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

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3-α5 chains, which disrupts trimerization, leading to glomerular basement membrane degeneration. Correcting the trimerization of α3/α4/α5 chain is a feasible therapeutic approach, but is hindered by lack of information on the regulation of intracellular α(IV) chain and the absence of high-throughput screening (HTS) platforms to assess α345(IV) trimer formation. Here, we developed sets of split NanoLuc-fusion α345(IV) proteins to monitor α345(IV) trimerization of wild-type and clinically-associated mutant α5(IV). The α345(IV) trimer assay, which satisfied the acceptance criteria for HTS, enabled the characterization of intracellular- and secretion-dependent defects of mutant α5(IV). siRNA-based and chemical screening targeting the endoplasmic reticulum identified several chemical chaperones that has potential to promote α5(IV) trimer formation. This split luciferase-based trimer formation assay is a functional HTS platform that realizes the feasibility of targeting α345(IV) trimers to treat Alport syndrome.