英国剑桥大学Tom L. Blundell、Shikang Liang等研究人员合作揭示抑制剂对DNA修复蛋白DNA-PKcs调节的结构基础。相关论文于2022年1月5日在线发表于国际学术期刊《自然》。

据研究人员介绍，DNA依赖性蛋白激酶催化亚单位（DNA-PKcs）在非同源末端连接中起着核心作用，这是人类检测和修复DNA双链断裂（DSB）的两个主要途径之一。DNA-PKcs在修复病理性DSB方面非常重要，这使得DNA-PKcs抑制剂与DSB诱导放疗和化疗相结合，成为有吸引力的癌症治疗药物。许多已经开发的DNA-PKcs选择性抑制剂显示出治疗各种癌症的潜力。

研究人员报道了从HeLa细胞核提取物中天然纯化的人类DNA-PKcs与腺苷-5′-(γ-硫)-三磷酸(ATPγS)和四种抑制剂（wortmannin、NU7441、AZD7648和M3814）的冷冻电镜结构，包括正在进行临床试验的候选药物。这些结构揭示了ATP在催化前与活性部位结合的分子细节，并提供了对竞争性抑制剂的作用方式和特异性的深入了解。值得注意的是，配体的结合导致PIKK调节域（PRD）的移动，从而揭示了p环和PRD构象之间的联系。对DNA依赖性蛋白激酶全酶的电泳迁移率测定和冷冻电镜研究进一步表明，配体的结合对全酶复合物的组装没有负向的异构作用或抑制作用，抑制剂通过与ATP的直接竞争发挥作用。

总的来说，这项研究描述的结构有望极大地帮助未来针对DNA-PKcs的合理药物设计工作，并表明冷冻电镜在结构指导药物开发中对大型和挑战性的目标具有潜力。

附：英文原文

Title: Structural insights into inhibitor regulation of the DNA repair protein DNA-PKcs

Author: Liang, Shikang, Thomas, Sherine E., Chaplin, Amanda K., Hardwick, Steven W., Chirgadze, Dimitri Y., Blundell, Tom L.

Issue&Volume: 2022-01-05

Abstract: The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5′-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.

DOI: 10.1038/s41586-021-04274-9

Source: https://www.nature.com/articles/s41586-021-04274-9