The hsp90 chaperones participate in the maturation of proteins involved in diverse cellular activities ranging from signaling to bacterial recognition. They are also key players in the response to cell stress events. Cytoplasmic Hsp90s guide the maturation of steroid hormone receptors and proto-oncogenic kinases, and hsp90 inhibitors disrupt this maturation and display potent anti-cancer activity. GRP94, the ER hsp90 paralog, chaperones proteins destined for transport to the cell surface, including the Toll-like receptors. Cells deficient in GRP94 are unresponsive to microbial stimuli. GRP94 has also been identified as a tumor rejection antigen and elicits suppression of tumor growth and metastasis. ? ? The activity of hsp90 chaperones is regulated by small molecule ligands such as ATP that bind to the N-terminal domain, as well as by their conformational state. Key questions that remain unanswered include how ligands exert their inhibitory or activation effects, how different hsp90 paralogs are selectively regulated, how changes in conformation lead to altered activity, and how client proteins interact with the chaperone. ? ? An understanding of the stereochemistry that underlies hsp90 biology and hsp90-targeted therapies requires the structural analysis of the relevant macromolecular species. Using X-ray crystallography, NMR spectroscopy, and biochemical- and cell-based assays, the experiments in this proposal will address how GRP94 is regulated by small molecule ligands and interacts with client proteins. We will solve and analyze the structure of the N-terminal domain of GRP94 alone and in complex with activators, broad-spectrum antagonists, and selective inhibitors. Novel selective inhibitors of GRP94 will be designed and synthesized based on these structures. We also propose to examine the interaction of GRP94 with peptides, building on recent work that identified a peptide-binding site in the N-domain. We also plan to investigate the structural basis for N-domain dimerization and oligomerization by constructing GRP94 and Hsp90 molecules lacking candidate interacting elements and determining their functional properties. Finally, the structures of multi-domain GRP94 constructs, including full-length variants, alone or in complex with client proteins will be determined in order to understand the interplay between the domains and the mechanism of substrate interaction. Structural findings will be correlated with measurements of ligand affinity and chaperone activity in both wild-type and mutationally altered molecules. ? ?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA095130-04
Application #
7228796
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Knowlton, John R
Project Start
2005-05-01
Project End
2009-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
4
Fiscal Year
2007
Total Cost
$300,000
Indirect Cost
Name
Hauptman-Woodward Medical Research Institute
Department
Type
DUNS #
074025479
City
Buffalo
State
NY
Country
United States
Zip Code
14203
Que, Nanette L S; Crowley, Vincent M; Duerfeldt, Adam S et al. (2018) Structure Based Design of a Grp94-Selective Inhibitor: Exploiting a Key Residue in Grp94 To Optimize Paralog-Selective Binding. J Med Chem 61:2793-2805
Huck, John D; Que, Nanette L; Hong, Feng et al. (2017) Structural and Functional Analysis of GRP94 in the Closed State Reveals an Essential Role for the Pre-N Domain and a Potential Client-Binding Site. Cell Rep 20:2800-2809
Ansa-Addo, Ephraim A; Thaxton, Jessica; Hong, Feng et al. (2016) Clients and Oncogenic Roles of Molecular Chaperone gp96/grp94. Curr Top Med Chem 16:2765-78
Gewirth, Daniel T (2016) Paralog Specific Hsp90 Inhibitors - A Brief History and a Bright Future. Curr Top Med Chem 16:2779-91
Maharaj, Kevin A; Que, Nanette L S; Hong, Feng et al. (2016) Exploring the Functional Complementation between Grp94 and Hsp90. PLoS One 11:e0166271
Patel, Hardik J; Patel, Pallav D; Ochiana, Stefan O et al. (2015) Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94. J Med Chem 58:3922-43
Seidler, Paul M; Shinsky, Stephen A; Hong, Feng et al. (2014) Characterization of the Grp94/OS-9 chaperone-lectin complex. J Mol Biol 426:3590-605
Taldone, Tony; Patel, Pallav D; Patel, Maulik et al. (2013) Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series. J Med Chem 56:6803-18
Hong, Feng; Liu, Bei; Chiosis, Gabriela et al. (2013) ?7 helix region of ?I domain is crucial for integrin binding to endoplasmic reticulum chaperone gp96: a potential therapeutic target for cancer metastasis. J Biol Chem 288:18243-8
Patel, Pallav D; Yan, Pengrong; Seidler, Paul M et al. (2013) Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2. Nat Chem Biol 9:677-84

Showing the most recent 10 out of 16 publications