title: A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation.
abstract: OBJECTIVE
  Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding.
  
  
  METHODS
  Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses.
  
  
  RESULTS
  Two stabilization assays of P23H mutant opsin were developed and validated, one based on β-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin.
  
  
  CONCLUSIONS
  Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.
authors.affiliation: Department of Pharmacology Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Drug Discovery Center, College of Medicine, University of Cincinnati, Cincinnati, Ohio, United States.
authors.forename: Hong
authors.initials: H
authors.lastname: Tang
authors.affiliation: Drug Discovery Center, College of Medicine, University of Cincinnati, Cincinnati, Ohio, United States.
authors.forename: William
authors.initials: W
authors.lastname: Seibel
authors.affiliation: Drug Discovery Center, College of Medicine, University of Cincinnati, Cincinnati, Ohio, United States.
authors.forename: Ruben
authors.initials: R
authors.lastname: Papoian
authors.affiliation: Department of Pharmacology Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Pharmacology and Toxicology, Georgia Regents University, Augusta, Georgia, United States.
authors.forename: Nevin A
authors.initials: NA
authors.lastname: Lambert
authors.affiliation: Department of Pharmacology Case Western Reserve University, Cleveland, Ohio, United States.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015151:Immunoblotting; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics*; D009154:Mutation; D010786:Photoreceptor Cells / Q000378:metabolism / Q000473:pathology; D012174:Retinitis Pigmentosa / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D012243:Rhodopsin / Q000235:genetics*
chemical_list: D050505:Mutant Proteins; D012243:Rhodopsin