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

17 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)647-658
Number of pages12
JournalJournal of Biomolecular Screening
Volume18
Issue number6
DOIs
StatePublished - Jul 2013

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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