PARP1在手性金属超分子配体诱导端粒G4稳定中的作用及在端粒靶向治疗中的应用研究

Telomeres are the structures at the end of eukaryotic linear chromosomes. Human telomeres consist of double stranded tandem repeats of the hexanucleotide sequence TTAGGG except for the terminal 3′ G-rich overhang. Telomeres can fold into t-loops that may result from the invasion of the 3′ overhang into duplex DNA or into G-quadruplex (G4) DNA, an unusual DNA conformation based on guanine quartets. Mammalian telomeres are associated with telomeric proteins or shelterin, forming a complex that functions to protect the ends of DNA from degradation and fusion, thus playing an essential role in controlling genomic stability. Uncapped telomeres, resulting from either loss of function of telomere-binding proteins or loss of telomeric DNA repeats, directly associate with many DNA damage response proteins and induce a response similar to that observed for DNA breaks. A wealth of published works revealed that uncapped telomeres can also be obtained by pharmacological G4 stabilization. Chiral metallo-supramolecular ligand NiP, is one of the most effective G4 ligands showing a high selectivity for quadruplex DNA structure. We revealed that this agent, by stabilizing G4 DNA at telomeres, impairs fork progression and/or telomere processing, resulting in telomere dysfunction. However, in view of clinical application, NiP is not active in vivo as a single agent. The poly-adenosine diphosphate (ADP) ribose polymerase (PARP) family consists of a complex number of enzymes that catalyze the poly-ADP ribosylation, a post-translational modification of proteins. PARP1, the founding member of the PARP family, and PARP2 have long been studied as DNA damage responsive enzymes required for maintaining genomic integrity. The intervention of PARP1 and PARP2 takes place early in the steps of the DNA repair process, as these enzymes bind to and are activated by DNA nicks. Upon poly-ADP ribosylation, PARPs recruit to the damaged site components of BER and NER machineries. Therefore, inhibition of PARP activity would prevent recruitment of repair enzymes to DNA breaks, hampering strand rejoining and consequent generation of permanent single/double-strand breaks, which in turn triggers cell death. Because of their involvement in repairing of DNA damage, PARP inhibitors are under investigation as chemo- and radio-sensitizers for cancer treatment . The inhibition of PARPs potentiates the activity of DNA-damaging agents such as alkylators, platinums, topoisomerase inhibitors and radiation in in vitro and in vivo models. The objective of this work was to study the involvement of PARPs during telomere damages aiming to identify compelling molecules that may have a synergistic effect in the response of tumors to telomere-based therapy. The impact of these findings is broad for basic and biomedical research as well as for clinical applications.

端粒是染色体末端的保护性结构，其末端的富G序列可以形成独特的G-四链体（G4）结构，识别并稳定这种结构的配体会诱导端粒结构的紊乱及DNA损伤反应，驱动细胞凋亡，是新型的端粒靶向的肿瘤治疗策略，但其体内抗肿瘤效果并不十分显著，需要借助与其他药物的协同作用。多聚-ADP-核糖聚合酶1（PARP1）是早期DNA损伤修复蛋白，其活性的抑制会产生持久的DNA切口，诱导细胞死亡，是多种DNA损伤类药物的敏化剂。我们推测，PARP1在G4配体诱导端粒紊乱的过程中被激活，且其活性的抑制会增强G4配体的作用效果。本课题以高选择性识别端粒G4结构的手性金属超分子配体为材料，分析其作用后对细胞内PARP1的活性及定位的影响，同时分析其活性抑制后在G4配体诱导的细胞毒性及端粒紊乱中的作用。最后，借助移植瘤模型，分析PARP1抑制剂与G4配体的体内协同抗肿瘤作用。本课题为开发新颖的端粒靶向的联合治疗策略提供新思路。

端粒是染色体末端的保护性结构，其末端的富G序列可以形成独特的G-四链体（G4）结构，识别并稳定这种结构的配体会诱导端粒结构的紊乱及DNA损伤反应，驱动细胞凋亡，是新型的端粒靶向的肿瘤治疗策略，但其体内抗肿瘤效果并不十分显著，需要借助与其他药物的协同作用。多聚-ADP-核糖聚合酶1（PARP1）是早期DNA损伤修复蛋白，其活性的抑制会产生持久的DNA切口，诱导细胞死亡，是多种DNA损伤类药物的敏化剂。我们推测，PARP1在G4配体诱导端粒紊乱的过程中被激活，且其活性的抑制会增强G4配体的作用效果。本课题以高选择性识别端粒G4结构的手性金属超分子配体为材料，分析其作用后对细胞内PARP1的活性及定位的影响，发现手性G4配体能够显著激活PARP的活性，并且免疫荧光和端粒-荧光原位杂交（Telo-FISH）实验证实，能够显著诱导PARP1蛋白在端粒的定位，说明这种激活作用发生在端粒部位。利用加入小分子抑制剂（GP115427）下调PARP的活性，发现其与手性配体的联合作用，明显抑制细胞增殖，诱导细胞周期G2-M期阻滞，而且Annexin V染色证实，两者联用诱导明显的细胞凋亡，同时也抑制细胞克隆形成。端粒紊乱效应研究发现，PARP1抑制剂明显增强手性配体诱导的端粒的打开（TdT assay），同时增强诱导端粒处的DNA损伤反应（γ-H2Ax,53BP1）,同时诱导端粒染色体末端的融合以及滞后链的复制合成，上调DNA损伤相关效应蛋白的表达（p-ATM, MRE11, Chk1, Chk2）。体内实验，利用乳腺癌移植瘤模型证实，PARP1抑制剂能增强NiP的体内抑瘤效果，延长裸鼠的生存期，同时降低肿瘤组织内Ki67以及PCNA的表达，即抑制肿瘤细胞增殖。同时TUNEL染色证实，两药联合作用诱导肿瘤组织内细胞凋亡，同时下调CD31的表达，说明抑制血管生成，同时诱导γ-H2Ax的表达的上调，说明PARP1抑制剂与手性金属配体联合作用，通过上调DNA损伤反应发挥作用。本课题的开展对进一步阐明稳定端粒G-四链体的配体的作用机制，寻找新的端粒靶向的作用位点，研发新的更有效的基于G-四链体配体的联合抗肿瘤策略具有重要的意义。