Target deconvolution of a selective HepG2 cytotoxic hit identifies GSTP1 and TRAP1 as candidate molecular targets

Hepatocellular carcinoma (HCC) remains a lethal malignancy with limited therapeutic options in advanced disease, highlighting the need to identify selective cytotoxic agents and biologically relevant molecular targets for early anticancer drug discovery. In this study, a focused in-house compound set was screened against HepG2, MCF-7, and MDA-MB-231 cancer cell lines using the MTT assay to identify compounds with selective activity toward HCC-derived cells. The most active hit, HTS00019, was then subjected to an integrated computational target-deconvolution workflow comprising SEA target prediction, reverse docking against cancer-relevant proteins, binding-mode analysis, 100 ns molecular dynamics simulations, MM-GBSA binding free energy estimation, and in silico toxicological profiling using ProTox-3.0. HTS00019 showed potent and selective cytotoxicity toward HepG2 cells, with an IC₅₀ value of 1.70 ± 0.22 µM and approximately 50-fold selectivity over MCF-7 and MDA-MB-231 cells. SEA and reverse docking analyses prioritized GST-family proteins and TRAP1 as likely candidate targets. Docking suggested favorable binding within the GSTP1 H-site and the ATP-associated region of TRAP1, while molecular dynamics simulations supported stable accommodation of the compound in both proteins, with stronger ligand positional stability in TRAP1. MM-GBSA analysis further favored TRAP1 binding, with a calculated ΔG _bind of -75.49 kcal mol^-1 compared with -59.98 kcal mol^-1 for GSTP1. Interaction analysis indicated hydrophobic and ionic contacts in GSTP1, whereas halogen bonding, π-cation, and hydrophobic interactions contributed to the predicted TRAP1 binding mode. In silico toxicological profiling classified HTS00019 as oral toxicity class 4 and flagged potential hepatotoxicity, mutagenicity, immunotoxicity, and carcinogenicity liabilities, while p53- and ATAD5-related stress pathway predictions were inactive. Overall, HTS00019 was identified as a selective HepG2 cytotoxic hit, with GSTP1 and TRAP1 emerging as computationally prioritized candidate targets. However, the absence of biochemical target validation and the predicted toxicological liabilities indicate that HTS00019 remains an early-stage hit requiring mechanistic confirmation and structural optimization.