Hsp70 proteins are a family of ancient and conserved molecular chaperones. They play an essential role in maintaining protein homeostasis, including facilitating protein folding and degradation, preventing protein aggregation, and participating in the stress response. Disruption of the cellular quality control machinery is associated with aging, cancer and neurodegenerative diseases. Hsp70 proteins are potential drug targets for treatment of diseases, particularly cancer.

PES (2-phenylethynesulfonamide or pifithrin-μ) has been reported to be an inhibitor of Hsp70. However, the mechanism of PES inhibition remained 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 stress inducible Hsp70 (hHsp70), resulting in covalent attachment of a PES molecule to each Cys residue. Glutathionylation of Cys-574 and Cys-603 has been previously shown to affect the structure and function of hHsp70. In this study, PES modification showed similar structural and functional effects on hHsp70 to glutathionylation. Further, it is 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.

The development of covalent inhibitors of hHsp70 targeting Cys-574 and Cys-603 is a promising strategy for drug design. Compound libraries could be screened for compounds that can covalently react with protein thiols, as these would be worth testing as candidates for hHsp70 inhibitors. Development of PES or other thiol reactive compounds could be further optimized for reaction efficiency and specificity, as a step towards drug development.

This study was carried out by the group of Prof. Sarah Perrett in the Institute of Biophysics, Chinese Academy of Sciences, and was published in the Journal of Biological Chemistry on 1 Jan 2021.

Figure: PES can undergo a Michael addition reaction with Cys-574 and Cys-603 in the SBDα of hHsp70, resulting in covalent attachment of a PES molecule to each Cys residue.

(Image by Dr. Sarah Perrett's group)

Article link: https://www.jbc.org/article/S0021-9258(20)00206-9/fulltext

Contact: Sarah Perrett

Institute of Biophysics, Chinese Academy of Sciences

Beijing 100101, China

Email: sperrett@ibp.ac.cn

(Reported by Dr. Sarah Perrett's group)