High-throughput screening detects pharmacologic compounds rescuing mutant myocilin secretion

Abstract

Purpose : Mutations in the MYOC gene encoding the protein myocilin cause autosomal dominant primary open-angle glaucoma (POAG) and are the major cause of juvenile POAG by disrupting proteostasis in trabecular meshwork (TM) cells. The purpose of this study was to discover new drug candidates capable of mitigating the dominant negative effects of misfolded myocilin by stabilizing the native structure or indirectly improving proteostasis, thereby alleviating TM cell stress and restoring TM function in regulating intraocular pressure.

Methods : Using a stable NIH3T3 cell line expressing human MYOC^Y437H (hMYOC^Y437H) with a C-terminal Gaussia luciferase (GLuc) reporter (hMYOC^Y437H-GLuc), a high-throughput GLuc reporter assay was performed in 384-well format to quantify mutant MYOC secretion. A drug repurposing library containing 3272 compounds was screened at 18 μM. The quality control parameter Z’>0.5 validates a robust HTS assay, where Z’=1–3*((σ₁₀₀+σ₀)/(μ₁₀₀–μ₀)). 100% and 0% controls (N=16 each) were 4-phenylbutyrate (4-PBA, 20 mM) and DMSO (0.18%), respectively. Controls were used to normalize the luminescence signal from each well to an Activity Score (%). Hit compounds with an Activity Score (%)>μ_all-wells+2σ were confirmed in triplicate at 18 µM for their induction of hMYOC^Y437H-GLuc secretion, but not GLuc enzymatic activity, followed by 10 doses in triplicate. Next, confirmed hits were validated in human TM (hTM) cells expressing the hMYOC^Y437H mutant. hMYOC^Y437H in secretion and cell lysate from hTM cells treated with each hit at EC₈₀ for 48 h were immunoblotted and quantified. Statistical significance was defined as p<0.05 via the Kruskal-Wallis test.

Results : We identified 66 hits via the HTS assay with activity scores ≥43.97%. Z'=0.63±0.08 demonstrates high HTS quality. Of these, 10 hits showed a dose-dependent increase for hMYOC^Y437H-GLuc secretion. Finally, 3 compounds were found to increase hMYOC^Y437H secretion in hTM cells that did not affect wild-type myocilin secretion.

Conclusions : We developed and validated a robust HTS assay and a workflow for large-scale drug discovery of small-molecule chaperones for treating MYOC-associated POAG. Importantly, we discovered 3 drug candidates that increase the secretion of a pathological MYOC mutant from human TM cells with 1000-fold higher potency than 4-PBA.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.