Structural and biological properties of novel G4-aptamer-drug conjugates as a vehicle for 5-fluoro-2′-deoxyuridine

One of the most appealing approaches for cancer treatment is targeted therapy, which is based on the use of drugs able to target cancer cells without affecting normal ones. This strategy lets to overcome the major limitation of conventional chemotherapy, namely the lack of specificity of anticancer drugs, which often leads to severe side effects, decreasing the therapy effectiveness. Delivery of cell-killing substances to tumor cells is one-way targeted drug therapy can work. Aptamer-drug conjugates represent a promising solution to minimize off-target effects, considering aptamers' remarkable selective binding capabilities [1]. Furthermore, recent advances in anticancer agents have highlighted the potential of G4-aptamers in clinical applications [2]. In order to enhance the therapeutic efficacy of the antineoplastic agent 5-fluoro-2′-deoxyuridine (FdU) in various cancer cells, a novel conjugate using the antiproliferative aptamer T30923 (INT) [3] as a drug vehicle was developed. Three derivatives composed of T30923 conjugated with different numbers of FdU units were synthesized, and their structural and biological properties were thoroughly characterized. The collected NMR, CD, and PAGE data strongly suggest that, similar to their unconjugated sequence, the resulting aptamer-drug conjugates showed the ability to fold into a dimeric G4 structure formed by the same G-quadruplexes, characterized by parallel strands, three all-anti-G-tetrads and three one-thymidine propeller loops. Moreover, antiproliferative properties, cellular uptake and endocytosis mechanism have been investigated showing that the coexistence of INT and (FdU)n not only strengthens the killing effect of free FdU by a synergistic effect, but also enables the conjugates to target and kill cancer cells selectively. In this way, it is possible to minimize side effects, that may arise from the non-specificity or cellular resistance of FdU alone, and also to increase the antiproliferative activity of both compounds.