Pharmacology of Hsp70 Activation

Abstract

The heat shock protein 90 and 70 (Hsp90 and Hsp70) chaperone system maintains protein quality control for over one hundred client proteins, including many implicated in neurodegenerative disorders, by surveilling substrate binding clefts and selectively targeting damaged proteins for degradation. Hsp90 stabilizes client proteins in their native state and prevents degradation, whereas Hsp70 facilitates ubiquitination by E3 ligases and promotes proteasomal degradation. Traditionally the chaperone system has been targeted with Hsp90 inhibitors, and while many Hsp90 inhibitors have entered clinical trials for cancer they have failed to progress due in part to toxicity. Activation of Hsp70 has recently emerged as an alternative approach, with numerous genetic studies demonstrating that activation of Hsp70-dependent ubiquitination increases the degradation of disease-causing client proteins in cellular and animal models. Studies on the pharmacological activation of Hsp70 have been limited by the small number of compounds known to activate Hsp70-dependent ubiquitination. Thus, an innovative workflow was developed to discover novel small molecule activators of Hsp70. A high-throughput Hsp70 thermal shift assay and Hsp90 counter screen were utilized to identify compounds that bind and thermostabilize Hsp70. To test the functional effect of Hsp70 thermostabilizers in vitro, a purified protein system for Hsp70-dependent ubiquitination of neuronal nitric oxide synthase (nNOS) and a highly sensitive ELISA for the measurement of nNOS ubiquitination were established. With the use of this workflow we successfully identified one novel small molecule activator of Hsp70-dependent ubiquitination. Moreover, in the course of these studies we discovered pharmacological activation of Hsp70 can selectively increase the ubiquitination of damaged nNOS over nNOS in its native state. The small molecule identified by this workflow increases the structural diversity of compounds known to activate Hsp70-dependent ubiquitination and may provide a valuable tool to advance the study of pharmacological activation of Hsp70 as a therapeutic strategy.