In silico discovery and experimental validation of natural derivatives as inhibitor of phosphoinositide Kinase, PIKfyve

Phosphoinositide kinase PIKfyve is a key regulator of endosomal trafficking and lysosomal function and is increasingly recognized as a therapeutic target in Cancer, neurodegeneration, and viral infections. However, the discovery of selective and safe inhibitors remains limited. Here, we integrated computational screening with biochemical validation to identify natural products (NPs) as potential PIKfyve inhibitors. From a library of over 3500 NPs, trilobatin-a dihydrochalcone glycoside-emerged as the most promising hit. It exhibited strong binding affinity in docking and MM-GBSA analyses, favourable pharmacokinetic and drug-likeness profiles, and acceptable safety indices. Molecular dynamics simulations (100 ns) confirmed the stability of its interactions with key residues in the active site, supported by persistent hydrogen bonding and robust electrostatic and hydrophobic contributions. Experimental kinase inhibition assays validated these findings, revealing that trilobatin inhibits PIKfyve activity in a dose-dependent manner with an IC₅₀ of ~ 0.29 µM. These results establish trilobatin as a potent non-morpholine scaffold distinct from conventional morpholine-based PIKfyve inhibitors, thereby expanding the chemical diversity available for lipid kinase targeting. Importantly, this study demonstrates the value of integrating in silico screening, ADMET prediction, and biochemical validation to accelerate early-phase drug discovery. Trilobatin thus represents a promising lead for further structure-activity optimization, cellular assays, and in vivo evaluation. Beyond its immediate relevance, this work underscores the potential of NPs with favourable pharmacological profiles as sources of novel scaffolds for selective kinase inhibition and highlights trilobatin as a compelling candidate for therapeutic development across PIKfyve associated pathologies.