Establishment of a High-Throughput Screening System for Membrane Expression of Mutant Nephrin

Background: Congenital Nephrotic syndrome of the Finnish type (CNF) is caused by a mutation in the NPHS1 gene that hinders the cell surface expression of nephrin and disrupts slit membrane formation. In this study, we constructed a novel evaluation system to search for compounds promoting the expression of nephrin on the cell membrane. Methods: To detect the membrane-expressed nephrin, we used the HiBiT nanoluciferase system by tagging Nephrin wild-type (wt) and mutants (mt) with HiBiT. For high throughput screening (HTS), we generated stable expression cell lines of HiBiT- wt and mutant nephrin (HiBiT-Neph) using the Flp-InTM System. Phosphorylated-Nephrin, which indicates the ability to be translocated to the surface, was also assessed by immunoblotting. Results: In contrast to wt HiBiT-Neph, most mutants did not produce luminescence and were not phosphorylated. Furthermore, among the mutants, we confirmed that the reduction of HiBiT activity in the S366R and E725D stable mutant cells met the HTS criteria. Using E725D stable cells, we screened several chemical chaperones and a natural products compound library. Chemical chaperone β-alanine, a CFTR corrector (Corr-4a), and 78 compounds from the library increased the HiBiT-Neph E725D. Cytotoxicity assays and further validation by immunoblotting of phosphorylated nephrin trimmed the candidate compounds to ten. Focusing on these compounds, we revealed their effects on post-translational modifications and degradation rates of Nephrin to promote the expression of nephrin mutants on the membrane. Conclusion: The HTS evaluation system identified compounds that increased mutant nephrin phosphorylation and cell surface expression and showed that this evaluation system is useful as a drug discovery platform for CNF. Moreover, the identified compounds help to elucidate the mechanisms of mutant nephrin cell regulation.