Click化学在生物医学研究中提供了有价值的工具，但其直接用于治疗仍然几乎未被探索。对于癌症治疗，类似核苷酸类似物（NA）如5-乙烯基-2'-脱氧尿嘧啶（VdU）可以代谢地被并入癌细胞DNA中，然后通过"点击"反应形成有毒产物。已经使用VdU和乙烯基蓝-四唑结合物（PINK）之间的反电子需求Diels-Alder（IEDDA）反应在培养细胞的细胞核标记中。在这里，我们报道了VdU和PINK的串联使用以诱导细胞毒性作用。细胞系随后用VdU和PINK处理，细胞存活率通过细胞充实度和3D肿瘤球测定评估。通过Western Blotting评估DNA损伤和凋亡，并通过流式细胞术分析细胞周期。使用彗星测定法测量双链DNA断裂（DSB）形成。通过荧光检测外化的磷脂酰丝氨酸残基评估凋亡。我们报道了VdU和PINK的组合在培养人类细胞中协同诱导细胞毒性。VdU和PINK的组合在2D和3D培养的癌细胞中明显降低了细胞存活率。机制上，这些化合物通过DSB形成导致DNA损伤，进而导致S期积累和凋亡。VdU和PINK的组合代表了一种新颖且有希望的DNA模板"点击"方法，可通过选择性诱导DNA损伤来治疗癌症。版权所有 © 2023 Elsevier Inc. 保留所有权利。

Click Chemistry is providing valuable tools to biomedical research, but its direct use in therapies remains nearly unexplored. For cancer treatment, nucleoside analogues (NA) such as 5-vinyl-2'-deoxyuridine (VdU) can be metabolically incorporated into cancer cell DNA and subsequently "clicked" to form a toxic product. The inverse electron-demand Diels-Alder (IEDDA) reaction between VdU and an acridine-tetrazine conjugate (PINK) has previously been used to label cell nuclei of cultured cells. Here, we report tandem usage of VdU and PINK to induce cytotoxicity.Cell lines were subsequently treated with VdU and PINK, and cell viability was measured via well confluency and 3D tumor spheroid assays. DNA damage and apoptosis were evaluated using Western Blotting and cell cycle analysis by flow cytometry. Double stranded DNA break (DSB) formation was measured using the comet assay. Apoptosis was assessed by fluorescent detection of externalized phosphatidylserine residues.We report that the combination of VdU and PINK synergistically induces cytotoxicity in cultured human cells. The combination of VdU and PINK strongly reduced cell viability in 2D and 3D cultured cancer cells. Mechanistically, the compounds induced DNA damage through DSB formation, which leads to S-phase accumulation and apoptosis.The combination of VdU and PINK represents a novel and promising DNA-templated "click" approach for cancer treatment via selective induction of DNA damage.Copyright © 2023 Elsevier Inc. All rights reserved.