Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363, Lodz, Poland.
BioMedChem Doctoral School of the University of Lodz and the Institutes of the Polish Academy of Sciences in Lodz, Lodz, Poland.
Sci Rep. 2024 Jun 7;14(1):13148. doi: 10.1038/s41598-024-63759-5.
Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue-alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure-activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate S and R diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist-AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the S-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial-mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the S-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.
最近的数据表明，细胞外 ATP 通过 P2Y2 依赖性信号通路影响伤口愈合效果。在目前的工作中，我们提出双修饰的 ATP 类似物-α-硫-β，γ-亚甲基-ATP 作为皮肤再生的潜在治疗剂。为了更好地了解构效关系，除了测试的 ATP 类似物外，还在测试目标化合物的适当单修饰衍生物，如α-硫-ATP 和β，γ-亚甲基-ATP，以了解它们在通过 P2Y2 受体参与 ATP 依赖性嘌呤能信号通路激活方面的作用。使用氧杂硫磷环方法作为单独的 S 和 R 非对映异构体获得了非对映纯的α-硫修饰-ATP 衍生物。然后测试了单修饰和双修饰的 ATP 类似物对人角质形成细胞活力和迁移的影响。通过将测试化合物对接至 P2Y2 受体，在计算机上分析 P2Y2 依赖性嘌呤能信号的参与，并通过研究人角质形成细胞 HaCaT 中的细胞内钙动员来进行实验。将获得的 ATP 类似物的作用与作为天然 P2Y2 激动剂的 ATP 的结果进行比较。为了确认 P2Y2 受体对观察到的作用的贡献，还在选择性 P2Y2 拮抗剂-AR-C118925XX 的存在下进行了测试。通过划痕愈合测定法和 Transwell 迁移试验在体外模型 HaCaT 和 MDA-MB-231 细胞上以及在体内使用斑马鱼系统分析了α-硫-β，γ-亚甲基-ATP 影响细胞迁移的能力。基于切除的斑马鱼尾巴的再生率来估计对组织再生的影响。体外和体内研究表明，S-α-硫-β，γ-亚甲基-ATP 类似物促进与再生相关的过程，使其成为增强伤口愈合的合适试剂。进行的研究表明它对细胞迁移、上皮-间充质转化和细胞内钙动员的影响。切除的斑马鱼尾巴的再生增强证实了这种 ATP 类似物的促再生活性。基于进行的研究，提出 S-α-硫-β，γ-亚甲基-ATP 作为治疗伤口愈合和皮肤再生的潜在治疗剂。
