The piperine and metabolite structures were optimized by ab initio calculation. The calculations were performed using the Gamess2018 quantum mechanics package with Hartree-Fock (HF) formalism and functional density theory (DFT) following the same method of our previous work. The protein structure was obtained from PDB 1TQN and prepared following the same method of our previous work. . Autodock tools 1.5.4 were used to prepare the protein, adding polar hydrogen bonds and Gasteiger charges. The Grid box was built to explore the whole protein (blind docking) with the grid box dimensions as 126×126×126 points with a spacing of 0.458 A˚ and centered at x = -19.213, y =-23.825, and z = -14.03. The protein binding sites were investigated with autodock4.2 using the Lamarckian Genetic Algorithm (LGA) in a total of 100 different conformations. The final poses were selected among the most negative energies.The affinity energy function obtained from our docking of piperine (-8.73 kcal/mol) is very close to the that reported in the literature (-8.55 kcal/mol), which gives us added confidence of the predicted pose of piperine within the active pocket of CYP3A4 (Figure and). The intermolecular distances between the clustered (see SI) low-energy binding pose of piperine and the N and Fe atoms of the heme group in CYP3A4 (distances are reported in Å in Figure ). This model is indicative that the benzo[d] dioxole motif is more likely to engage with the heme group and undergo an oxidation event in the body. For instance, metabolites e.g M8, M12, M18, M3 as detected in a hepatocyte incubation which evaluates both phase I (oxidative) and phase II (conjugation) events. M5-2 and M6 from our work demonstrates an oxidative event in this fragment of piperine. We next considered whether the metabolites identified from our electrochemical screen (Scheme 1) would be feasible to exist in the CYP active pocket, post metabolic oxidation. To achieve this we explored the clustered binding patterns of our piperine metabolites compare to piperine itself within the cytochrome (Figure ). The findings from molecular docking, reveal that metabolites M2 and M3 dock precisely in the same region as piperine with an increase in energy score for M3 and an decrease of energy scores for M2 (as shown in Table ). This suggests that upon metabolism, the M2 metabolite exhibit diminished affinity for the protein. One of the highly specific interactions observed is the π-π followed by π-cation interaction, owing to its high level of molecular organization. These interactions are recognized as highly specific molecular recognition interactions in protein-ligand complex, often considered as complex identity interactions. Analyzing the results of molecular docking (Figure ), both metabolites M2 and M3 exhibit π-π interactions with the heme group of the protein. It is interesting note that M3 presented an increasing of binding affinity compared to piperine while M2 presented a reduced affinity for the protein compared to piperine. These results strongly suggest that these piperine derivatives might act as protein inhibitors post-metabolism, unless the decreased affinity caused M2 to vacate the binding site because of competition with other biological molecules. Moreover, Besides π-π interaction, the amino acids performed non specific interactions with piperine, M2 and M3 with no hydrogen bonds presented. Despite M6 not binding directly at the piperine site, it still engages in a π-cation interaction with the iron of the heme group and forms a hydrogen bond between the hydroxyl group of M6 and the heme group (2.9 Å). While M6 binds to the heme group, it's noteworthy that the π-cation interaction is less specific than π-π interactions, and the metabolite does not bind to the piperine interaction site. This suggests that this metabolite may not have the potential to inhibit the protein. Metabolites M5-1 and M5-2 do not interact with the heme group in the two lowest energy poses. The only specific interaction observed is a hydrogen bond formed by metabolite M5-2 with R212 (2.82 Å), the others interactions are non-specific. Besides that these three metabolites presented lower affinity to protein compared to piperine. This suggest that upon metabolism, the metabolites might leave the protein when in contact with others biological components.