Jang Dae Song, Penthala Narsimha R, Apostolov Eugene O, Wang Xiaoying, Fahmi Tariq, Crooks Peter A, Basnakian Alexei G
Department of Pharmacology & Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
J Biomol Screen. 2015 Feb;20(2):202-11. doi: 10.1177/1087057114555828. Epub 2014 Oct 17.
Deoxyribonuclease I (DNase I), the most active and abundant apoptotic endonuclease in mammals, is known to mediate toxic, hypoxic, and radiation injuries to the cell. Neither inhibitors of DNase I nor high-throughput methods for screening of high-volume chemical libraries in search of DNase I inhibitors are, however, available. To overcome this problem, we developed a high-throughput DNase I assay. The assay is optimized for a 96-well plate format and based on the increase of fluorescence intensity when fluorophore-labeled oligonucleotide is degraded by the DNase. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z' ≥ 0.5), and operationally simple, and it has low operator, intraassay, and interassay variability. The assay was used to screen a chemical library, and several potential DNase I inhibitors were identified. After comparison, 2 hit compounds were selected and shown to protect against cisplatin-induced kidney cell death in vitro. This assay will be suitable for identifying inhibitors of DNase I and, potentially, other endonucleases.
脱氧核糖核酸酶I（DNase I）是哺乳动物中活性最强且含量最丰富的凋亡内切核酸酶，已知其介导细胞的毒性、缺氧及辐射损伤。然而，目前既没有DNase I抑制剂，也没有用于筛选大量化学文库以寻找DNase I抑制剂的高通量方法。为克服这一问题，我们开发了一种高通量DNase I检测方法。该检测方法针对96孔板形式进行了优化，基于荧光团标记的寡核苷酸被DNase降解时荧光强度的增加。与其他内切核酸酶相比，该检测方法对DNase I高度敏感、可靠（Z'≥0.5）且操作简单，并且具有较低的操作者、批内和批间变异性。该检测方法用于筛选化学文库，鉴定出了几种潜在的DNase I抑制剂。经过比较，选择了2种命中化合物，并证明它们在体外可保护细胞免受顺铂诱导的肾细胞死亡。该检测方法将适用于鉴定DNase I抑制剂，以及潜在地鉴定其他内切核酸酶的抑制剂。
