Ligands for Glaucoma-Associated Myocilin Discovered by a Generic Binding Assay

• Authors: Susan D. Orwig, Georgia Institute of Technology; Pamela V. Chi, Georgia Institute of Technology; Yuhong Du, Emory University; Shannon E. Hill, Georgia Institute of Technology; Marchello A. Cavitt, Georgia Institute of Technology; Amrithaa Suntharalingam, University of South Florida; Katherine C. Turnage, Georgia Institute of Technology; Chad A. Dickey, University of South Florida; Stefan France, Georgia Institute of Technology; Haian Fu, Emory University; Raquel L. Lieberman, Georgia Institute of Technology
• Journal Title: ACS Chemical Biology
• Volume: Volume 9, Number 2
• Publisher: American Chemical Society | 2014-02-01, Pages 517-525
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1021/cb4007776
• Subjects: Chemistry, Biochemistry; Health Sciences, Pharmacology
• Keywords: Science & Technology; Life Sciences & Biomedicine; Biochemistry & Molecular Biology; HIGH-THROUGHPUT; OLFACTOMEDIN DOMAIN; PROTEIN STABILITY; ISOTHERMAL DENATURATION; INDUCED-APOPTOSIS; MECHANISMS; SURFACE; DESIGN; DETECT; STRESS

Abstract

Mutations in the olfactomedin domain of myocilin (myoc-OLF) are the strongest link to inherited primary open angle glaucoma. In this recently identified protein misfolding disorder, aggregation-prone disease variants of myocilin hasten glaucoma-associated elevation of intraocular pressure, leading to vision loss. Despite its well-documented pathogenic role, myocilin remains a domain of unknown structure or function. Here we report the first small-molecule ligands that bind to the native state of myoc-OLF. To discover these molecules, we designed a general label-free, mix-and-measure, high throughput chemical assay for restabilization (CARS), which is likely readily adaptable to discover ligands for other proteins. Of the 14 hit molecules identified from screening myoc-OLF against the Sigma-Aldrich Library of Pharmacologically Active Compounds using CARS, surface plasmon resonance binding studies reveal three are stoichiometric ligand scaffolds with low micromolar affinity. Two compounds, GW5074 and apigenin, inhibit myoc-OLF amyloid formation in vitro. Structure-activity relationship-based soluble derivatives reduce aggregation in vitro as well as enhance secretion of full-length mutant myocilin in a cell culture model. Our compounds set the stage for a new chemical probe approach to clarify the biological function of wild-type myocilin and represent lead therapeutic compounds for diminishing intracellular sequestration of toxic mutant myocilin.