Implications of Protein Interaction in the Speciation of Potential VIVO-Pyridinone Drugs

The interaction of [(VO)-O-IV-(empp)(2)] (empp(-)= 1-methyl-2-ethyl-3-hydroxy-4-(1H)-pyridinonato)with hen egg white lysozyme (HEWL) reveals covalent and noncovalentbinding to protein of different vanadium-containing fragments: [(VO)-O-IV-(empp)-(H2O)](+), [(VO)-O-IV-(empp)-(H2O)(2)](+), cis-[(VO)-O-IV-(empp)(2)(H2O)], and even a trinuclear oxidovanadium-(V)complex, [V-V O-3(6)(empp)(3)(H2O)]. Vanadium complexes (VCs) are promising agents for thetreatment,among others, of diabetes and cancer. The development of vanadium-baseddrugs is mainly limited by a scarce knowledge of the active speciesin the target organs, which is often determined by the interactionof VCs with biological macromolecules like proteins. Here, we havestudied the binding of [(VO)-O-IV-(empp)(2)] (whereHempp is 1-methyl-2-ethyl-3-hydroxy-4-(1H)-pyridinone),an antidiabetic and anticancer VC, with the model protein hen eggwhite lysozyme (HEWL) by electrospray ionization-mass spectrometry(ESI-MS), electron paramagnetic resonance (EPR), and X-ray crystallography.ESI-MS and EPR techniques reveal that, in aqueous solution, both thespecies [(VO)-O-IV-(empp)(2)] and [(VO)-O-IV-(empp)-(H2O)](+), derived from the first one upon the lossof a empp(-) ligand, interact with HEWL. Crystallographic data,collected under different experimental conditions, show covalent bindingof [(VO)-O-IV-(empp)-(H2O)](+) to the sidechain of Asp48, and noncovalent binding of cis-[(VO)-O-IV-(empp)(2)(H2O)], [(VO)-O-IV-(empp)-(H2O)](+), [(VO)-O-IV-(empp)-(H2O)(2)](+), and of an unusual trinuclear oxidovanadium-(V)complex, [V-V O-3(6)(empp)(3)(H2O)], with accessible sites on the protein surface.The possibility of covalent and noncovalent binding with differentstrength and of interaction with various sites favor the formationof adducts with the multiple binding of vanadium moieties, allowingthe transport in blood and cellular fluids of more than one metal-containingspecies with a possible amplification of the biological effects.