Nadanaciva Sashi, Murray James, Wilson Casey, Gebhard David F, Will Yvonne
Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut, USA.
Curr Protoc Toxicol. 2011 May;Chapter 3:Unit3.11. doi: 10.1002/0471140856.tx0311s48.
Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.
损害线粒体DNA（mtDNA）或mtDNA编码蛋白合成的化合物会降低氧化磷酸化所必需的13种蛋白的水平，导致线粒体ATP生成减少。由于mtDNA和mtDNA编码蛋白的更新率较低，这些化合物引起的毒性在24至72小时细胞毒性试验中很少被发现。在此，我们描述了三种高通量筛选试验，用于检测影响mtDNA编码蛋白水平的化合物。所有这三种试验都测量两种蛋白的水平，一种是在线粒体核糖体上合成的mtDNA编码蛋白，另一种是在胞质核糖体上合成的核DNA编码蛋白。第一种试验通过定量图像分析测量这两种蛋白的水平，需要一个高内涵成像系统。第二种试验是一种细胞内免疫测定法，利用红外染料检测这两种蛋白，因此需要一个LI-COR奥德赛系统。第三种试验是一种细胞内免疫测定法，利用比色法检测这两种蛋白，需要一个吸光酶标仪。
