Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma

• Type of Material: Article
• Authors: Dustin J.E. Huard, Georgia Institute of Technology; Vincent M. Crowley, University of Kansas; Yuhong Du, Emory University; Ricardo A. Cordova, University of South Florida; Zheying Sun, University of South Florida; Moya O. Tomlin, Georgia Institute of Technology; Chad A. Dickey, University of South Florida; John Koren, III, University of South Florida; Laura Blair, University of South Florida; Haian Fu, Emory University; Brian S.J. Blagg, University of Notre Dame; Raquel L. Lieberman, Georgia Institute of Technology
• Language: English
• Date: 2018-04-01
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2018 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/acschembio.7b01083
• Title of Journal or Parent Work: ACS Chemical Biology
• ISSN: 1554-8929
• Volume: 13
• Issue: 4
• Start Page: 933
• End Page: 941
• Grant/Funding Information: This work was supported by NIH grants R01EY021205 to R. Lieberman, R01EY024232 to B. Blagg and C. Dickey, as well as NCATS award UL1TR000454 to R. Lieberman, H. Fu and Y. Du. V. Crowley is supported by F99CA212467 and M. Tomlin by the Petit Scholars Program (Georgia Tech).
• Keywords: OPEN-ANGLE GLAUCOMA; TRABECULAR MESHWORK CELLS; OLFACTOMEDIN DOMAIN; TERMINAL HSP90 INHIBITORS; NON-SECRETION; MUTANT MYOCILIN; Biochemistry & Molecular Biology; Science & Technology; ER STRESS; Life Sciences & Biomedicine; AGGREGATED MYOCILIN; ENDOPLASMIC-RETICULUM; IN-VITRO
• Research Categories: Biogeochemistry; Biology, Molecular

Abstract

Gain-of-function mutations within the olfactomedin (OLF) domain of myocilin result in its toxic intracellular accumulation and hasten the onset of open-angle glaucoma. The absence of myocilin does not cause disease; therefore, strategies aimed at eliminating myocilin could lead to a successful glaucoma treatment. The endoplasmic reticulum Hsp90 paralog Grp94 accelerates OLF aggregation. Knockdown or pharmacological inhibition of Grp94 in cells facilitates clearance of mutant myocilin via a non-proteasomal pathway. Here, we expanded our support for targeting Grp94 over cytosolic paralogs Hsp90α and Hsp90β. We then developed a high-throughput screening assay to identify new chemical matter capable of disrupting the Grp94/OLF interaction. When applied to a blind, focused library of 17 Hsp90 inhibitors, our miniaturized single-read in vitro thioflavin T -based kinetics aggregation assay exclusively identified compounds that target the chaperone N-terminal nucleotide binding site. In follow up studies, one compound (2) decreased the extent of co-aggregation of Grp94 with OLF in a dose-dependent manner in vitro, and enabled clearance of the aggregation-prone full-length myocilin variant I477N in cells without inducing the heat shock response or causing cytotoxicity. Comparison of the co-crystal structure of compound 2 and another non-selective hit in complex with the N-terminal domain of Grp94 reveals a docking mode tailored to Grp94 and explains its selectivity. A new lead compound has been identified, supporting a targeted chemical biology assay approach to develop a protein degradation-based therapy for myocilin-associated glaucoma by selectively inhibiting Grp94.