Isoform-selective Hsp90 inhibition rescues model of hereditary open-angle glaucoma

Abstract

The heat shock protein 90 (Hsp90) family of molecular chaperones regulates protein homeostasis, folding, and degradation. The ER-resident Hsp90 isoform, glucose-regulated protein 94 (Grp94), promotes the aggregation of mutant forms of myocilin, a protein associated with primary open-angle glaucoma. While inhibition of Grp94 promotes the degradation of mutant myocilin in vitro, to date no Grp94-selective inhibitors have been investigated in vivo. Here, a Grp94-selective inhibitor facilitated mutant myocilin degradation and rescued phenotypes in a transgenic mouse model of hereditary primary open-angle glaucoma. Ocular toxicities previously associated with pan-Hsp90 inhibitors were not evident with our Grp94-selective inhibitor, 4-Br-BnIm. Our study suggests that selective inhibition of a distinct Hsp90 family member holds translational promise for ocular and other diseases associated with cell stress and protein misfolding.

Figure 1

4-Br-BnIm interacts with the ATP-binding pocket of Grp94. (a) Chemical structure of Grp94-selective inhibitor, 4-Br-BnIm. (b) Crystal structure of the N-terminal domain of Grp94 in complex with 4-Br-BnIm. 4-Br-BnIm bound in the ATP binding pocket of the Grp94 NΔ41 construct, based on a 2.7 Å resolution crystal structure (see Supplementary Table 1). Grey: not observed in electron density. Black dash: H-bonding interactions. Red ball: modeled water molecules. Green: chloride substituent.

Figure 2

4-Br-BnIm rescued IOP and reduced myocilin accumulation in Tg-MYOC^Y437H mice. (a) IOP was measured biweekly over 12 weeks of treatment with vehicle or 4-Br-BnIm. Statistical analysis was carried out using one-way ANOVA with Bonferroni post-hoc test. *P < 0.05, ***P < 0.001. WT + vehicle (n = 3) WT + 4-Br-BnIm (n = 3), Tg-MYOC^Y437H + vehicle (n = 6), Tg-MYOC^Y437H + 4-Br-BnIm (n = 7). (b) Representative images depicting myocilin accumulation (red) in the trabecular meshwork (TM). TM, Schlemm’s canal (SC) and ciliary body (CB) are labeled. DAPI is used as a nuclear counterstain (blue). Scale Bar = 20 µm. (c) Quantification of myocilin levels. Statistical analysis was carried out using one-way ANOVA with Bonferroni post-hoc test. **P < 0.01, F = 5.77, df = 14. WT + vehicle (n = 2) WT + 4-Br-BnIm (n = 3), Tg-MYOC^Y437H + vehicle (n = 5), Tg-MYOC^Y437H + 4-Br-BnIm (n = 5).

Figure 3

4-Br-BnIm reversed the light-adapted response phenotype in transgenic Tg-MYOC^Y437H mice and protects RGCs. (a) Light-adapted electroretinography (LA-ERG) tracings of WT and Tg-MYOCY437H mice without and with 4-Br-BnIm treatment. (b) Quantification of the PhNR from LA-ERG tracings. Statistical analysis was carried out using one-way ANOVA with Bonferroni post-hoc test. *P < 0.05, F = 2.48, df = 27. n values represent individual mice. WT + vehicle (n = 2), WT + 4-Br-BnIm (n = 3), Tg-MYOC^Y437H + vehicle (n = 5), Tg-MYOC^Y437H + 4-Br-BnIm (n = 4). (c) Representative images showing DAB stained NeuN positive cells in a 350 µm length of RGC layer from the midperipheral region of the retina from each treatment group. Scale bars = 20 µm. (d) A representative whole eye section from a WT vehicle mouse, boxes identify the region of retina used in the quantitation of RGC density. (e) Quantification of RGC density normalized to a WT vehicle treated mouse. Statistical analysis was carried out using two-tailed unpaired t-test. *P < 0.05, t = 3.63, df = 4. WT + 4-Br-BnIm (n = 3), Tg-MYOC^Y437H + vehicle (n = 3), Tg-MYOC^Y437H + 4-Br-BnIm (n = 3).

Figure 4

4-Br-BnIm does not induce Hsp70 in Tg-MYOC^Y437H mice. (a) Representative images depicting Hsp70 levels (red fluorescence), as observed by fluorescent immunostaining and multiphoton microscopy, in the trabecular meshwork (TM) of mouse tissue. TM and ciliary body (CB) are labeled. DAPI is used as a nuclear counterstain (blue). Scale Bar = 50 µm. (b) Quantification of Hsp70 levels normalized to WT vehicle-treated controls. Error bars represent mean ± SEM. Eyes assessed: WT + vehicle (n = 2), WT + 4-Br-BnIm (n = 3), Tg-MYOC^Y437H + vehicle (n = 7), Tg-MYOC^Y437H + 4-Br-BnIm (n = 4). No significant difference was observed between groups as determined by one-way ANOVA analysis, F = 2.8, df = 15. (c) Western Blot analysis and quantitation of Hsp70 levels following vehicle, 17-AAG, and 4-Br-BnIm treatment to HTM cells.