An Irreversible Inhibitor of HSP72 that Unexpectedly Targets Lysine‐56

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. Simply irreversible: The stress‐inducible molecular chaperone, HSP72, is an important target in oncology but few validated inhibitors have emerged. To overcome the high affinity and abundance of its endogenous substrates, an irreversible nucleotide‐competitive inhibitor, which unexpectedly targeted lysine‐56, was discovered by rational design.