TY - JOUR
T1 - A high-throughput cell-based gaussia luciferase reporter assay for identifying modulators of fibulin-3 secretion
AU - Hulleman, John D.
AU - Brown, Steven J.
AU - Rosen, Hugh
AU - Kelly, Jeffery W.
N1 - 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.).
PY - 2013/7
Y1 - 2013/7
N2 - 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.
AB - 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.
KW - Gaussia luciferase
KW - LOPAC
KW - Malattia Leventinese
KW - fibulin-3
KW - fibulin-3-dependent gliomas
KW - high-throughput chemical screening
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U2 - 10.1177/1087057112469405
DO - 10.1177/1087057112469405
M3 - Article
C2 - 23230284
AN - SCOPUS:84879475121
SN - 1087-0571
VL - 18
SP - 647
EP - 658
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 6
ER -