PES inhibits human-inducible Hsp70 by covalent targeting of cysteine residues in the substrate-binding domain

Hsp70 proteins are a family of ancient and conserved chaperones. They play important roles in vital cellular processes, such as protein quality control and the stress response. Hsp70 proteins are a potential drug target for treatment of disease, particularly cancer. PES (2-phenylethynesulfonamide or pifithrin-μ) has been reported to be an inhibitor of Hsp70. However, the mechanism of PES inhibition is still unclear. In this study we found that PES can undergo a Michael addition reaction with Cys-574 and Cys-603 in the SBDα of human HspA1A (hHsp70), resulting in covalent attachment of a PES molecule to each Cys residue. We previously showed that glutathionylation of Cys-574 and Cys-603 affects the structure and function of hHsp70. In this study, PES modification showed similar structural and functional effects on hHsp70 to glutathionylation. Further, we found that susceptibility to PES modification is influenced by changes in the conformational dynamics of the SBDα, such as are induced by interaction with adjacent domains, allosteric changes, and mutations. This study provides new avenues for development of covalent inhibitors of hHsp70.