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, ATP. Irreversible inhibition is an important strategy for tackling proteins with high-affinity substrates, as shown in the recent renaissance being led by drugs targeting the tyrosine kinase, EGFR, in refractory cancers. 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 ligand, 1, to synthesize a cysteine-targeting irreversible inhibitor, 2. 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. By developing a method to measure the covalent kinetic parameters, kinact and KI, from a fluorescence polarisation assay, we were able to design a second-generation covalent inhibitor, 3. This showed greater reversible affinity, and reactivity that is more suited to the harder lysine nucleophile. The discovery of the first lysine-selective targeted covalent inhibitors of HSP72 offer a potential new design paradigm in the development of cell-active HSP72 chemical probes. We plan to use these compounds to develop chemical probes for use in activity-based protein profiling experiments, in order to better understand the role of HSP72 in cells.

Design and synthesis of lysine-targeted covalent inhibitors of HSP72

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.