Management of Hsp90-Dependent Protein Folding by Small Molecule Targeting the Aha1 Co-Chaperone

Abstract

The core cytosolic Hsp90 chaperone/co-chaperone complex plays a critical role in proteostasis management of human health and disease. To identify novel compounds that alter the ability of the Hsp90 co-chaperone Aha1 to modulate the ATPase activity found in multiple folding diseases ranging from steroid hormone receptor (SHR) sensitive prostate cancer to tauopathies associated with neurodegenerative diseases, we employed a high throughput screening (HTS) assay to monitor selectively Aha1stimulated Hsp90 (ASH) ATPase activity. The ASH assay identified SEW04784 (SEW), a small molecule that disrupts ASH activity without inhibiting the basal Hsp90 ATPase activity. NMR analysis reveals that SEW binds to the C-terminal domain of Aha1 to disrupt its asymmetric binding to Hsp90 leading to abrogation of its chaperoning activity of Hsp90. SEW exhibits therapeutic potential by blocking the transcriptional activity of prostate cancer (PCa) associated variants of the androgen receptor (AR) in a cell-based model of PCa. Additionally, SEW exhibits the ability to clear toxic, phosphorylated tau aggregated species associated with tauopathies. By not directly impacting the basal ATPase function of the abundant and ubiquitous Hsp90, SEW could provide a therapeutic approach for mitigation of client-specific proteostatic disease.of TPR containing co-chaperones via its MEEVD motif 5,14 . The Hsp90 dimer undergoes a series of structural rearrangements during its ATP-binding and hydrolysis cycle. In the nucleotide-free (apo) form, the C-terminally dimerized Hsp90 adopts an open conformation, which closes upon ATP binding and hydrolysis, a process that promotes protein folding and is assisted by the action of its multiple co-chaperones 5,14 , including the accelerator of Hsp90 ATPase (Aha1) 17-32 .Aha1 is a two domain, Hsp90 co-chaperone conserved from yeast to man 21,23,[33][34][35] . It competes with other Hsp90 co-chaperones for binding to Hsp90 and contributes to the functional activation of client proteins, including kinases, steroid hormone receptors and transcription factors 1,4,5,33,36,37 , by stimulating the ATPase activity of Hsp90 [38][39][40] . We have previously shown that Aha1 binding to Hsp90 is asymmetric. It bridges the middle domain of one of the Hsp90 protomers in the Hsp90 dimer with the N-terminal domain of the other Hsp90 molecule to promote the N-terminal dimerization of Hsp90 and thereby stimulate its ATPase activity [38][39][40] . The silencing of Aha1 decreases client protein activation and increases cellular sensitivity to Hsp90 inhibitors 38,41,42 . In cystic fibrosis (CF), a reduction of the Aha1 expression levels in vivo corrects the trafficking and functional defect associated with the deletion of Phe508 (F508del) variant of the cystic fibrosis transmembrane conductance regulator (CFTR), the most abundant CF-associated mutation 32 . These data suggest an important role for reduced ATPase activity in the stabilization of mutant CFTR, a hypothesis that we posited can be extended to other Hsp90-associated disea...

The role and therapeutic potential of Hsp90, Hsp70, and smaller heat shock proteins in peripheral and central neuropathies

“…163,164 A quinaldine red ATPase assay was used to identify one final compound, SEW04784 that binds to the C-terminal domain of Aha1 and disrupts its interaction with Hsp90. 165 This compound inhibits the Aha1-stimulated ATPase activity of Hsp90, but not Hsp90's basal ATPase activity.…”

“…Both of these compounds restored chloride channel activity in cells expressing mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein and may be further developed to treat cystic fibrosis, as Aha1 appears to play a disruptive role in CFTRΔ508 degradation during cystic fibrosis pathology 163,164 . A quinaldine red ATPase assay was used to identify one final compound, SEW04784 that binds to the C‐terminal domain of Aha1 and disrupts its interaction with Hsp90 165 . This compound inhibits the Aha1‐stimulated ATPase activity of Hsp90, but not Hsp90's basal ATPase activity.…”