Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls

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Humans are commonly exposed to α,β-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.g. dimethylfumarate, DMFU, a front-line drug for the treatment of multiple sclerosis and psoriasis). These compounds undergo rapid Michael addition reactions with amine, imidazole and thiol groups on biological targets, with reaction at protein Cys residues being a major reaction pathway. However, the cellular targets of these species (the ‘adductome’) are poorly understood due to the absence of readily identifiable tags or reporter groups (chromophores/fluorophores or antigens) on many α,β-unsaturated carbonyls. Here we report a ‘proof of concept’ study in which we synthesize novel α,β-unsaturated carbonyls containing an alkyne function introduced at remote sites on the α,β-unsaturated carbonyl compounds (e.g. one of the methyl groups of dimethylfumarate). The presence of this tag allows ‘click-chemistry’ to be used to visualize, isolate, enrich and characterize the cellular targets of such compounds. The probes show similar selectivity and reactivity to the parent compounds, and compete for cellular targets, yielding long-lived (stable) adducts that can be visualized in intact cells (such as primary human coronary artery smooth muscle cells), and extracted and enriched for subsequent target analysis. It is shown using this approach that dimethylfumarate forms adducts with multiple intracellular targets including cytoskeletal, organelle and nuclear species, with these including the rate-limiting glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This approach should be amenable to use with multiple α,β-unsaturated carbonyls and a wide variety of targets containing nucleophilic sites.

Original languageEnglish
Article number102299
JournalRedox Biology
Volume52
Number of pages8
ISSN2213-2317
DOIs
Publication statusPublished - 2022

Bibliographical note

Funding Information:
This work was supported by grants from the Novo Nordisk Foundation ( NNF13OC0004294 and NNF20SA0064214 to MJD), and a Lundbeck Foundation Fellowship ( 102-6816/20-3000 ) to LFG.

Publisher Copyright:
© 2022 The Authors

    Research areas

  • Click chemistry, Dimethylfumarate, Electrophile, GAPDH, Keap-1, Michael adduct, Unsaturated carbonyls

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