Evaluation of the binding of four anti-tumor Casiopeínas® to human serum albumin

Abstract

The metal complexes designated by Casiopeínas® are mixed-ligand CuII-compounds some of them having promising antineoplastic properties. We report studies of binding of Cu(glycinato)(4,7-dimethyl-1,10-phenanthroline) (Cas-II-Gly (1)), Cu(acetylacetonato)(4,7-dimethyl-1,10-phenanthroline) (Cas-III-Ea (2)), Cu(glycinato)(4,4′-dimethyl-2,2′-bipyridine) (Cas-IV-Gly (3)) and Cu(acetylacetonato)(4,4′-dimethyl-2,2′-bipyridine) (Cas-III-ia (4)) to human serum albumin (HSA) by circular dichroism (CD), Electron paramagnetic resonance (EPR) and fluorescence spectroscopy. The results indicate that HSA may bind up to three molecules of the tested Casiopeínas. This is confirmed by inductively coupled plasma – atomic absorption spectroscopy measurements of samples of HSA-Casiopeínas after passing by adequate size-exclusion columns. The binding of Cas-II-Gly to HSA was also confirmed by MALDI-TOF mass spectrometric experiments. In the physiological range of concentrations the Casiopeínas form 1:1 adducts with HSA, with conditional binding constants of ca. 1×109 (1), 4×107 (2), 1×106 (3) and 2×105 (4), values determined from the CD spectra measured, and the fluorescence emission spectra indicates that the binding takes place close to the Trp214 residue. Overall, the data confirm that these Casiopeínas may bind to HSA and may be transported in blood serum by this protein; this might allow some selective tumor targeting, particularly in the case of Cas-II-Gly. In this work we also discuss aspects associated to the reliability of the frequently used methodologies to determine binding constants based on the measurement of fluorescence emission spectra of solutions containing low concentrations of proteins such as HSA and BSA, by titrations with solutions of metal complexes.