Protein metalation by metal-based drugs: reactions of cytotoxic gold compounds with cytochrome c and lysozyme

Protein metalation processes are crucial for the mechanism of action of several anticancer metallodrugs and warrant deeper characterisation. We have explored the reactions of three cytotoxic gold(III) compounds-namely [(bipy(2Me))(2)Au-2(mu-O)(2)][PF6](2) (where bipy(2Me) is 6,6'-dimethyl-2,2'-bipyridine) (Auoxo6), [(phen(2Me))(2)Au-2(mu-O)(2)][PF6](2) (where phen(2Me) is 2,9-dimethyl-1,10-phenanthroline) (Au(2)phen) and [(bipy(dmb)-H)Au(OH)][PF6] [where bipy(dmb)-H is deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine] (Aubipyc)-with two representative model proteins, i.e. horse heart cytochrome c and hen egg white lysozyme, through UV-visible absorption spectroscopy and electrospray ionisation mass spectrometry (ESI MS) to characterise the inherent protein metalation processes. Notably, Auoxo6 and Au(2)phen produced stable protein adducts where one or more naked gold(I) ions are protein-coordinated; very characteristic is the case of cytochrome c, which upon reaction with Auoxo6 or Au(2)phen preferentially forms tetragold adducts with four protein-bound gold(I) ions. In turn, Aubipyc afforded monometalated protein adducts where the structural core of the gold(III) centre and its +3 oxidation state are conserved. Auranofin yielded protein derivatives containing the intact auranofin molecule. Additional studies were performed to assess the role played by a reducing environment in protein metalation. Overall, the approach adopted provides detailed insight into the formation of metallodrug-protein derivatives and permits trends, peculiarities and mechanistic details of the underlying processes to be highlighted. In this respect, electrospray ionisation mass spectrometry is a very straightforward and informative research tool. The protein metalation processes investigated critically depend on the nature of both the metal compound and the interacting protein and also on the solution conditions used; thus, predicting with accuracy the nature and the amounts of the adducts formed for a given metallodrug-protein pair is currently extremely difficult.