Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, New Delhi 110 007, India.
Biochemistry. 2010 Sep 28;49(38):8388-97. doi: 10.1021/bi1005927.
Quadruplex-specific molecules can serve as suitable drugs in cancer therapy. We have synthesized a pair of furan-based cyclic homooligopeptides, ligand 1 and ligand 2, to specifically target G-quadruplexes. We have shown by CD spectroscopy and UV melting that these ligands can effectively induce G-quadruplex structures in the G-rich 22-mer c-MYC DNA sequence and further stabilize the structure. Equilibrium binding constants measured by isothermal titration calorimeter methods indicate a high affinity of the ligands for the quadruplex structures (K ∼ 10(7) M(-1)) and no affinity for the duplex DNA, demonstrating that these ligands are selective for G-quadruplex structures. Surface plasmon resonance was also used to compute the binding while fluorescence resonance energy transfer-based assay was additionally used to confirm the selectivity. Moreover, using real time PCR we observed up to 90% downregulation of c-MYC transcripts after 24 h of ligand treatment in HeLa cells. Using a luciferase assay we show the downregulation of the protein levels. Fluorescent-assisted cell sorter-based cell cycle analysis showed a prominent arrest of cells in the sub-G1 stage upon treatment of ligands that leads toward apoptosis. Altogether, these experiments support the hypothesis that the present molecules are effective in specifically binding and stabilizing quadruplexes and provide a suitable scaffold to develop into a quadruplex-targeting therapeutic agent.
四链体特异性分子可以作为癌症治疗的合适药物。我们合成了一对基于呋喃的环状同寡肽配体 1 和配体 2，以专门针对 G-四链体。我们通过 CD 光谱和 UV 熔融实验表明，这些配体可以有效地诱导富含 G 的 22 -mer c-MYC DNA 序列中的 G-四链体结构，并进一步稳定结构。通过等温滴定量热法测量的平衡结合常数表明，配体对四链体结构具有高亲和力（K∼10(7)M(-1)），而对双链 DNA 没有亲和力，表明这些配体对 G-四链体结构具有选择性。表面等离子体共振也用于计算结合，而荧光共振能量转移测定法也用于确认选择性。此外，使用实时 PCR，我们观察到在用配体处理 HeLa 细胞 24 小时后，c-MYC 转录物的下调高达 90%。使用荧光素酶测定法，我们显示了蛋白水平的下调。荧光辅助细胞分选的细胞周期分析表明，在用配体处理后，细胞在亚 G1 期明显停滞，导致细胞凋亡。总的来说，这些实验支持了这样的假设，即目前的分子在特异性结合和稳定四链体方面是有效的，并为开发四链体靶向治疗剂提供了合适的支架。
