Chen Yuanyuan, Tang Hong, Seibel William, Papoian Ruben, Li Xiaoyu, Lambert Nevin A, Palczewski Krzysztof
Department of Pharmacology Case Western Reserve University, Cleveland, Ohio, United States.
Drug Discovery Center, College of Medicine, University of Cincinnati, Cincinnati, Ohio, United States.
Invest Ophthalmol Vis Sci. 2015 Apr;56(4):2553-67. doi: 10.1167/iovs.14-16298.
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.
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.
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.
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.
P23H突变视蛋白的固有不稳定性约占常染色体显性遗传性视网膜色素变性病例的10%。我们的目的是开发一套全面可靠的筛选策略，以评估突变视紫红质的稳定或增强降解是否能挽救表达这种突变蛋白的视杆光感受器。这些策略有望揭示活性化合物，并阐明生物学重要过程的分子机制，如抑制靶标降解或增强靶标折叠。
开发并验证了基于细胞的生物发光报告基因检测方法，用于高通量筛选促进P23H突变视蛋白稳定或降解的化合物。免疫印迹和基于图像的分析进一步补充了此类检测。
开发并验证了两种P23H突变视蛋白的稳定检测方法，一种基于β-半乳糖苷酶互补性，另一种检测涉及生物发光共振能量转移（BRET）技术。此外，还采用了另外两种评估突变蛋白降解的检测方法，一种基于发光消失，另一种采用ALPHA免疫检测。细胞成像揭示了突变视紫红质的细胞定位，而免疫印迹则确定了P23H突变视蛋白聚集和糖基化的变化。
我们的研究结果表明，这些初步的高通量筛选及后续检测能够识别活性治疗化合物，即使对于像突变视紫红质这样的困难靶点也是如此。这些检测易于扩展，其功能已通过模型化合物得到验证。由自动成像和经典免疫检测支持的高通量筛选，可以进一步表征这种重要视觉系统蛋白生物合成和降解中的多个步骤和途径。
