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.

Figure 1.

Effects of Hsp90 paralogs on mutant myocilin aggregation. a) Schematic of possible interactions between Hsp90 paralogs and aggregating mutant myocilin in a cellular context. b) Effects of Hsp90α, Hsp90β, and Grp94 siRNA knockdown on mutant myocilin levels, as well as ER stress markers Grp78 and pIRE1α. c) All Hsp90s enhance the rate of OLF aggregation in vitro. d) Post-assay analysis of aggregates from (c) by SDS-PAGE (right) reveals Hsp90α and Hsp90β co-aggregation with OLF. S = supernatant; W = wash; P = aggregate pellet. Left: Hsp90α or Hsp90β at the start of the aggregation experiment.

Figure 2.

Assay development. a) Plate-to-plate reproducibility of optimized, single-point readout, 384-well plate format miniaturized assay. A Zʹ score of 0.5 or greater was achieved per plate. Inhibitor 4-Br-BnIm was used as a positive control. b) Post-assay analysis of aggregates by SDS-PAGE reveals dose-responsive rescue of Grp94 from co-aggregation with OLF. S, W, P as in Figure 1.

Figure 3.

Blind screen of Blagg lab library. a) ThT fluorescence at time = 0 hours reveals intrinsic fluorescence of compounds 1, 5, 8, and 17 at 25 μM (purple line). b) Raw data from ThT fluorescence at 18 h suggests hit compounds are 1–7, 9–11. Average signals for OLF (solid black line) and OLF+Grp94 (solid blue line) controls are presented with ± 3 standard deviations (paired color bars). Data for odd-numbered compounds/replicates designated by black spheres; even-numbered compound data are shown by magenta spheres. c) Calculated percent inhibition scores using different methods. Heat gradient and compound intrinsic fluorescence adjustments classify compounds 5 and 10 as hits. d) Representative post-assay aggregate analysis SDS-PAGE gels. Grp94 co-aggregation with OLF seen in control (0, left) and non-hits (15, right). Hits (1, middle) resulted in partial rescue of Grp94. S, W, P as in Figure 1. For post-assay analysis of all compounds, see Supplementary Figure S3b–h.

Figure 4.

Dose-responsive rescue of Grp94 from co-aggregation with OLF. a-b) Representative post-assay SDS-PAGE analysis of hit compound (2, a) and negative control (15, b), with control conditions given at the left of each gel. S = supernatant; W = wash; P = pellet/aggregate. c) Densitometric analysis of post-dose-response aggregation assay bands on SDS-PAGE reveals that compounds identified as hits increase the percentage of soluble Grp94 by inhibiting chaperone activity. Data reflect at least 2 experiments and 2 gels per experiment. Error bars indicate standard deviation. Raw data appear in Supplementary Figures S4 and S5.

Figure 5.

Cellular profiling of hits. a) Western blot analysis of lysates from iHEK cells reveals that treatment with 2 results in dose-dependent clearance of mutant myocilin. Representative of two independent experiments is presented. b) Quantification of intracellular myocilin bands in (a). c-e) Compound 2 does not evoke the ER stress response (c, Hsp70; d, Grp78; e, pIRE1α) . f) Comparison of cellular responses to highest dose (30 μM) of compounds tested for compounds 2, 4, and 9; p <0.05, p<0.01, p<0.001, and p<0.0001 by Bonferoni Multiple Comparisons Post-hoc test. GAPDH serves as a load control and error bars are ± SEM.

Figure 6.

Crystal structures of 2 (a-d) and 4 (e-h) bound to NΔ41. a, e) Surface representation of NΔ41 nucleotide binding pocket bound by inhibitors, shown as sticks. b, f) Zoomed view, with direct protein-inhibitor interactions highlighted. The final 2F_o-F_c map (blue mesh), contoured at 1.0 σ for ligands and 1.3 σ for amino acid residues, are superimposed with F_o-F_c densities (green mesh) contoured at 2.5 σ after initial molecular replacement (see Supporting Information). Inhibitors and residues are depicted as sticks, waters are red spheres. c, g) NΔ41-inhibitor interactions mediated by hydrogen bonds (dashes) in zoomed view. d, h) NΔ41-inhibitor interactions (dashed lines, hydrogen bonds or cation-π interactions; dashed crescents, hydrophobic interactions. Panels a-c, e-g were prepared with PyMOL (http://www.pymol.org). Panels d, h were generated with ChemDraw (version 16.0.1.4, Perkin Elmer).