A high-throughput drug screening strategy for detecting rhodopsin P23H mutant rescue and degradation

Yuanyuan Chen, Hong Tang, William Seibel, Ruben Papoian, Xiaoyu Li, Nevin A Lambert, Krzysztof Palczewski

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

PURPOSE. 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 highthroughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and imagebased analyses. RESULTS. Two stabilization assays of P23H mutant opsin were developed and validated, one based on b-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. Highthroughput 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.

Original languageEnglish (US)
Pages (from-to)2553-2567
Number of pages15
JournalInvestigative Ophthalmology and Visual Science
Volume56
Issue number4
DOIs
StatePublished - Jan 1 2015

Fingerprint

Opsins
Preclinical Drug Evaluations
Rhodopsin
Mutant Proteins
Immunoassay
Galactosidases
Retinal Rod Photoreceptor Cells
Retinitis Pigmentosa
Energy Transfer
Luminescence
Glycosylation
Immunoblotting
Proteolysis
Technology
Proteins

Keywords

  • GPCR
  • Misfolded protein
  • Ocular pharmacology
  • Phototransduction
  • References
  • Retinal degeneration
  • Rhodopsin
  • Rod photoreceptors

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

A high-throughput drug screening strategy for detecting rhodopsin P23H mutant rescue and degradation. / Chen, Yuanyuan; Tang, Hong; Seibel, William; Papoian, Ruben; Li, Xiaoyu; Lambert, Nevin A; Palczewski, Krzysztof.

In: Investigative Ophthalmology and Visual Science, Vol. 56, No. 4, 01.01.2015, p. 2553-2567.

Research output: Contribution to journalArticle

Chen, Yuanyuan ; Tang, Hong ; Seibel, William ; Papoian, Ruben ; Li, Xiaoyu ; Lambert, Nevin A ; Palczewski, Krzysztof. / A high-throughput drug screening strategy for detecting rhodopsin P23H mutant rescue and degradation. In: Investigative Ophthalmology and Visual Science. 2015 ; Vol. 56, No. 4. pp. 2553-2567.
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AB - PURPOSE. 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 highthroughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and imagebased analyses. RESULTS. Two stabilization assays of P23H mutant opsin were developed and validated, one based on b-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. Highthroughput 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.

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