Cytotoxic effects of Metvan (cis-[VO(OSO)(Mephen)], where Mephen = 4,7-dimethyl-1,10-phenanthroline) and its analogues with 1,10-phenanthroline (phen) and 2,2'-bipyridine (bpy) ligands in cultured human lung cancer (A549) cells have been re-investigated in conjunction with reactivity of the V(IV) complexes in neutral aerated aqueous solutions and in cell culture medium. All the V(IV) complexes underwent rapid oxidation to the corresponding V(V) species (cis-[V(O)L]), followed by release of free ligands (shown by electrospray mass spectrometry). Decomposition of V(IV) complexes in cell culture medium within minutes at 310 K was confirmed by UV-Vis and EPR spectroscopies. High cytotoxicities (low μM or sub-μM IC range in 72 h assays) were observed for the phen and Mephen complexes, but they were not different from that of the corresponding free ligands, which confirmed that the original V(IV) complexes played no significant role in the observed biological activities. The cytotoxicities of the ligands were most likely due to their complexation of redox-active essential metal ions, such as Cu(II) and Fe(II), in the medium, and their increased cellular uptake, leading to oxidative stress-related cell death. These results emphasize the need to assess the stability of metal-based drugs under the conditions of biological assays, particularly when biologically active ligands, such as 1,10-phenanthroline and its derivatives, are used. These ligands have high systemic toxicities in vivo and their release in the GI tract and blood makes the complexes unsuitable for use as anti-cancer drugs.

Mixed Lineage Kinase domain-Like pseudokinase (MLKL) is implicated in a broad range of diseases due to its role as the ultimate effector of necroptosis and has therefore emerged as an attractive drug target. Here, we describe the development of PROteolysis TArgeting Chimeras (PROTACs) as a novel approach to knock down MLKL through chemical means. A series of candidate degraders were synthesized from a high-affinity pyrazole carboxamidebased MLKL ligand leading to the identification of a PROTAC molecule that effectively degraded MLKL and completely abrogated cell death in a TSZ model of necroptosis. By leveraging the innate ability of these PROTACs to degrade MLKL in a dose-dependent manner, the quantitative relationship between MLKL levels and necroptosis was interrogated. This work demonstrates the feasibility of targeting MLKL using a PROTAC approach and provides a powerful tool to further our understanding of the role of MLKL within the necroptotic pathway.