandem mass tag technology (TMT) continues to increase the throughput of CETSA MS, allowing its use for structure–activity relationship (SAR) studies with a limited number of molecules. In this review, we discussed the differences between different label-free methods to study target engagement, but our focus was on CETSA and recent advances in the CETSA method.

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Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay

Cited by 22 publications

References 29 publications

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21

Tracking the PROTAC degradation pathway in living cells highlights the importance of ternary complex measurement for PROTAC optimization

Current Advances in CETSA

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