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

• Type of Material: Article
• 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
• Language: English
• Date: 2014-02-01
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2013 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/cb4007776
• Title of Journal or Parent Work: ACS Chemical Biology
• ISSN: 1554-8929
• Volume: 9
• Issue: 2
• Start Page: 517
• End Page: 525
• Grant/Funding Information: This work was funded by grants from NIH (R01EY021205) and Pew Scholar in Biomedical Sciences program to R. L. L.; U. S. Department of Education (Graduate Assistance in Areas of National Need P200A060188) to S.D.O; NSF Graduate Research Fellowship (DGE-1148903) and Georgia Tech Presidential Fellowship to M. A. C.; and a Georgia Tech (Presidential Undergraduate Research Award) and Merck Fellowship to P. V. C.
• Keywords: INDUCED-APOPTOSIS; MECHANISMS; Biochemistry & Molecular Biology; OLFACTOMEDIN DOMAIN; SURFACE; Life Sciences & Biomedicine; Science & Technology; PROTEIN STABILITY; HIGH-THROUGHPUT; DESIGN; DETECT; STRESS; ISOTHERMAL DENATURATION
• Research Categories: Health Sciences, Pharmacology; Chemistry, Biochemistry

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.