Binding of potential antitumor Casiopeínas® with small proteins

Abstract

Casiopeínas® are a family of patented CuII anticancer compounds. Cas II-gly and Cas VII-gly are formed by 4,7-dimethyl-1,10-phenanthroline (Me₂phen) or 1,10-phenanthroline (phen), respectively, and the bidentate glycinato ligand (Gly), plus a nitrate anion acting as a counterion. In biological fluids, they can keep their identity or form mixed species and adducts with several bioligands, in particular with proteins. In this study, the binding of Cas II-gly, and Cas VII-gly for comparison, to small proteins like myoglobin (Mb), ubiquitin (Ub), and lysozyme (Lyz) was evaluated through combination of instrumental (ESI-MS and EPR) and computational (dockings) methods. Simulations of the peak signals in the ESI-MS spectra allow confirming the formation of the adducts. The results indicated that in all systems adducts with formula Protein–[Cu^II(Me₂phen)]_n with n = 1-3 are formed after the replacement of glycinato in the equatorial positions by side-chain donors. Dockings shows that the three proteins use different donor sets to bind the fragment Cu^II(Me₂phen)²⁺: (N_His, N_His) or (N_His, COO^–_Asp/Glu) for Mb, N_His68 or (COO^–_Glu/Asp, COO^–_Glu/Asp) for Ub, (COO^–_Glu/Asp, CO) or only monodentate O donor for Lyz. The computational exploration of the protein structure reveals that more than one metal fragments can bind to the macromolecule. At the moment, it is not clear whether the formation of the adducts improves or worsens the action of Casiopeínas®. However, the obtained results suggest that, at the low copper concentrations found in the organism, the species Protein–[Cu^II(Me₂phen)]_n coexist with [Cu^II(Me₂phen)(Gly)]⁺ and the fragment Cu^II(Me₂phen)²⁺ which – in turn – could dissociate, at least partially, to Cu²⁺ ion and free ligand Me₂phen. Therefore, a mixture of species could be responsible of the biological activity of Casiopeínas®.