A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation

作者：Chen Yuanyuan; Tang Hong; Seibel William; Papoian Ruben; Li Xiaoyu; Lambert Nevin A; Palczewski Krzysztof^*

来源：Investigative Ophthalmology & Visual Science, 2015, 56(4): 2553-2567.

DOI：10.1167/iovs.14-16298

• rhodopsin
• rod photoreceptors
• phototransduction
• misfolded protein
• GPCR
• retinal degeneration
• ocular pharmacology

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 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 beta-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.

• 出版日期2015-4