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Chris W. Lee, Christopher Eckman; A High-Throughput Screening Assay for the Identification of Pharmacological Chaperones (PCs) to Treat Myocilin-related Primary Open-Angle Glaucoma (POAG). Invest. Ophthalmol. Vis. Sci. 2011;52(14):3097.
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The purpose of this project is to establish a high-throughput screening (HTS) assay to identify small pharmacological molecules that can act as ligands for misfolded myocilin protein and hence, may potentially be use as pharmacological chaperone to alleviate molecular phenotypes associated with POAG-causing mutations inherited on the MYOC gene.
To identify myocilin-specific PCs, we propose to establish a HTS-based, ultra-sensitive myocilin binding assay utilizing the new Corning Epic® system. The Epic® is one of the few label-free, HTS platforms designed for identification of ligands for all functional classes of protein utilizing the property of Surface Plasmon Resonance. In an effort to develop a PC-based treatment for POAG, we have produced recombinant human myocilin carboxyl-terminal fragment (CTF) with a 6His tag from a human cell line and to use the purified protein as a bait to screen for specific ligands. We have also established cell-based disease models with pathogenic phenotypes induced by mutated and misfolded myocilin, to test whether the identified ligands can alleviate the phenotypes.
The established Epic® myocilin binding assay was capable of detecting specific binding events from positive controls in a dose-dependent manner. The positive controls include antibodies which are known to target the myocilin CTF or the 6His tag. A small molecule binder, sulconazole nitrate (461Da) an imidazole-related compound known to bind to 6His tag, was included as a positive control. A significant binding signal to myocilin-CTF was observed at sub-micromolar concentration. The data demonstrate that the assay can reliably detect specific binding of antibody or chemical compound to the purified myocilin CTF-6His protein. In addition, our established TM cell-based disease models showed significant inductions of pathogenic phenotypes such as inclusions formation associated with 5 POAG mutations in myocilin protein.
The data strongly suggest that the Epic® myocilin binding assay can reliably detect specific binding of antibody or chemical compound to the purified myocilin CTF-6His protein. We believe that translation of this HTS platform to identify myocilin targeted ligands holds promise to identify potential PCs to treat POAG.
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