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Updated: May 16, 2026
Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate
Published on: June 25, 2019
A high-throughput cell-based Gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion
John D Hulleman1, Steven J Brown, Hugh Rosen
1Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA. hulleman@scripps.edu
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.
Insights
Researchers screened compounds to find molecules that alter fibulin-3 secretion, potentially aiding treatment for Malattia Leventinese (ML), a macular dystrophy. One compound selectively reduced the secretion of mutated fibulin-3, offering a promising therapeutic avenue.
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- A fibulin-3 R345W mutation causes Malattia Leventinese (ML), a macular dystrophy, through inefficient secretion and increased intracellular levels.
- The exact mechanism by which R345W fibulin-3 causes ML remains unclear, necessitating further investigation into secretion pathways.
Purpose of the Study:
- To identify small molecules that modulate fibulin-3 secretion using a high-throughput screening approach.
- To develop tools for investigating the molecular basis of fibulin-3-related macular dystrophies.
Main Methods:
- Developed ARPE19 retinal cell lines with inducible expression of wild-type (WT) or R345W fibulin-3 fused to enhanced Gaussia luciferase (eGLuc2).
- Screened the Library of Pharmacologically Active Compounds using a Gaussia luciferase (GLuc) assay for high-throughput screening.
- Performed counterscreens and secondary assays with untagged fibulin-3 to confirm compound selectivity and efficacy.
Main Results:
- The cell lines and GLuc assay proved suitable for high-throughput chemical screening.
- Two estrogen-related compounds enhanced fibulin-3 secretion; numerous others reduced it.
- Phorbol 12-myristate 13-acetate selectively reduced R345W fibulin-3 secretion while minimally affecting WT fibulin-3 secretion.
Conclusions:
- Identified small molecules that can alter fibulin-3 secretion, providing potential tools for studying ML pathogenesis.
- The selective inhibition of R345W fibulin-3 secretion by phorbol 12-myristate 13-acetate presents a promising therapeutic strategy for ML.
