Metallodrugs and Proteins: Expanding the Bioanalytical Toolkit to Investigate the Metallation of Biomolecules

Abstract

Metal-based anticancer agents are a widely used class of chemotherapeutics. Ru complexes show increasing promise over traditional Pt-based drugs as selective treatment options. Organometallic compounds with π-bound ligands are highly versatile in terms of structure and biomolecule binding profiles. To investigate reactions with biomolecules, a bioanalytical toolkit, primarily comprised of crystallography and mass spectrometry, was employed to understand the broad range of interactions observed of metal-based anticancer agents. To characterise interactions between cisplatin and human serum albumin, matrix-matched standards were prepared for use in LA–ICP-MS after separation with polyacrylamide gel electrophoresis. The use of such standards made from HSA and cisplatin adducts proved to be an effective way to analyse the amount of platinum bound to albumin. Dimeric Ru/Os(arene) compounds were investigated for their interactions with the model protein hen egg white lysozyme (HEWL). Protein crystallography, ion mobility mass spectrometry, differential scanning calorimetry, and dynamic light scattering revealed that the metal centre and the labile ligands determines the amount of adducts formed. To extend the study of the interactions between HEWL and metal complexes, Me- and Bn-substituted N-heterocyclic carbene (NHC) complexes were investigated. Binding studies between [RuII(η6-p-cymene)(NHC)Cl2] and HEWL revealed that p-cymene was cleaved concomitant to bidentate coordination of Ru to Arg14-His15 and oxidation of RuII to RuIII, as confirmed by electron paramagnetic resonance spectroscopy. To determine the structure-(re)activity profiles of NHC complexes with alternate metal centres, Os/Rh/Ir compounds were investigated. The Rh(η5-pentamethylcyclopentadienyl) analogue also bound at His15 upon substitution of the NHC ligand. In contrast, the Os(η6-p-cymene) and Ir(η5-pentamethylcyclopentadienyl) analogues underwent chlorido/side chain ligand exchange with Asn103 and Asp101, respectively. The complexes were investigated for their inhibition of thioredoxin reductase as a potential target and only the Rh analogues were effective. Furthermore, the lipophilicity influenced the cytotoxicity of all studied NHC complexes. These results suggest that small changes to organometallic complexes affect the biological activity and interactions with proteins. [RuII(η6-p-cymene)(N-fluorophenyl-2-pyridinecarbothioamide)Cl]Cl is highly cytotoxic and was shown to be selective towards the large scaffold protein plectin. Molecular mass spectrometry was used to determine which part of plectin forms adducts with the Ru complex. The most prominent binding partner was identified as the construct containing the first two spectrin repeats. The bioanalytical studies used here to explore the interactions between metal-based anticancer agents and proteins demonstrates that small changes to the metal centre or the attached ligands affect the amounts and type of adducts formed. The investigations lay the foundation for the development of more specific metallodrugs.