A high-throughput cell-based gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion

John D. Hulleman, Steven J. Brown, Hugh Rosen, Jeffery W. Kelly

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)647-658
Number of pages12
JournalJournal of Biomolecular Screening
Issue number6
StatePublished - Jul 2013
Externally publishedYes

Bibliographical note

Funding Information:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Lita Annenberg Hazen Foundation, the Skaggs Institute for Chemical Biology, Grant UL1 RR025774 from the Scripps Translational Science Institute (J.W.K.), Grant AG018917 from the National Institutes of Health (J.W.K.), and Grant U54 MH0845121 from the National Institutes of Health (H.R.).


  • fibulin-3
  • fibulin-3-dependent gliomas
  • Gaussia luciferase
  • high-throughput chemical screening
  • Malattia Leventinese


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