Tandem mass tag-based thermal proteome profiling for the discovery of drug-protein interactions in cancer cells

Johnson, Fraser; Hughes, Christopher S.; Liu, Alvin; Lockwood, William W.; Morin, Gregg B.

doi:10.1016/j.xpro.2022.102012

Cited by 2 publications

(2 citation statements)

References 28 publications

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“…Utilizing TPP, we identified several proteins as target candidates of the natural compound prodigiosin. By offering an unimpaired approach that is not dependent on pre-functionalization of the small molecule or of proteins, TPP represents a highly versatile target identification strategy [ 31 ]. Prioritizing candidates for target validation can be challenging due to multiple possible candidates.…”

Section: Discussionmentioning

confidence: 99%

“…To avoid focussing on false positive targets, Johnson et al proposed orthogonal target identification methods like computational target prediction [ 31 ]. To our knowledge, only the N-terminal GRASP domain of GRASP55 (residues 1–207) has been successfully crystallized so far [ 41 , 67 ], hindering reliable computational modelling of a direct prodigiosin-GRASP55 interaction.…”

Section: Discussionmentioning

confidence: 99%

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The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

Berning,

Lenz,

Bergmann

et al. 2023

Cell Commun Signal

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Background The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. Methods We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. Results We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. Conclusion Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

Berning,

Lenz,

Bergmann

et al. 2023

Cell Commun Signal

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

Van Vranken,

Li,

Mintseris

et al. 2024

Preprint

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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.

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Tandem mass tag-based thermal proteome profiling for the discovery of drug-protein interactions in cancer cells

Cited by 2 publications

References 28 publications

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin

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

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