Cancer Research UK Drug Discovery Unit, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK.
Anal Biochem. 2013 Sep 1;440(1):1-5. doi: 10.1016/j.ab.2013.05.003. Epub 2013 May 18.
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the hydrolysis of phosphodiester bonds between the DNA 3'-phosphate and tyrosine residues and plays a major role in the repair of stalled topoisomerase I-DNA covalent complexes. Given this role, Tdp1 is of interest as a potential target for anticancer therapy. Inhibiting Tdp1 in combination with clinically used Top1 inhibitors may potentiate the effects of the latter and help to overcome some of the chemoresistance issues currently observed. In addition, Tdp1 can function during DNA repair to remove a variety of other 3' adducts from DNA such as phosphoglycolates and abasic or apurinic/apyrimidinic (AP) sites. Here we describe a new mix-and-read homogeneous fluorogenic assay for the measurement of the AP-site cleavage activity of Tdp1 that is compatible with high-throughput screening. The application of such an assay will open up further avenues for the discovery of novel Tdp1 inhibitors.
酪氨酰 DNA 磷酸二酯酶 1（Tdp1）催化 DNA 3'-磷酸和酪氨酸残基之间的磷酸二酯键的水解，在修复停滞的拓扑异构酶 I-DNA 共价复合物中发挥主要作用。鉴于其作用，Tdp1 作为一种潜在的抗癌治疗靶点受到关注。与临床使用的 Top1 抑制剂联合抑制 Tdp1 可能会增强后者的作用，并有助于克服目前观察到的一些化疗耐药问题。此外，Tdp1 在 DNA 修复过程中可以从 DNA 中去除多种其他 3' 加合物，如磷酸甘油酸和碱基缺失或无碱基/嘧啶缺失（AP）位点。在这里，我们描述了一种新的混合读取均相荧光测定法，用于测量 Tdp1 的 AP 位点切割活性，该方法与高通量筛选兼容。这种测定法的应用将为发现新型 Tdp1 抑制剂开辟更多途径。
