Post-translational modification and conformational state of Heat Shock Protein 90 differentially affect binding of chemically diverse small molecule inhibitors

Abstract

Heat shock protein 90 (Hsp90) is an essential molecular chaperone in eukaryotes that facilitates the conformational maturation and function of a diverse protein clientele, including aberrant and/or over-expressed proteins that are involved in cancer growth and survival. A role for Hsp90 in supporting the protein homeostasis of cancer cells has buoyed interest in the utility of Hsp90 inhibitors as anti-cancer drugs. Despite the fact that all clinically evaluated Hsp90 inhibitors target an identical nucleotide-binding pocket in the N domain of the chaperone, the precise determinants that affect drug binding in the cellular environment remain unclear, and it is possible that chemically distinct inhibitors may not share similar binding preferences. Here we demonstrate that two chemically unrelated Hsp90 inhibitors, the benzoquinone ansamycin geldanamycin and the purine analog PU-H71, select for overlapping but not identical subpopulations of total cellular Hsp90, even though both inhibitors bind to an amino terminal nucleotide pocket and prevent N domain dimerization. Our data also suggest that PU-H71 is able to access a broader range of N domain undimerized Hsp90 conformations than is geldanamycin and is less affected by Hsp90 phosphorylation, consistent with its broader and more potent anti-tumor activity. A more complete understanding of the impact of the cellular milieu on small molecule inhibitor binding to Hsp90 should facilitate their more effective use in the clinic.

Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions

“…Studies also indicate that the sensitivity of a tumor to pharmacologic inhibition may not be driven by the expression of total HSP90 in the tumor cell, but rather by the presence of actively chaperoning HSP90 species [91,98,99]. These species, whose presence are regulated by co-chaperone recruitment and likely also by post-translational modifications, maintain multiple oncogenic pathways dependent on HSP90 activity.…”

“…These species, whose presence are regulated by co-chaperone recruitment and likely also by post-translational modifications, maintain multiple oncogenic pathways dependent on HSP90 activity. In fact, recent proteomic and biochemical studies support the notion that tumor selection in the HSP90 field should be based on more sophisticated functional proteomic insights that measure not only the network of tumor-driving HSP90 clientele but also the abundance and the biochemical nature of the HSP90 fraction available for inhibitor binding [91,98,99]. …”

Structural and functional basis of protein phosphatase 5 substrate specificity

“…It is well-established that increased binding of Hsp90 to its inhibitors is associated with sensitivity of cells to Hsp90 drugs (24,25). We have previously shown that overexpression of the yeast PP5 ortholog (Ppt1) sensitizes cells to the Hsp90 inhibitor geldanamycin (8).…”

Selective targeting of the stress chaperome as a therapeutic strategy

“…These epigenetic modifiers may guide HSP90 to sample multiple distinct conformations that ultimately determine the fate of bound substrate (i.e. proper folding and activation or degradation), and as we are now appreciating, the interaction of a small molecule with HSP90 [8, 56, 57]. …”

“…These inhibitors bind to the ATP binding pocket of HSP90 and prevent HSP90 from cycling between the ADP and ATP bound conformations thus impairing the chaperone activity [41]. Though N-terminal HSP90 inhibitors share a similar binding site their exact mode of binding may be different, and the result is the wide ranging effects on biology [8, 56]. The specific binding mode as well as a discussion on the residues that many of these compounds interact with has recently been analyzed in detail [85].…”