Hengel Sarah R, Malacaria Eva, Folly da Silva Constantino Laura, Bain Fletcher E, Diaz Andrea, Koch Brandon G, Yu Liping, Wu Meng, Pichierri Pietro, Spies M Ashley, Spies Maria
Department of Biochemistry, University of Iowa, Iowa City, United States.
Department of Environment and Health, Section of Experimental and Computational Carcinogenesis, Istituto Superiore di Sanita, Rome, Italy.
Elife. 2016 Jul 19;5:e14740. doi: 10.7554/eLife.14740.
The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing.
DNA修复蛋白RAD52是一种对BRCA缺陷型肿瘤至关重要的新兴治疗靶点。RAD52的缺失与肿瘤抑制因子BRCA1、BRCA2和PALB2的缺陷具有合成致死性。RAD52还参与停滞复制叉的恢复。鉴于单链DNA结合活性是RAD52细胞功能的基础，我们开展了一项高通量筛选活动，以鉴定破坏RAD52-单链DNA相互作用的化合物。先导化合物在生化分析中被确认为RAD52抑制剂。计算分析预测这些抑制剂结合在RAD52寡聚环的单链DNA结合凹槽内。通过计算机筛选活动验证了抑制剂-RAD52复合物的性质，最终发现了另一种RAD52抑制剂。对我们的抑制剂进行的细胞研究表明，RAD52-单链DNA相互作用使其在停滞复制叉处发挥功能，并且抑制RAD52-单链DNA结合在细胞杀伤中与BRCA2或MUS81缺失具有累加作用。
