Covalent Enzyme Inhibition through Fluorosulfate Modification of a Non-Catalytic Serine Residue.

Irreversible enzyme inhibitors often utilize the reaction of a protein cysteine residue with an appropriately positioned electrophile (an acrylamide for example) in the ligand template. Cysteine residues are not always available for site-specific protein labeling and therefore new approaches are needed to expand the toolkit of appropriate electrophiles that target alternative amino acids. We have previously described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger enzyme, DcpS) using inhibitors armed with sulfonyl fluoride electrophiles that additionally enabled the subsequent development of clickable probe technology to measure drug-target occupancy in live cells. Here we describe a fluorosulfate-containing inhibitor (FS-p1) with excellent chemical and metabolic stability that reacted selectively with a non-catalytic serine residue in the same active site of DcpS, as confirmed by peptide mapping experiments. Our results, that include metabolic and chemical stability characterization, suggest that non-catalytic serine targeting using fluorosulfate electrophilic warheads could be a suitable strategy for the development of covalent inhibitor drugs and chemical probes.