Efflux phenomena, caused by transport proteins, are often responsible for the poor bioavailability of natural products, which lead to difficulties in their clinical application . Adenosine triphosphate binding cassette (ABC) transporters constitute a large family of proteins involved in membrane transport of a wide variety of substrates 2 . Among the 48 human ABC-transporters identified, ABCB1, also called MDR-1 or Pglycoprotein (P-gp), is the best characterized for its involvement in the drug discovery process . The physiological role of P-gp is to protect sensitive tissues from the accumulation of toxic xenobiotics but also mediates multidrug resistance in cancer cells . As P-gp is expressed in membrane barriers, such as the intestine, it can affect the bioavailability of orally administered drugs , including natural products . Thus, the discovery of effective P-gp inhibitors/modulators has been the focus of many research studies, to the extent that the European Medicines Agency and the USA Food and Drug Administration have proposed their own guidelines on how to conduct this kind of research . Three generations of P-gp inhibitors have been developed so far, but, unfortunately, none of them was effective in clinical facing). Moreover, they have been resolved in the presence of compounds of clinical use (such as taxol and zosuquidar), providing useful insights for structure-based drug discovery. Indeed, in 2018, Kim and Chen reported the molecular structure of human P-gp in the ATP-bound, outward-facing conformation . ATP binding causes the reorientation of the drug-binding cavity, leading to the release of the substrate into the extracellular space. Right after this publication, Alam and co-workers described the UIC2-bound structure of human P-gp with the ligand-free or zosuquidar-bound binding cavity . Remarkably, this was the first paper reporting the tridimensional structure of P-gp bound to a drug of current clinical use, zosuquidar, a third generation P-gp inhibitor, which reveals an intermediate conformation in which the gap between the two NBD is partially closed. In 2019, the same research group used lipidic nanodiscs to reconstitute a substrate-bound (with the chemotherapeutic agent taxol) and an inhibitor-bound (with zosuquidar) P-gp intermediate conformations . This work suggested that only minor structural differences occurred in the binding of substrate and inhibitor to the same P-gp site, while structural differences were amplified along the structure, leading to altered conformations of the NBD, which are responsible for ATP hydrolysis and substrate release. Moreover, it confirmed the ability of P-gp to accommodate two molecules of zosuquidar within the same drug-binding pocket, as reported in their previous publication. Additionally, the analysis of the ligand binding pocket of the taxol-and zosuquidar-bound structures confirmed that most of the ligand-interacting residues were aromatic and hydrophobic, with only one hydrogen bond found between the quinoline moiety of zosuquidar and Tyr953, and four hydrogen bonds between taxol and Tyr307, Tyr310, Gln725 and Gln991. Eventually, cryo-EM has been used to determine the structure of the vincristine-, elacridar-, and tariquidar-bound human P-gp intermediate conformation . More recently, the structure of human P-gp in complex with encequidar, a novel potent dual P-gp and CYP3A4 inhibitor, was also reported . All the structures confirmed that inhibitors bound in pairs, except for the substrate vincristine that was in a 1:1 stoichiometry with P-gp, in agreement with that found for the other substrate taxol. The authors suggested that the peculiar binding mechanism of the inhibitors could explain why these molecules can act as substrates at low concentrations by binding the central pocket, and as inhibitor at higher concentration when a second molecule engages also the access tunnel. Interestingly, hydrogen bonds with Tyr310 and Tyr953 were found for 3 out of 4 inhibitors, but they were not presents in the substrates-bound structures. The aim of this work was to set up an in silico model for the VS of curated natural compounds libraries from the ZINC database, to be used for the identification of hit compounds with potential P-gp inhibitory activity. In vitro assays on selected compounds provide full validation of the VS approach.