Development of a Cell-Based Fluorescence Polarization Biosensor Using Preproinsulin to Identify Compounds That Alter Insulin Granule Dynamics.
Diabetes currently affects 9.3% of the U.S. population totaling $245 billion annually in U.S. direct and indirect healthcare costs. Current therapies for diabetes are limited in their ability to control blood glucose and/or enhance insulin sensitivity. Therefore, innovative and efficacious therapies for diabetes are urgently needed. Herein we describe a fluorescent insulin reporter system (preproinsulin-mCherry, PPI-mCherry) that tracks live-cell insulin dynamics and secretion in pancreatic β-cells with utility for high-content assessment of real-time insulin dynamics. Additionally, we report a new modality for sensing insulin granule packaging in conventional high-throughput screening (HTS), using a hybrid cell-based fluorescence polarization (FP)/internal FRET biosensor using the PPI-mCherry reporter system. We observed that bafilomycin, a vacuolar H(+) ATPase inhibitor and inhibitor of insulin granule formation, significantly increased mCherry FP in INS-1 cells with PPI-mCherry. Partial least squares regression analysis demonstrated that an increase of FP by bafilomycin is significantly correlated with a decrease in granularity of PPI-mCherry signal in the cells. The increased FP by bafilomycin is due to inhibition of self-Förster resonant energy transfer (homo-FRET) caused by the increased mCherry intermolecular distance. FP substantially decreases when insulin is tightly packaged in the granules, and the homo-FRET decreases when insulin granule packaging is inhibited, resulting in increased FP. We performed pilot HTS of 1782 FDA-approved small molecules and natural products from Prestwick and Enzo chemical libraries resulting in an overall Z'-factor of 0.52 ± 0.03, indicating the suitability of this biosensor for HTS. This novel biosensor enables live-cell assessment of protein-protein interaction/protein aggregation in live cells and is compatible with conventional FP plate readers.
1 Biomanufacturing Research Institute and Technology Enterprise, Department of Pharmaceutical Sciences, North Carolina Central University , Durham, North Carolina.
Na Young
NY
Yi
1 Biomanufacturing Research Institute and Technology Enterprise, Department of Pharmaceutical Sciences, North Carolina Central University , Durham, North Carolina.
Qingping
Q
He
1 Biomanufacturing Research Institute and Technology Enterprise, Department of Pharmaceutical Sciences, North Carolina Central University , Durham, North Carolina.
Thomas B
TB
Caligan
1 Biomanufacturing Research Institute and Technology Enterprise, Department of Pharmaceutical Sciences, North Carolina Central University , Durham, North Carolina.
Ginger R
GR
Smith
2 The Neuroscience Center and Department of Cell Biology & Physiology, UNC Chapel Hill School of Medicine , Chapel Hill, North Carolina.
Lawrence J
LJ
Forsberg
2 The Neuroscience Center and Department of Cell Biology & Physiology, UNC Chapel Hill School of Medicine , Chapel Hill, North Carolina.
Jay E
JE
Brenman
1 Biomanufacturing Research Institute and Technology Enterprise, Department of Pharmaceutical Sciences, North Carolina Central University , Durham, North Carolina.
Jonathan Z
JZ
Sexton
D000818:Animals; D001688:Biological Products / Q000633:toxicity; D015374:Biosensing Techniques / Q000379:methods*; D002478:Cells, Cultured; D005454:Fluorescence Polarization / Q000379:methods* / Q000639:trends; D005456:Fluorescent Dyes / Q000032:analysis; D057166:High-Throughput Screening Assays / Q000379:methods* / Q000639:trends; D006801:Humans; D007328:Insulin / Q000032:analysis* / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis*; D011498:Protein Precursors / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D000097573:Red Fluorescent Protein
D001688:Biological Products; D005456:Fluorescent Dyes; D007328:Insulin; D008164:Luminescent Proteins; D011498:Protein Precursors; D000097573:Red Fluorescent Protein; C013737:preproinsulin