Background/Objectives: Advanced-stage hepatocellular carcinoma is characterized by a very poor prognosis; thus, highly effective medication is still needed. Often overexpressed heat shock protein 90 is a promising target due to its pivotal role in carcinogenesis. Methods: Antiproliferative effects of 246TMP-3SF5 on HepG2 and HuH-7 cells were assessed by crystal violet staining. Apoptosis was evaluated via subG1 peak, caspase-3 activity and PARP cleavage, and ferroptosis via ROS, glutathione and malondialdehyde levels. Migration was assessed by scratch assay, and in ovo models were used to study angiogenesis and drug effects on microtumors. CAM vascular networks were quantified by semi-automatic segmentation, morphometric and box-counting fractal analysis. Molecular docking and molecular dynamics simulation of heat shock protein 90 were carried out using Autodock Vina and Gromacs respectively. Results: Profound dose- and time-dependent antiproliferative effects of 246TMP-3SF5 against HCC cell lines were observed, revealing low micromolecular IC₅₀ values and selectivity for carcinoma cells with selectivity indices > 1. A significant increase in the sub-G1 peak, key effector caspase-3 activity, as well as cleavage of PARP strongly suggested apoptosis playing a crucial role in the antiproliferative effects. Additionally, HuH-7 cells revealed an elevation of reactive oxygen species and both cell lines showed significant glutathione depletion concomitant with an increase in malondialdehyde concentration upon treatment. The observed effect could be partially reversed by applying ferrostatin-1, suggesting ferroptosis as an additional relevant mode of action. Changes in the cell cycle as well as impaired tumor cell migration were observed. Upon treatment, angiogenesis was impaired and mass of microtumors was significantly reduced. Quantitative CAM analysis showed that vascularized area fraction increased in controls but decreased under both 17-AAG and 246TMP-3SF5. Likewise global mean vessel width decreased relative to controls, while box-counting dimension was reduced under 246TMP-3SF5. Molecular docking and molecular dynamics simulation analysis predicts 246TMP-3SF5 to be binding in catalytic site of heat shock protein 90. Conclusions: 246TMP-3SF5 is a promising novel inhibitor meriting further research as a potential treatment against hepatocellular carcinoma.

Background/Objectives: Advanced-stage hepatocellular carcinoma is characterized by a very poor prognosis; thus, highly effective medication is still needed. Often overexpressed heat shock protein 90 is a promising target due to its pivotal role in carcinogenesis. Methods: Antiproliferative effects of novel synthesized inhibitor 246TMP-3SF5 on HepG2 and HuH-7 were analyzed through crystal violet staining. Apoptosis was assessed by measurement of subG1 peak, caspase-3 activity and cleavage of PARP. Ferroptosis was evaluated through reactive oxygen species, glutathione and malondialdehyde levels. Scratch assays were used to assess cancer cell migration and in-ovo models to analyze effects of 246TMP-3SF5 on angiogenesis and microtumors. Molecular docking and molecular dynamics simulation into heat shock protein 90 were carried out using Autodock Vina and Gromacs respectively. Results: Profound dose- and time-dependent antiproliferative effects of 246TMP-3SF5 against HCC cell lines were observed, revealing low micromolecular IC50 values. A significant increase in subG1 peak, key effector caspase-3 activity as well as cleavage of PARP strongly suggested apoptosis playing a crucial role in the antiproliferative effects. Additionally, HuH-7 cells revealed an elevation of reactive oxygen species and both cell lines showed significant glutathione depletion concomitant with malondialdehyde concentration increased upon treatment. The observed effect could be partially reversed applying ferrostatin-1, suggesting ferroptosis as an additionally relevant mode of action. Changes in the cell cycle as well as impaired tumor cell migration were observed. Upon treatment angiogenesis was impaired and mass of microtumors was significantly reduced. Molecular docking revealed interaction with catalytic site of heat shock protein 90. Conclusions: 246TMP-3SF5 is a promising novel inhibitor meriting further research as potential treatment against hepatocellular carcinoma.