A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome

作者：Omachi Kohei; Kamura Misato; Teramoto Keisuke; Kojima Haruka; Yokota Tsubasa; Kaseda Shota; Kuwazuru Jun; Fukuda Ryosuke; Koyama Kosuke; Matsuyama Shingo; Motomura Keishi; Shuto Tsuyoshi; Suico Mary Ann^*; Kai Hirofumi^*

来源：Cell Chemical Biology, 2018, 25(5): 634-+.

DOI：10.1016/j.chembiol.2018.02.003

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen alpha 3-alpha 5 chains (alpha 3-alpha 5(IV)), which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of alpha 3/alpha 4/alpha 5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular alpha(IV) chain and the absence of high-throughput screening (HTS) platforms to assess alpha 345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion alpha 345(IV) proteins to monitor alpha 345(IV) trimerization of wild-type and clinically associated mutant alpha 5(IV). The alpha 345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular-and secretion-dependent defects of mutant alpha 5(IV). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote alpha 345(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting alpha 345(IV) trimers to treat Alport syndrome.