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

Cell Chem Biol. 2018 May 17;25(5):634-643.e4. doi: 10.1016/j.chembiol.2018.02.003. Epub 2018 Mar 8.

Abstract

Alport syndrome is a hereditary glomerular disease caused by mutation in type IV collagen α3-α5 chains (α3-α5(IV)), 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). Small interfering RNA-based and chemical screening targeting the ER identified several chemical chaperones that have potential to promote α345(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.

Keywords: Alport syndrome; chemical chaperone; high-throughput screening; split nano-luciferase; type IV collagen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoantigens / chemistry*
  • Autoantigens / genetics
  • Collagen Type IV / chemistry*
  • Collagen Type IV / genetics
  • Drug Evaluation, Preclinical / methods*
  • HEK293 Cells
  • High-Throughput Screening Assays / methods
  • Humans
  • Nephritis, Hereditary / drug therapy*
  • Nephritis, Hereditary / genetics
  • Point Mutation
  • Protein Multimerization / drug effects*

Substances

  • Autoantigens
  • COL4A5 protein, human
  • Collagen Type IV
  • type IV collagen alpha3 chain