A chemical compound inhibiting the Aha1–Hsp90 chaperone complex

Abstract

The eukaryotic Hsp90 chaperone machinery comprises many co-chaperones and regulates the conformation of hundreds of cytosolic client proteins. Therefore, it is not surprising that the Hsp90 machinery has become an attractive therapeutic target for diseases such as cancer. The compounds used so far to target this machinery affect the entire Hsp90 system. However, it would be desirable to achieve a more selective targeting of Hsp90-co-chaperone complexes. To test this concept, in this-proof-of-principle study, we screened for modulators of the interaction between Hsp90 and its co-chaperone Aha1, which accelerates the ATPase activity of Hsp90. A FRET-based assay that monitored Aha1 binding to Hsp90 enabled identification of several chemical compounds modulating the effect of Aha1 on Hsp90 activity. We found that one of these inhibitors can abrogate the Aha1-induced ATPase stimulation of Hsp90 without significantly affecting Hsp90 ATPase activity in the absence of Aha1. NMR spectroscopy revealed that this inhibitory compound binds the N-terminal domain of Hsp90 close to its ATP-binding site and overlapping with a transient Aha1-interaction site. We also noted that this inhibitor does not dissociate the Aha1-Hsp90 complex but prevents the specific interaction with the N-terminal domain of Hsp90 required for catalysis. In consequence, the inhibitor affected the activation and processing of Hsp90-Aha1-dependent client proteins We conclude that it is possible to abrogate a specific co-chaperone function of Hsp90 without inhibiting the entire Hsp90 machinery. This concept may also hold true for other co-chaperones of Hsp90.

Proteomic interrogation of HSP90 and insights for medical research

“…As a consequence, several groups are trying to develop inhibitors that either target the C-terminal domain [93–97] or the middle domain [98] or aim at blocking interactions of HSP90 with co-chaperones, e.g. inhibiting interactions with the kinase adapter CDC37 [99–102] or inhibiting the ATPase stimulation by Aha1 [103]. The goal of these efforts is either to have inhibitors that block Hsp90 action without inducing the deleterious heat shock response and/or have more selective inhibitors with more restricted and better predictable effects.…”

The proteostasis network provides targets for neurodegeneration

“…A number of small molecules have been reported to attenuate the Aha1–HSP90 interaction; KU‐177 [7] , a truncated derivative of the natural product noviobiocin, a C‐terminal domain inhibitor of HSP90, was reported to reduce tau aggregation through inhibition of the Aha1–HSP90 protein–protein interaction (Shelton et al, ). Stiegler et al reported Ham‐1 [8] —an apparent allosteric inhibitor of HSP90—that abolished Aha1‐mediated stimulation of HSP90 ATPase activity but did not significantly decrease native HSP90 ATPase activity nor dissociate the protein complex (Stiegler et al, ). Ihrig and Obermann conducted a screen for inhibitors of the Aha1–HSP90 interaction using AlphaScreen Technology and identified A12 [9] and A16 [10] as hit compounds but did not demonstrate which protein partner the compounds primarily interacted with (Ihrig & Obermann, ).…”

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

“…The molecular silencing of most Hsp90 regulatory co-chaperones impedes the ability of 90inh to mediate the clearance of toxic p-tau species 93 , suggesting that it is the Hsp90 chaperone/co-chaperone complex that facilitates clearance and not Hsp90 in isolation. Indeed, Aha1 is critical for the Hsp90-mediated aggregation of p-tau species and that inhibiting its Hsp90 binding with small molecules targeting Hsp90 to prevent binding to Aha1 17,20 , or those that are presumed to interact directly with Aha1 cannot only prevent the formation of de novo aggregates but also promote the clearance of p-tau 18 .…”

“…While this mutual exclusion observation could be explained by the co-chaperone activity of Aha1, which shifts the binding equilibrium of the Hsp90/Aha1 interaction to the non-bound state, Aha1 was able to prevent the aggregation of both FLuc and Rhodanese in vitro and in vivo in the absence of Hsp90, suggesting that it possesses direct chaperoning activity. While the ability of Aha1 to bind to its clients has been shown to require the first 22 amino acids located at the NTD of Aha1 17,39 , which is unaffected by SEW, the ATPase stimulating activity in vitro required the full-length protein. These data suggest that while SEW04784 is unlikely to disrupt the ability of Aha1 to bind to its client proteins, it potentially could also impact the chaperoning activity of Aha1, thereby promoting the clearance of bound clients, such as p-tau and the AR, through ubiquitination, as shown tau in response to co-incubation with IU1-47 117 or for Aha1-bound, heat denatured FLuc in the absence of chaperone-mediated refolding 25 .…”

“…To circumvent this issue, new Hsp90 inhibitors have been developed to target co-chaperone binding. These include novobiocin and other compounds 51,[63][64][65][66][67][68][69] that competes for binding to the C-terminal domain of Hsp90 and blocks the binding of TPR containing co-chaperones, and the recently-identified small molecules that disrupt the binding of Aha1 by directly interacting with Hsp90 17,20 as well as compounds indirectly inferred to interact only with Aha1 18 . The potential for small molecule regulators of Hsp90 co-chaperone sensitive disease remains to be established in the clinic.…”