Unveiling Novel Molecular Candidate Targets of Loratadine, Raloxifene, and Sorafenib in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains a major global health burden. We have recently demonstrated the synergistic cytotoxicity elicited by the combination of loratadine, raloxifene, and sorafenib in liver cancer cells. In this study, we investigated the potential mechanistic basis of this novel multi-drug regimen by identifying candidate protein targets of the three drugs. Potential protein targets of each drug were identified using the Drug Affinity Responsive Target Stability (DARTS) assay in protein extracts from the HCC cell line HuH7, and molecular docking was performed to predict binding modes and explore the structural feasibility of these drug–target interactions. Bioinformatics analyses were conducted to predict biological functions, molecular interactions, and clinical relevance. We identified 147 potential candidate DARTS-stabilized proteins for loratadine, 191 for sorafenib, and 157 for raloxifene; a proportion of these candidate proteins maintained a statistically significant association with overall survival in HCC patients: 20.14% for loratadine, 22.93% for raloxifene, and 24.47% for sorafenib. Cross-referencing with public databases confirmed several previously documented targets—complementing our experimental approach—while revealing numerous previously unreported target candidates. Docking analyses predicted favorable binding interactions between the drugs and several proteins, such as the putative complex between loratadine and N-glycanase 1 (NGLY1), whose expression correlates with reduced overall survival in HCC patients. Functional enrichment analysis showed that several targets are involved in hallmarks of cancer. Clinically relevant candidate targets of loratadine, raloxifene, and sorafenib were identified. However, as these candidate protein targets were identified exclusively in HuH7 cells, they may not fully generalize across all HCC models without further validation. Nevertheless, our results suggest that loratadine, raloxifene and sorafenib modulate key pathways involved in tumorigenesis. These findings strengthen the use of this combination as a promising therapeutic strategy for HCC and provide new opportunities for the identification of novel targets, prognostic markers, and molecular vulnerabilities suitable for pharmacological intervention in HCC and other malignancies and diseases.