Kinetics and mechanistic study of competitive inhibition of thymidine phosphorylase by 5-fluoruracil derivatives

• 3 derivatives of 5-fluorouracile, included or not in liposome formulations, were investigated.
• Inhibition, equilibrium and kinetic constants (KI, KM, kcat) were determined.
• Docking simulations were also performed.
• all compounds act as competitive inhibitors of the target enzyme.
• the length of the spacer does not affect the inhibitory efficacy.

In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N3 position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3′-azido-2′-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer.

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