In-depth characterization of Staurosporine induced proteome thermal stability changes

Chernobrovkin, Alexey; Lengqvist, Johan; Körner, Cindy; Amadio, Daniele; Friman, Tomas; Molina, Daniel Martinez

doi:10.1101/2020.03.13.990606

Cited by 8 publications

(13 citation statements)

References 13 publications

(15 reference statements)

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“…In FECH has also been reported to bind [2Fe-2S] iron-sulphur complexes 40 . We found that BSJ-03-204 stabilized FECH in both intact cells and lysate and FECH has been reported by others as an off-target for many small molecules, including kinase inhibitors [34][35][36] . Moreover, even…”

Section: Discussionsupporting

confidence: 71%

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

Chernobrovkin

Cázares-Körner

Friman

et al. 2020

Preprint

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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 to 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. IMiDs) 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).

show abstract

Section: Discussionsupporting

confidence: 71%

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

Chernobrovkin

Cázares-Körner

Friman

et al. 2020

Preprint

Self Cite

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“…By applying the recently developed one-pot, or compressed, format, 38–40 we have explored a wider concentration range (lysate experiment) as well as the time dependence of the binding and degradation profile ( Fig. 2 ).…”

Section: Resultsmentioning

confidence: 99%

“…50 We found that BSJ-03-204 stabilized FECH in both intact cells and lysate, and FECH has been reported by others as an off-target for many small molecules, including kinase inhibitors. 40,44,45 Moreover, even higher pomalidomide concentrations altered the thermal stability of several other redox-associated proteins. This may be in line with previous reports on antioxidative activity of IMiDs, 41 and teratogenic mode of action, in which increased levels of reactive oxygen species were detected upon thalidomide treatment.…”

Section: Discussionmentioning

confidence: 99%

A Tale of Two Tails: Efficient Profiling of Protein Degraders by Specific Functional and Target Engagement Readouts

et al. 2021

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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).

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“…We have applied the CETSA combined with quantitative LC-MS-based proteomics (CETSA MS) to profile compound-induced protein thermal stability changes for 22 compounds in intact HepG2 cells. The experiments were performed in compressed 21 format (also known as one-pot format 22 ). HepG2 cells were treated with 30 µM of each compound and incubated for 60 min at 37 °C in serum-free saltbased medium.…”

Section: Resultsmentioning

confidence: 99%

“…The resulting protein abundance in the soluble fraction corresponded to the area under the protein’s melting curve. 21 The experiment was repeated to yield three biological replicates. Single-compound concentration and application of compressed (one-pot) experimental design allowed for the assessment of reliable protein stability changes at a relatively high throughput of ~6 h acquisition time per compound.…”

Section: Resultsmentioning

confidence: 99%

CETSA MS Profiling for a Comparative Assessment of FDA-Approved Antivirals Repurposed for COVID-19 Therapy Identifies TRIP13 as a Remdesivir Off-Target

et al. 2021

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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.

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In-depth characterization of Staurosporine induced proteome thermal stability changes

Cited by 8 publications

References 13 publications

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

A Tale of Two Tails: Efficient Profiling of Protein Degraders by Specific Functional and Target Engagement Readouts

CETSA MS Profiling for a Comparative Assessment of FDA-Approved Antivirals Repurposed for COVID-19 Therapy Identifies TRIP13 as a Remdesivir Off-Target

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