PDF(Pubmed)
Abstract：
ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.
摘要：
ADP-核糖基转移酶PARP1和PARP2通过检测DNA损伤并诱导聚ADP-核糖基化依赖性染色质松弛和修复蛋白的募集在DNA修复机制中起主要作用。催化PARP抑制剂用作抗癌药物,尤其是在由致敏突变引起的肿瘤的情况下。最近,一项研究表明，组蛋白PAR化因子（HPF1）与PARP1/2形成联合活性位点。HPF1与PARP1/2的相互作用改变了从天冬氨酸/谷氨酸到丝氨酸的修饰位点,这已被证明是DNA损伤背景下的关键ADP核糖基化事件。因此,破坏PARP1/2-HPF1相互作用可能是药物开发阻断PARP1/2活性的替代策略.在这项研究中,我们描述了一种基于FRET的高通量筛选试验,以筛选抗PARP-HPF1相互作用的抑制剂库.我们优化了FRET信号的条件，并通过多种方式竞争FRET对验证了相互作用。该测定是稳健的并且易于自动化。有效的筛选显示了该测定的强大性能，我们发现了两种化合物二甲基丙烯紫草素和碱宁，对PARP1/2-HPF1相互作用具有μM抑制效力。该测定将有助于发现针对HPF1-PARP1/2复合物的抑制剂并开发潜在的新的有效抗癌剂。
