Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small-molecule/protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ϵ-amino group in lysine, this strategy generates a number of specific challenges. Herein, we review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.

The stress‐inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validating HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of its endogenous nucleotide substrates. We hypothesized this could be overcome using a cysteine‐targeted irreversible inhibitor. Using rational design, we adapted a validated 8‐N‐benzyladenosine ligand for covalent bond formation and confirmed targeted irreversible inhibition. However, no cysteine in the protein was modified; instead, we demonstrate that lysine‐56 is the key nucleophilic residue. Targeting this lysine could lead to a new design paradigm for HSP72 chemical probes and drugs.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (S VI -Fs) as reactive groups for chemical biology applications. S VI -Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of S VI -Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of S VI -Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the S VI -F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of S VI -F used. This work highlights how S VI -F reactivity can be used as a tool to expand the liganded proteome.