TY - JOUR

T1 - Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls

AU - Morozzi, Chiara

AU - Sauerland, Max

AU - Gamon, Luke F.

AU - Manandhar, Asmita

AU - Ulven, Trond

AU - Davies, Michael J.

N1 - 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

PY - 2022

Y1 - 2022

N2 - 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.

AB - 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.

KW - Click chemistry

KW - Dimethylfumarate

KW - Electrophile

KW - GAPDH

KW - Keap-1

KW - Michael adduct

KW - Unsaturated carbonyls

U2 - 10.1016/j.redox.2022.102299

DO - 10.1016/j.redox.2022.102299

M3 - Journal article

C2 - 35358849

AN - SCOPUS:85127202798

VL - 52

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

M1 - 102299

ER -