Proteomics-Based Studies Identify the Metallothionein-2A as a Potential Target of Thioxo-Tetrahydropyrimidine Derivatives in Hepatocellular Carcinoma

Novel tetrahydropyrimido-pyrimidine diones (THPP) (compounds 2d, 3d, 4d, and 19) and dihydropyrimidinones (DHPM) (compounds 2b, 4b, and 5b) have been synthesized and tested for their antioxidative capacities by assays based on their radical scavenging properties with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, respectively. Reducing properties were determined using a Ferric Reducing Antioxidant Power assay (FRAP). Most of the THPPs exhibited more potent antioxidant effects compared to DHPMs. Anticancer activities were measured against brain (SW-1088, U87 MG), lung (A-549, H460), breast (MCF7, MDA-MB-231), liver (HepG2, HuH-7), and colon cancer (HCT 116, HT-29) cell lines. Among these, THPP derivatives (3d and 4d) have shown the most prominent anticancer activity in all tested cancer cell lines, with the highest potency observed in hepatocellular carcinoma cell lines HepG2 (IC₅₀ = 24.32 ± 1.38 and 27.69 ± 4.93 μM, respectively) and HuH-7 (IC₅₀ = 9.77 ± 0.18 and 8.35 ± 0.28 μM, respectively). Apoptosis-inducing effects of compounds 3d and 4d were confirmed through Hoechst 33342/propidium iodide (PI) staining, Annexin V/PI flow cytometry, and Western blot analysis of cleaved PARP. Proteomic profiling and pathway enrichment analyses further suggested that both compounds 3d and 4d induced apoptosis, disrupted cell cycle checkpoints, and impaired DNA damage response pathways. Notably, Metallothionein-2A (MT-2A), a zinc-binding protein involved in stress response and cancer progression, was identified as a commonly downregulated target of these compounds. Molecular docking further suggested that 3d and 4d interact with key cysteine residues within the Zn-(II)-binding domain of MT-2A, potentially impairing its function. In silico ADMET predictions indicated that both 3d and 4d possess favorable pharmacokinetic properties and acceptable toxicity profiles, supporting their potential as drug-like compounds. Collectively, these findings support compounds 3d and 4d as promising lead candidates for hepatocellular carcinoma therapy.