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

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.

Stable calcium-free myocilin olfactomedin domain variants reveal challenges in differentiating between benign and glaucoma-causing mutations

“…are instead sequestered intracellularly (15,30,37,38), specifically within the ER (39), not the cytosol (40). Misfolded myocilin is recognized by the molecular chaperone Grp94 as not competent for secretion, but instead of undergoing efficient degradation, aggregated mutant myocilin accumulates, leading to ER stress, TM cell death (8,15,41), and a cascade that results in early onset ocular hypertension, the causal risk factor for glaucoma.…”

Physiological function of myocilin and its role in the pathogenesis of glaucoma in the trabecular meshwork (Review)

“…It has been reported that the inhibition of Grp94 is an effective approach to the treatment of glaucoma (79,98), supporting the co-aggregation of Grp94 with mutant myocilin and leading to retention within the ER (Fig. 4) (80).…”

“…However, recent findings (42,78) have suggested that the dominant negative effect may be involved in the pathogenic role of myocilin in glaucoma. Notably, the majority of experimental evidence support gain-of-function as a pathogenic mechanism involved in myocilin mutation-associated glaucoma (21,23,36,37,41,42,67,73,76,79-84).…”

“…4) (80). Not only does Grp94 accelerate the myocilin-OLF aggregation rate, but it also enhances co-precipitation with OLF (79,80,99). Therefore, Grp94 inhibition facilitates mutant myocilin clearance as an anti-glaucoma therapy (79,97,100).…”

“…Not only does Grp94 accelerate the myocilin-OLF aggregation rate, but it also enhances co-precipitation with OLF (79,80,99). Therefore, Grp94 inhibition facilitates mutant myocilin clearance as an anti-glaucoma therapy (79,97,100). 4-Br-BnIm, a Grp94 inhibitor, significantly clears aggregated myocilin caused by overexpression mutations (97), and alleviates mutant myocilin-induced toxicity against TM cells (27,79) via a secondary autophagic pathway to facilitate clearance (27).…”