Identification of Potent Modulators of Human Caseinolytic Protease P for the Treatment of Hepatocellular Carcinoma: Molecular Docking, Density Functional Theory Calculation, Molecular Dynamic Simulation Studies, and In vitro Validation

Background: Human caseinolytic protease P (HsClpP) is a mitochondrial protease playing a vital role in mitochondrial protein quality control and represents a promising therapeutic target for hepatocellular carcinoma (HCC). We aim to identify HsClpP modulators as potential therapeutics for HCC through a combination of in silico approaches and in vitro assays. Methods: Molecular docking, Prime MM-GBSA calculation, density functional theory (DFT) analysis, protein–ligand interaction studies, molecular dynamic (MD) simulations, and in vitro assays were used in this study. Results: Molecular docking and Prime MM-GBSA analyses revealed that F2018-4488 and F2752-1178 bind effectively to HsClpP, with favorable binding free energies. Moreover, DFT analysis suggested that these compounds have smaller energy gaps between frontier molecular orbitals (FMOs), implying enhanced chemical reactivity and bioactivity. Furthermore, MD simulations confirmed complex stability. Finally, the in vitro assay showed that compounds F2018-4488 and F2752-1178 inhibited the growth of HepG2 cells with IC 50 values of 15.94 ± 10.23 µM and 54.15 ± 25.25 µM, respectively. Conclusion: This study has preliminarily indicated that F2018-4488 and F2752-1178 may serve as potential modulators of HsClpP and therapeutic candidates for HCC treatment, providing a start point for future drug development.