The stress-inducible molecular chaperone, HSP72, is an important therapeutic target in oncology, but inhibiting this protein with small molecules has proven particularly challenging. Validation of HSP72 inhibitors in cells is difficult owing to competition with the high affinity and abundance of the endogenous substrate, ADP. Irreversible inhibition is an important strategy for tackling proteins with high-affinity substrates, with the recent renaissance being led by drugs targeting the tyrosine kinase, EGFR. With the clear clinical potential that a HSP72 inhibitor could offer and the lack of cell-active chemical probes to study its role in cancer, we proposed that a nucleotide-competitive targeted covalent inhibitor could overcome many of these challenges. Our original rational design hypothesis was to exploit the 8-N-benzyladenosine ligands to synthesize a cysteine-targeting irreversible inhibitor. The time-dependent HSP72 inhibition and intact protein mass spectrometry data showed we were successful, but trypsin-digest mass spectrometry clearly contradicted our conclusion that any cysteine was the nucleophilic residue responsible. Using x-ray crystallography and site-directed mutagenesis to search for the key residue, we discovered that the highly conserved lysine-56 was responsible for the covalent bond formation. The discovery of the first lysine-selective targeted covalent inhibitor of HSP72, 1, offers a potential new design paradigm in the development of cell-active HSP72 inhibitors. Co-crystal structures have been used to design new compounds with greater reversible affinity, and electrophiles that are more suited to the harder lysine nucleophile.

Design and synthesis of lysine-56 targeting covalent inhibitors of HSP72

253rd National Meeting (2017)

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