Biochemical Assays for Evaluating Anticancer Activity and Validating Mechanisms of Action in Coordination/Organometallic Compounds (Review)

Research on metal-based coordination and organometallic compounds is flourishing due to their potential to overcome drug resistance, reduce systemic toxicity, and target diverse cellular pathways. Driven by the success of cisplatin and other Pt-based drugs, transition metal complexes such as Pt(II/IV), Ru(II/III), Au(I/III), Cu(I/II), and Pd(II) have been widely investigated for their ability to interact with biomolecular targets, including DNA, proteins, and enzymes. However, the development of effective anticancer metallodrugs requires rigorous mechanistic validation, as this field is often hindered by overinterpretation and poorly designed studies. This review emphasizes the necessity of multi-assay strategies, integrating classical cytotoxicity and apoptosis assays with advanced methods such as CETSA and TPP, to clarify mechanisms of action. By correlating assay outcomes with molecular mechanisms, including redox modulation, apoptosis, proteasome inhibition, and non-apoptotic pathways such as ferroptosis and necroptosis, researchers can design more selective and multitargeted agents. This approach aims to enhance reproducibility, prevent overinterpretation, and accelerate mechanism-based drug development.