Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma
Digital Object Identifier (DOI)
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.
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Citation / Publisher Attribution
ACS Chemical Biology, v. 13, issue 4, p. 933-941
Scholar Commons Citation
E. Huard, Dustin J.; Crowley, Vincent M.; Du, Yuhong; Cordova, Ricardo A.; Sun, Zheying; Tomlin, Moya O.; Dickey, Chad A.; Koren, John; Blair, Laura; Fu, Haian; Blagg, Brian S. J.; and Lieberman, Raquel L., "Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma" (2018). Molecular Medicine Faculty Publications. 104.