Paralog-selective Hsp 90 inhibitors define tumor-specific regulation of Her 2

Although the Hsp90 chaperone family, comprised in humans of four paralogs, Hsp90α, Hsp90β, Grp94 and Trap-1, has important roles in malignancy, the contribution of each paralog to the cancer phenotype is poorly understood. This is in large part because reagents to study paralogspecific functions in cancer cells have been unavailable. Here we combine compound library screening with structural and computational analyses to identify purine-based chemical tools that are specific for Hsp90 paralogs. We show that Grp94 selectivity is due to the insertion of these compounds into a new allosteric pocket. We use these tools to demonstrate that cancer cells use individual Hsp90 paralogs to regulate a client protein in a tumor-specific manner and in response to proteome alterations. Finally, we provide new mechanistic evidence explaining why selective Grp94 inhibition is particularly efficacious in certain breast cancers. The Hsp90 family of molecular chaperones regulates and maintains cell homeostasis under proteotoxic stress and pathogenic pressure1. In humans, Hsp90α and Hsp90β in the cytoplasm, Grp94 in the endoplasmic reticulum and Trap-1 in the mitochondria are the four © 2009 Nature America, Inc. All rights reserved. Correspondence and requests for materials should be addressed to G.C. 6These authors contributed equally to this work. *chiosisg@mskcc.org or gewirth@hwi.buffalo. edu Accession codes. Protein Data Bank (PDB): Structure factors and coordinates were deposited under accession codes 3O2F for Grp94N-PU-H54 and 3O0I for Hsp90N-PU-H54. Author contributions P.D. P., P.Y. , H.J.P. and C.Y. performed the chemistry and chemical biology experiments, and T.T. and W.S. provided reagents. P.M.S., N.S. Q. and D.T. G. carried out the crystal structure determination and analysis. All of the authors participated in the design and analysis of various experiments, and G.C. , D.T. G. and P.D. P. wrote the paper. Competing financial interests The authors declare competing financial interests: details accompany the online version of the paper. Additional information Supplementary information, chemical compound information and chemical probe information is available in the online version of the paper. Reprints and permissions information is available online at http://www.nature. com/reprints/index.html. NIH Public Access Author Manuscript Nat Chem Biol. Author manuscript; available in PMC 2014 November 01. Published in final edited form as: Nat Chem Biol. 2013 November ; 9(11): 677–684. doi:10.1038/nchembio.1335. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript Hsp90 paralogs2,3. Hsp90s are characterized by a distinct ‘Bergerat fold’ in the N-terminal ATP-binding domain (NTD)4. Binding and release of the nucleotide drives the Hsp90 catalytic cycle and the refolding of client proteins. Occupancy of this pocket by smallmolecule inhibitors inactivates Hsp90 chaperone function. Pan-Hsp90 inhibitors have demonstrated potent reversal of disease phenotypes when tested in models of cancer, neurodegeneration, infection and inflammatory disease5. Some of these compounds have moved to the clinic for the treatment of cancers6. Despite considerable interest in the use of Hsp90 inhibitors for the treatment of disease, little is known about the contribution of each paralog to the observed therapeutic benefit. To date, published studies have used pan-Hsp90 inhibitors to inactivate all of the Hsp90s and the processes that depend on them, making it impossible to correlate the role of individual paralogs with the biological effects. This is unsatisfying as the chaperoning roles of these Hsp90s do not overlap2,3,7,8. Much of our inability to study individual paralogs in cancer cells stems from a lack of suitable tools. Strategies that address the biology of Hsp90s and their individual paralogs in an endogenous cellular environment where the chaperones are limiting but not absent (that is, in unengineered cancer cell lines and in primary samples) are needed. Ideally, this gap would be filled by chemical tools that probe a protein’s function in a controlled manner. Such tools would complement traditional approaches by aiding the molecular characterization of biomolecules both in vitro and within their natural biological contexts. The discovery of paralog-specific Hsp90 inhibitors is challenging because of the high degree of conservation in their ATP-binding cavities, the pocket to which the known synthetic ligands bind7,8. Most reported Hsp90 inhibitors bind equally well to the majority of these paralogs9,10. Crystal structures of apo-Hsp90α NTD or nucleotideor inhibitor-bound Hsp90α and Hsp90β NTDs are essentially superimposable11,12. In addition, though different poses were observed for some inhibitors when bound to Hsp90 and Grp94, these have not yet resulted in appreciable selectivity and specific cellular activity through individual paralog inhibition7,13. Paradoxically, despite the high degree of sequence conservation in their ATP-binding pockets, crystallographic and biochemical studies have shown that, when bound to nucleotides, Hsp90α, Hsp90β, Grp94 and Trap-1 adopt distinctly different conformations and hydrolyze ATP with notably different rates14–17. The overall structure and conformational flexibility of the paralogs thus have an important role in configuring their ATP-binding sites. Here, we take advantage of the conformational distinctions between the Hsp90 paralogs and use the chemical diversity of the purine-scaffold class18,19 to identify Hsp90 paralog– specific ligands. We explain the source of paralog binding specificity using structural and modeling analyses. We then use several of the identified paralog-selective inhibitors to provide new insights into the tumor-specific chaperoning of a client protein by individual Hsp90s.