Ten Non-N-Terminal-Domain Inhibitors of the Hsp90 Chaperone Machine Fail to Elicit Canonical Proteome-wide Responses...

Heat shock protein 90's (Hsp90) roles in cancer have prompted the development of small-molecule inhibitors that target its N-terminal ATP-binding pocket. Moreover, numerous alternative Hsp90 inhibitors (AHI) have been developed to target other sites of the Hsp90 chaperone complex. Alternative Hsp90 inhibition is typically validated in living cells by assaying the inhibitor's impact on a small number of select Hsp90 client proteins. Here, we expand these traditional characterizations by orders of magnitude, measuring changes in the proteomes of Jurkat cells treated with 10 different AHI compounds (ailanthone, celastrol, clorobiocin, coumermycin A1, daurisoline, derrubone, gambogic acid, garcinol, β-lapachone, and α-mangostin). By comparing these changes to those induced by the N-terminal Hsp90 inhibitor NVP-AUY922, we find that none of our 10 AHI compounds induce the canonical changes characteristic of N-terminal Hsp90 inhibition. Instead, six compounds appear to antagonize mitochondrial processes, while the other four demonstrate individual mechanistic profiles. We conclude that none of these 10 AHI compounds induce apoptosis in Jurkat cells via Hsp90 inhibition. Because these compounds have been previously advanced as Hsp90 inhibitors, results here indicate that insular client depletion assays are inadequate to the task of validating Hsp90 inhibition in living cells.