Many proteins have pairs of spatially proximal cysteine residues which act as redox switches via disulfide bond formation/reduction. These switches can often have profound effects on the biological activity of the proteins.
Dibromomaleimide and dibromopyridazinedione derivatives were previously reported to be effective in crosslinking two cysteines. Here, the selective targeting of dithiols over single cysteines was attempted using two approaches. One approach used a bicyclic electrophile which it was hoped would give direct selective reaction. The second strategy was to effect non-selective cysteine functionalization followed by selective cleavage with thiol of adducts on isolated cysteines. Unfortunately, the bicyclic electrophiles examined failed to label the protein models, whilst in the second strategy, thiol cleavage removed both crosslinked adducts and non-crosslinked single cysteine adducts.
Interestingly, monobromopyridazinedione was found to react with lysine. It labelled the lysines in somatostatin and this lysine modification was stable under thiol cleavage conditions. Thus, these experiments revealed a potential strategy for selective labelling of lysine residues in the presence of competing cysteines.
Covalent modification of proteins was also explored using a chemical probe based on a series of inhibitors previously reported to inhibit NF-κB expression. These inhibitors were previously reported to covalently bind to proteins via a Michael receptor warhead and they had been shown in our lab to prevent cyclin D1 down-regulation after DNA damaging treatment of mammalian cells. An alkyne handle was introduced to the molecule previously synthesized in our group to allow “click reaction” with an azide-tagged biotin/fluorophore to isolate targets for subsequent identification. Competition assays between the probe and the parental inhibitors in both cell lysates and cells were conducted. The inhibitors and the probe acted in competition in cell lysates, but had a synergistic effect on labelling in cell.Open Acces
