期刊
ACS CHEMICAL BIOLOGY
卷 13, 期 4, 页码 933-941出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.7b01083
关键词
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资金
- NIH [R01EY021205, R01EY024232]
- NCATS [UL1TR0004S4]
- Petit Scholars Program (Georgia Tech)
- [F99CA212467]
- NATIONAL CANCER INSTITUTE [K00CA212467, F99CA212467, R01CA213566] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES [UL1TR000454] Funding Source: NIH RePORTER
- NATIONAL EYE INSTITUTE [R01EY024232, R01EY021205] Funding Source: NIH RePORTER
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 Hsp90 alpha and Hsp90 beta. 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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