In many cases, the mechanistic claims appear to be inferred from molecular docking or single-pathway marker assays, without complementary target validation methods (e.g., knockdown, rescue experiments, or target engagement assays). This raises a major concern about the reliability of mechanistic attribution.
For instance, multiple hybrids are reported to induce apoptosis or cell cycle arrest, and these outcomes are linked to specific molecular targets. Yet in several cited works, the primary evidence comes from docking simulations or indirect protein expression changes, which are insufficient to confirm direct target inhibition. Given the structural diversity of the hybrids and the broad range of cancer cell lines tested, how do the authors distinguish between off-target cytotoxicity, general stress responses, and true mechanism-specific effects?