Identification of galectin-1 and other cellular targets of alpha, beta-unsaturated carbonyl compounds, including dimethylfumarate, by use of click-chemistry probes

alpha,beta-Unsaturated carbonyls are a common motif in environmental toxins (e.g. acrolein) as well as therapeutic drugs, including dimethylfumarate (DMFU) and monomethylfumarate (MMFU), which are used to treat multiple sclerosis and psoriasis. These compounds form adducts with protein Cys residues as well as other nucleophiles. The specific targets ('adductome') that give rise to their therapeutic or toxic activities are poorly understood. This is due, at least in part, to the absence of antigens or chromophores/fluorophores in these compounds. We have recently reported click-chemistry probes of DMFU and MMFU (Redox Biol., 2022, 52, 102299) that allow adducted proteins to be visualized and enriched for further characterization. In the current study, we hypothesized that adducted proteins could be 'clicked' to agarose beads and thereby isolated for LC-MS analysis of DMFU/MMFU targets in primary human coronary artery smooth muscle cells. We show that the probes react with thiols with similar rate constants to the parent drugs, and give rise to comparable patterns of gene induction, confirming similar biological actions. LC-MS proteomic analysis identified similar to 2970 cellular targets of DMFU, similar to 1440 for MMFU, and similar to 140 for the control (succinate-probe) treated samples. The most extensively modified proteins were galectin-1, annexin-A2, voltage dependent anion channel-2 and vimentin. Other previously postulated DMFU targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cofilin, p65 (RELA) and Keapl were also identified as adducted species, though at lower levels with the exception of GAPDH. These data demonstrate the utility of the click-chemistry approach to the identification of cellular protein targets of both exogenous and endogenous compounds.

Automated summary

Alpha, beta-unsaturated carbonyls are commonly found in environmental toxins and therapeutic drugs. However, the specific targets and mechanisms of these compounds are poorly understood. Recently, a click-chemistry method has been developed to visualize and enrich adducted proteins for further characterization. LC-MS proteomic analysis has identified the cellular targets of DMFU and MMFU.