Discovery of novel RIPK3 inhibitors by virtual screening and bioactivity evaluation.

As the most common chronic neurodegenerative disease, Alzheimer's disease (AD) has limited effective treatment options, highlighting the critical urgency of developing new therapeutic drugs. Receptor-interacting protein kinase 3 (RIPK3) is a key regulatory protein in the process of cell necroptosis and is also a potential target for AD. Existing inhibitors are not suitable for clinical treatment of AD due to problems such as insufficient activity, high toxicity, and poor selectivity. Therefore, the discovery of novel RIPK3 inhibitors is necessary and urgent. This study proposes and applies an integrated virtual screening strategy based on molecular fingerprint similarity, pharmacophore models, molecular docking, and molecular dynamics (MD) simulation. Using this workflow, we selected 54 compounds from approximately 9.7 million compounds for in vitro biological validation. In vitro kinase activity assays showed that three compounds (8019 - 0682, STK930700, and T72029) exhibited good inhibitory activity against RIPK3, with IC50 values of 1.684 µM, 3.002 µM, and 7.592 µM. Among them, 8019 - 0682 and T72029 demonstrated good cytoprotective effects in the TNF-α-induced L929 cell necroptosis model, with EC50 values of 2.135 µM and 15.891 µM. Finally, MD simulations and binding free energy calculations for the three compounds revealed a crucial hydrogen bond interaction with the M97 residue in the RIPK3 hinge region. In summary, this study proposes a combined virtual screening strategy and offers three promising lead compounds targeting RIPK3 for AD treatment.