Proximity-induced SuFEx increases the potency of cytosolic nucleotidase inhibitors and reveals a rare example of covalently targeted histidine.

Structure-guided design is one of the most validated solutions for targeting proteins with specific ligands for therapeutic purposes. Nevertheless, it remains challenging to target enzymes with low affinity for their natural ligands and specificities that overlap with those of other proteins. Cytosolic 5'-nucleotidases - involved in the metabolism of nucleic acid derivatives - are an example of such a family. Here we illustrate how precisely designed covalent inhibitors represent a potential solution for selective nucleotidase targeting. We employed the sulfur-fluoride exchange (SuFEx) to develop a covalent inhibitor of cytosolic nucleotidase IIIB (cNIIIB). Using the known inhibitor (7-benzylguanosine monophosphate, BnGMP) and computational methods, we designed and synthesized a series of SuFExable inhibitors. One compound indeed covalently bound cNIIIB, which increased the inhibition potency by over 100-fold. The formation of a covalent S-N bond with a non-catalytic His110 residue was confirmed through MS and N NMR. The selectivity of the compound in the context of other protein that recognises similar ligands was also confirmed. The study expands the principle of covalent inhibition of nucleotide processing enzymes. It also represents a rare example of histidine tagging by SuFEx. This may facilitate the broader application of SuFEx chemistry in biochemistry and medicinal chemistry.