Binding of potential antitumor Casiopeínas® to small proteins

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) and 1,10-phenanthroline (phen), respectively, and the bidentate glycinato ligand (Gly), along with a nitrate anion acting as a counterion. In biological fluids, they can maintain their identity or form mixed species and adducts with several bioligands, particularly proteins. In this study, the binding of Cas II-gly, and, for comparison, Cas VII-gly, to small proteins such as myoglobin (Mb), ubiquitin (Ub), and lysozyme (Lyz) was evaluated through a combination of instrumental (ESI-MS and EPR) and computational (dockings) methods. Simulations of the peak signals in the ESI-MS spectra confirmed the formation of the adducts. The results indicated that in all systems, adducts with the formula protein-[CuII(Me2phen)]n (with n = 1-3) were formed after the replacement of glycinato in the equatorial positions by side-chain donors. Docking studies showed that the three proteins used different donor sets to bind the CuII(Me2phen)2+ fragment: (NHis, NHis) or (NHis, COO−Asp/Glu) for Mb; NHis68 or (COO−Glu/Asp, COO−Glu/Asp) for Ub; and (COO−Glu/Asp, CO) or only a monodentate O donor for Lyz. Computational exploration of the protein structure revealed that more than one metal fragment could bind to the macromolecule. At present, it is not clear whether the formation of the adducts improves or worsens the activity of Casiopeínas®. However, the results suggested that, at the low copper concentrations found in the organism, the species protein-[CuII(Me2phen)]n coexist with [CuII(Me2phen)(Gly)]+ and the fragment CuII(Me2phen)2+, which - in turn - could partially dissociate into Cu2+ ions and free Me2phen ligands. Therefore, a mixture of species could be responsible for the biological activity of Casiopeínas®.