Competitive binding of anticancer drugs 5-fluorouracil and cyclophosphamide with serum albumin: Calorimetric insights

• Binding of 5-fluorouracil and cyclophosphamide studied with serum albumin
• The drugs bind weakly with serum albumin.
• Electrostatic and hydrophobic interactions participate in binding.
• 5-Fluorouracil can displace cyclophosphamide from the binding site on protein.
• The drugs bind at Site II of sub-domain IIIA.

BackgroundIsothermal titration calorimetry (ITC) has emerged as an excellent method to characterize drug–protein interactions. 5-Fluorouracil and cyclophosphamide have been used in combination for the treatment of breast carcinoma, though individually these drugs have also been useful in treating other types of cancer. A quantitative understanding of binding of these drugs with the transport protein under different conditions is essential for optimizing recognition by the protein and delivery at the target.MethodsThe values of binding constant, enthalpy, and entropy of binding have been determined by using ITC. Fluorescence and circular dichroism spectroscopies have been used to obtain further support to calorimetric observations, monitor conformational changes in the protein and establishing stoichiometry of association.ResultsThe thermodynamic parameters have enabled a quantitative understanding of the affinity of 5-fluorouracil and cyclophosphamide with bovine serum albumin. The nature of binding has been unraveled based on effect of ionic strength, tetrabutyl-ammonium bromide, and sucrose which interfere in ionic, hydrophobic, and hydrogen bonding interactions. The binding site has been identified by using site marker warfarin in combination with 5-fluorouracil and cyclophosphamide. Further, the experiments have been done to establish whether both the drugs share the same binding site, and the effect of antibiotic drug carbenecillin and anti-inflammatory drug naproxen on their association.General significanceTuning optimum association of drugs with the transport vehicles for effective drug delivery requires identification of the nature of interacting groups in terms of energetics of interactions. Such studies employing ITC have direct significance in rational drug design. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

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