Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada.
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD, USA.
SLAS Discov. 2019 Dec;24(10):969-977. doi: 10.1177/2472555219875934. Epub 2019 Sep 26.
Retinitis pigmentosa (RP) is a degenerative retinal disease, often caused by mutations in the G-protein-coupled receptor rhodopsin. The majority of pathogenic rhodopsin mutations cause rhodopsin to misfold, including P23H, disrupting its crucial ability to respond to light. Previous screens to discover pharmacological chaperones of rhodopsin have primarily been based on rescuing rhodopsin trafficking and localization to the plasma membrane. Here, we present methods utilizing a yeast-based assay to screen for compounds that rescue the ability of rhodopsin to activate an associated downstream G-protein signaling cascade. We engineered a yeast strain in which human rhodopsin variants were genomically integrated, and were able to demonstrate functional coupling to the yeast mating pathway, leading to fluorescent protein expression. We confirmed that a known pharmacological chaperone, 9- retinal, could partially rescue light-dependent activation of a disease-associated rhodopsin mutation (P23H) expressed in yeast. These novel yeast strains were used to perform a phenotypic screen of 4280 compounds from the LOPAC1280 library and a peptidomimetic library, to discover novel pharmacological chaperones of rhodopsin. The fluorescence-based assay was robust in a 96-well format, with a Z' factor of 0.65 and a signal-to-background ratio of above 14. One compound was selected for additional analysis, but it did not appear to rescue rhodopsin function in yeast. The methods presented here are amenable to future screens of small-molecule libraries, as they are robust and cost-effective. We also discuss how these methods could be further modified or adapted to perform screens of more compounds in the future.
色素性视网膜炎（RP）是一种退行性视网膜疾病，通常由 G 蛋白偶联受体视紫红质的突变引起。大多数致病性视紫红质突变导致视紫红质错误折叠，包括 P23H，破坏其对光的关键响应能力。以前发现视紫红质药理学伴侣的筛选主要基于挽救视紫红质的运输和定位到质膜。在这里，我们提出了利用酵母为基础的测定来筛选化合物的方法，这些化合物可以恢复视紫红质激活相关下游 G 蛋白信号级联的能力。我们设计了一种酵母菌株，其中人类视紫红质变体在基因组上整合，并且能够证明与酵母交配途径的功能偶联，导致荧光蛋白表达。我们证实，一种已知的药理学伴侣，9-视黄醛，可以部分挽救与疾病相关的视紫红质突变（P23H）在酵母中表达的光依赖性激活。这些新型酵母菌株被用于对来自 LOPAC1280 文库和肽模拟文库的 4280 种化合物进行表型筛选，以发现视紫红质的新型药理学伴侣。基于荧光的测定在 96 孔格式中具有稳健性，Z' 因子为 0.65，信号与背景比大于 14。选择了一种化合物进行进一步分析，但它似乎没有挽救酵母中的视紫红质功能。这里提出的方法适用于未来的小分子文库筛选，因为它们具有稳健性和成本效益。我们还讨论了如何进一步修改或改编这些方法，以便在未来筛选更多的化合物。
