Separate and simultaneous binding effects through a non-cooperative behavior between cyclophosphamide hydrochloride and fluoxymesterone upon interaction with human serum albumin: Multi-spectroscopic and molecular modeling approaches

This study was designed to examine the interaction of two anti-breast cancer drugs, i.e., fluoxymesterone (FLU) and cyclophosphamide (CYC), with human serum albumin (HSA) using different kinds of spectroscopic, zeta potential and molecular modeling techniques under imitated physiological conditions. The RLS technique was utilized to investigate the effect of the two anticancer drugs on changes of the protein conformation, both separately and simultaneously. Our study suggested that the enhancement in RLS intensity was attributed to the formation of a new complex between the two drugs and the protein. Both drugs demonstrated a powerful ability to quench the fluorescence of HSA, and the fluorescence quenching action was much stronger when the two drugs coexisted. The quenching mechanism was suggested to be static as confirmed by time-resolved fluorescence spectroscopy results. The effect of both drugs on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and demonstrated a conformational change of HSA with the addition of both drugs. The binding distances between HSA and the drugs were estimated by the Förster theory, and it was revealed that nonradiative energy transfer from HSA to both drugs occurred with a high probability. According to CD measurements, the influence of both drugs on the secondary structure of HSA in aqueous solutions was also investigated and illustrated that the α-helix content of HSA decreased with increasing drug concentration in both systems. Moreover, the zeta-potential experiments revealed that both drugs induced conformational changes on HSA. Docking studies were also performed and demonstrated that a reduction of the binding affinity between the drugs and HSA occurred in the presence of both drugs.

Contour diagrams of HSA–FLU. There were two “humps” in the three-dimensional fluorescence spectrum for HSA in which the peaks were marked peaks 1 and 2. The excitation wavelength of peak 1 was 280 nm and it chiefly revealed the spectral characteristic of the Tyr and Trp residues, due to the fluorescence of the Phe residue being negligible when HSA was excited at this wavelength.Figure optionsDownload as PowerPoint slideHighlights
► We studied the simultaneous interaction of cyclophosphamide hydrochloride and fluoxymestrone with HSA.
► They may compete in binding to HSA and increase uncontrolled toxic effects.
► We determined the critical induced aggregation concentration of both drugs on HSA.
► The binding mechanism of drugs as separate and simultaneous to HSA has been compared.
► The binding site of both drugs as simultaneous effects on HSA has been determined.