We used a combination of cell-based and cell-free assays to evaluate in a comprehensive manner the in vitro inhibition of P-gp activity of the selected compounds. First, we performed a cytotoxicity assay using cytotoxic P-gp substrates colchicine and vincristine . Fig. shows the ability of compounds 4, 5, 6, 13, 14, and 18 to reduce the cell viability of colchicine-and vincristine-stimulated Caco-2 cells, compared to colchicine or vincristine alone (normalized as 100%). Results from nearly all compounds show a similar reduction of cell viability in the range of 30-40 %, compatible with an increased intracellular accumulation of the cytotoxic drugs due to the inhibition of their efflux transport. Consistent results were obtained in the subsequent intracellular calcein accumulation assay (Fig. ), with compounds 4, 5, 6, 13, 14, and 18 showing a significant increase of calcein-mediated intracellular fluorescence intensity compared to the untreated control (normalized as 1). Compound 27 also showed a small but statistically significant increase in fluorescence intensity not observed in the cytotoxicity assay. The calcein assay was validated using zosuquidar as a positive control (Fig. ). To evaluate the direct interaction with P-gp, a cell-free human recombinant P-gp-containing membrane-based ATPase activity assay was also performed to determine the IC50 value for each selected compound (Table ). Compounds 2, 20, 23, 27, and 29 were found to be inactive (IC50 > 50 µM), compound 17 had an IC50 value of about 20 µM, and compounds 4, 5, 6, 8, 13, 14, and 18 showed IC50 values lower than 10 µM (Table ). Interestingly, 8 out of the top 10 compounds ranked by consensus scoring were found to be active, with compound 2 being the only exception. These results strongly correlate with our in silico predictions, suggesting that the combination of the initial enrichment factor obtained with Vina and the subsequent rescoring with the 3D common features pharmacophoric models was successful in identifying P-gp inhibitors and reduce the number of false positives from the VS. Compound 6 resulted to be the most active P-gp inhibitor, with an IC50 of 1.28 ± 0.11 µM, and, together with 2, 4, 13, and 14, was a derivative of the natural anti-fungal agent griseofulvin. Interestingly, the possible P-gp binding activity of griseofulvin has been speculated, even if more experimental support is required to delineate this hypothesis more concisely . Nevertheless, to the best of our knowledge, none of the derivatives identified in this work were already known for their P-gp inhibitory activity. Compound 6 (Fig. ) overlaps with the position 2 of the zosuquidar binding site (Fig. ), except for the 4-hydroxy-3methoxyphenyl moiety, which interacts with residues involved in the binding of the DTP moiety of zosuquidar in position 1. Consistently with the lipophilic nature of the P-gp binding site, most of the interactions were represented by van der Waals contacts with hydrophobic residues. Three important anchor points were represented by the hydrogen bonds between: a) the benzofurane moiety and Tyr310; b) the carbonyl group on the propanamide moiety and Tyr307; and c) the hydroxy group of the 4-hydroxy-3-methoxyphenyl moiety with the Trp232. A very similar interaction pattern was observed for the other griseofulvin derivatives, with the only exception of 13 that did not establish hydrogen bonds with Trp232, while its nitro group establishes two weak hydrogen bonds with the amino group of Asn842 and the backbone carbonyl of Asn721 (Fig. ). Although similar binding poses were observed for the griseofulvin derivatives, the presence of the oxindole moiety in 2 significantly impaired its activity in vitro. Among flavonoid derivatives (5, 17, and 29), 5 is the only one with an interesting in vitro activity, while 17 showed very weak activity, and 29 was inactive. Interestingly, a weak P-gp inhibitory activity of 17 (scutellarin) has been previously reported , thus supporting our data. Compound 5 showed a preference for position 2 of the zosuquidar binding site, except for the 3,4dimethoxyphenyl moiety that occupied the portion of the binding site where the DTP moiety of zosuquidar in 1 was bound. Hydrogen bonds with key residues Tyr307, Trp232, and Asn842 were among the interactions of 5 (Fig. ). Interestingly, 17 lacked the polar interaction with Tyr307 found in the majority of inhibitor-bound structures, which might correlate with the reduced activity (Fig. ). Compound 8, a nitrotyrosinate steroid derivative, also showed preference for position 2 of the zosuquidar binding site, establishing hydrogen bond with Tyr307 and Tyr310, with the nitrophenol group reaching the portion of the binding site occupied by the DTP moiety of zosuquidar in position 1 and engaging Trp232 in a hydrogen bond (Fig. ). Differently, compound 18 assumed a mixed pose, with the methoxyphenyl and the carboxyl moieties overlapping the DTP moiety of zosuquidar in position 1, and the rest of the molecules overlapping position 2 of zosuquidar. Two important anchor points in the predicted binding mode were the hydrogen bonds between the carbonyl group in the pyranyl moiety and Tyr307, and the methoxy-oxopropyl moiety and Asn842 (Fig. ).