Towards a combined skin cancer treatment: the macrocyclic effect of pillar[5]arene containing 6-mercaptopurine fragments, integrated with an exfoliating serine protease and 5-fluorouracil.

A novel approach for the highly specific and complex skin cancer treatment is proposed, leveraging the combined action of serine proteases for exfoliation of the keratinized skin layer and a small-molecule antitumor drug integrated into a multifunctional macrocyclic scaffold. We report the first synthesis of a water-soluble pillararene containing ten fragments of 6-mercaptopurine. The selective cytotoxic effect of this compound on cancer cells (A431) was demonstrated. UV-Vis and fluorescence spectroscopy, together with microcalorimetric methods, confirmed the formation of associations between the pillararene and the exfoliating serine protease (subtilisin) (lgКass = 6.42, stoichiometry 1:3 (subtilisin/macrocycle)), and the antitumor drug 5-fluorouracil (lgКass = 4.07, stoichiometry 1:1). The interaction between the pillar[5]arene, subtilisin, and 5-fluorouracil was investigated using dynamic light scattering and transmission electron microscopy, revealing the formation of stable aggregates with diverse morphologies. The three-component assembly comprising the thiopurine-functionalized pillar[5]arene, 5-fluorouracil, and subtilisin enhanced the enzymatic activity of subtilisin by 220% compared to the free enzyme. MTT assays demonstrated that self-assembled pillar[5]arene/subtilisin/5-fluorouracil system exerted selective cytotoxicity towards A431 tumor cells while exhibiting lower toxicity to normal fibroblasts (HSF). Molecular docking studies further supported the anticancer potential of the three-component assembly, suggesting that competitive binding with proteins involved in tumor progression may facilitate the release and cellular uptake of 5-fluorouracil.