title: Ligands for glaucoma-associated myocilin discovered by a generic binding assay.
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.
authors.affiliation: School of Chemistry & Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive NW, Atlanta, Georgia 30332-0400, United States.
authors.forename: Susan D
authors.initials: SD
authors.lastname: Orwig
authors.forename: Pamela V
authors.initials: PV
authors.lastname: Chi
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Du
authors.forename: Shannon E
authors.initials: SE
authors.lastname: Hill
authors.forename: Marchello A
authors.initials: MA
authors.lastname: Cavitt
authors.forename: Amrithaa
authors.initials: A
authors.lastname: Suntharalingam
authors.forename: Katherine C
authors.initials: KC
authors.lastname: Turnage
authors.forename: Chad A
authors.initials: CA
authors.lastname: Dickey
authors.forename: Stefan
authors.initials: S
authors.lastname: France
authors.forename: Haian
authors.initials: H
authors.lastname: Fu
authors.forename: Raquel L
authors.initials: RL
authors.lastname: Lieberman
mesh_terms: D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D016326:Extracellular Matrix Proteins / Q000737:chemistry; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005902:Glaucoma, Open-Angle / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D006023:Glycoproteins; D008024:Ligands; D054852:Small Molecule Libraries; D000099179:Myocilin; C081451:olfactomedin