title: A high-throughput cell-based Gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion.
abstract: An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3-related diseases.
authors.affiliation: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA. hulleman@scripps.edu
authors.forename: John D
authors.initials: JD
authors.lastname: Hulleman
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Brown
authors.forename: Hugh
authors.initials: H
authors.lastname: Rosen
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
mesh_terms: D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004967:Estrogens / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008268:Macular Degeneration / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D012160:Retina / Q000187:drug effects / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology
chemical_list: C119630:EFEMP1 protein, human; D004967:Estrogens; D016326:Extracellular Matrix Proteins; D054852:Small Molecule Libraries; D008156:Luciferases