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Piperine is an alkaloid present in black pepper, a commonly used spice. We have tested its cytoxicity/cell viability in MDCK cell line which expresses high number of P-gp. Piperine treated MDCK cells survive for longer time than piperine untreated cells i.e. this molecule has survival advantage when cells are deprived of nutrition and under stress (Figure-2). We have also tested effect of piperine in MCF-7 a breast cancer cell line and U87 and U373 brain tumor cell line where it has not shown any undesired effect upto 100ÂµM.(data has not shown). Han Y et al has also shown in Caco-2 cell line that piperine is non toxic upto 10-100 ÂµM and increase bioavailability of digioxin in vitro and invivo  Han.
Figure-2: Survival benefit of piperine over MDCK cell line. Untreated cells have shown decrease in percent cell viability after 90 h while piperine exposed cells have not shown cyotoxic effect but also mange to survive for longer time.
Piperine is also known as a molecule which enhances bioavailability of curcumin, the yellow pigment of turmeric, rifampin, phenytoin, propanol and several other which enhance P-gp's substrate bioavailability by unknown mechanism [1-5]. To quantify this effect of piperine cells were treated with rhodamine and monitored by FACS. At higher dose of piperine, rhodamine accumulation increased by two fold (figure-3a & 3b). This confirms that piperine is a modulator of P-gp. Further analyzing drug accumulations qualitatively on treatment with piperine, the cell were analyzed by fluorescence microscopy (figure 4a & 4b). Rhodamine accumulation in mitochondria and cytosol was to found to be higher comparable to control at 100 ÂµM concentration of piperine.
Figure-3:Quantativae accumulation of rhodamine on treatment of piperine measured by FACS.(a) Bar diagram showing increased accumulation of rhodamine on treatment of 20 -140Âµg of piperine. (b) Overlay picture of FACS at 120-140 Âµg of piperine treatment.
Figure-4: Rhodamine accumulations on treatment of piperine (a) cells were treated only with rhodamine and (b) rhodamine and piperine. Rhodamine accumulation is clearly observed in the surrounding of nuclear membrane as well as in cytosol. All pictures were taken at 40X on inverted microscope.
3.3.2 Simulation for binding mode of piperine
Wet experiments suggest that piperine is non cytotoxic and causes accumulation of fluorescent dye rhodamine. These findings and published reports indicate that piperine might be binding in close vicinity to ATP binding site as steroid and flavonides bind . In order to find out binding mode of piperine and how it accommodates diverse sets of modulators/inhibitors, its cavity volume and binding mode of ADP, ATP, and AMP-PNP were studied. Cavity of NBD simulation has shown its volume is 628Å (Fig-5a). ATP binds in the middle of the cavity and left huge space in both side of the ATP/ADP. To find out binding mode of piperine it was docked to the NBD domain of P-gp. Binding mode of piperine and residues lie in close to 5Å was compared with ADP, ATP, AMP-PNP (ATP Analogs) complexes with P-gp protein. As a result it has shown similar mode of binding it might be due to analogy with purine ring of ATP/ADP (Fig-5b).
Fig. 5 (a) Co-crystallized ADP (PDB ID-2hyd) in the cavity (in green shade) of the NBD and the residues around 6Å of ADP showing interactions. (b) Docked conformation of piperine showing the alignment with co-crystallized ADP: The symbols R, A and D denotes aromatic and H-bond acceptor and H-bond donar (R24 -1.73, N22-1.26, A6-1.21, D14 -1.21, R23 -0.66, A13 -0.17, A8 -0.38)
3.3.3 Docking study of known modulator
Docking simulation was carried out using Glide module of Schrodinger ligand and receptors were prepared as described in methods section. All these modulators docked into the cavity of NBD except high molecular weight macrocyclic ligands (Clarthromycin, Sirolimus, Tacrolimus Valinomycin and Erthromycin). Midazolam is only ligand which docked but unable scored high at the region where purine ring of ATP/and ADP get interact. Rest of the modulators has shown high affinity at the site of binding of purine rnig of ATP/ADP. AMP-PNP are an analog of ATP has shown different mode of interaction. The adenine moiety has shown binding in the region where aromatic site of most of the modulators get interact. This indicates if a ligand able to bind either of the two regions of NBD domain could be a potential pg-p modulators. All the third generations of modulators have shown that they occupied both aromatic sites having spatial distance of 11.026Å (fig-6).
Fig-6: Binding mode of ATP, ADP and AMP-PNP. Showing conformation of purine moiety which could take either of two conformations having spatial distance of 11.026Å
Amino acids frequency graph were prepared for all docked poses located at periphery of 3Çº of modulator in nucleotide binding domain to know which amino acids are more frequently involve in interactions. Amino acids frequency graph (Figs. 7a & 7b) has shown that SER-381, TYR-349, GLY-377, ILE-367, GLY-379, LEU-380, GLN-422, GLU-353, SER-376 of chain-A and SER-379, LYS-477, LEU-478, LEU-463, VAL-476, GLN-482 of chain-B play significant role in binding at the nucleotide binding domain. Presence of these residues at binding site could be used as reference for docking and virtual screening for next generation of p-gp inhibitors. Significant residues in docked conformations at the nucleotide binding site indicate that molecules have effective interaction and can work as p-gp inhibitors.
Fig. 7 Represent frequency of amino acids lies in close to 3Çº of ligand at the nucleotide binding site of (a) chain-A (b) Chain-B.
Table-1: Glide dock score of 1st and 2nd generation of modulators
Glide Dock Score
Glide Dock Score
Figure8: All Docked poses are shown in this figure with energy based score of pharmacophore features. An orange ring represented by R alphabet is a most favorable regions calculated from XP descriptors of Glide module. R372 -1.80, R383 -1.26, R359 -1.26, N311 -1.24, R388 -1.23, R416 -1.21, R370 -1.01
3.3.4 Classification of the P-gp modulators: Since Pg-modulators are very diverse set of active molecules therefore we prepared hierarchical cluster based on linear finger prints and Tanimoto similarity using the program Canvas ( Canvas Schrodinger LLC, NewYork; NY) to cluster into seven classes. These clusters are Cluster_1: ADP, ATP and AMP-PNP Cluster_2: Itraconazole, ketaconazole and Chloropromazine Cluster_3: Ritonavir, Nicardpine, Terfenadine, Tetrabenzene, Tamoxifen and Bepridil Cluster_4: LY335979, Quanidine, Vinblastine and Quinine Cluster_5 Cortisol, Progestreron, Clarthromycin, Sirolimus, Tacrolimus and Erthromycin Cluster_6: XR9576, GF120918, R101933, Biricodar(VX-710), Reserpine, Aestimizole and Amiodarone Cluster_7: OC144-093and Piperine. Disulfiram and Dipridamole do not qualify to any cluster. All these clusters were docked with SP glide and refined with XP glide scoring function using energy based descriptors. Energy based pharmacophore (e-pharmacophore) shown in the figure 9(a-f). The most favorable interaction has shown by all clusters are aromatic regions R35, R42, R58, R87, R18 of cluster 2,3,4,6 and 7 of the NBD where purine ring of ATP /ADP binds. These aromatic regions R35, R42, R58, R87, R18 has shown by orange ring have spatial distance in range of of 11-13Å with negative ionizable center (N shown figure 9a).
Fig:9 e-pharmacophore of the p-gp modulators (a)Cluster_1 (b) Cluster_2 (c) Cluster_3 (d) Cluster_4 (e) Cluster_6 (f) Cluster_7
Table2: e-Pharmacophore features the modulators
Cluster_1: N62 -1.26, A16-1.21, D41,-1.21, R74 -1.21,D45 -0.96, D46 -0.73,D50 -0.70
Cluster_2: R35 -1.64, A15-1.45, R36 -1.13, R42 -0.83, R37- 0.82, A16-0.68, A15 -0.65
Cluster_3: R42 -1.98, R33-1.74,R40 -1.28, R35-1.21, R33-1.12, D13-0.70,H20 -1.32
Cluster_4: R58 -1.56, R52 -1.12, D18 -0.89, R60 -.84, A11 -70, D19 -0.70, R59 -0.44
Cluster_6: R87-1.75, R106 -1.60, H76 -1.1, R86-0.84, R103-0.81, A5-0.67, A17 -0.67
Cluster_7: R18 -1.73, R19 -.64, A2 -0.63, D6 -0.63, R15 -0.60, A3 -0.38, R17 -0.33
* :R41-1.79,R48-1.48,R49-1.41,R47-0.94, H35- 0.84, A10- 0.65 R45 -60
*Pharmacophore feature of the p-gp modulators (LY335979, GF120918, Biricodar-vx-710) gave best glide dock score
3.3.5 E-Pharmacophore model for P-gp modulators: The e-pharmacophore feature of the cluster-1 suggests that one negative and a aromatic feature with the spatial distance of 11-13 Å is needed to be a class of absolute pg-p modulators that could be an analog of ADP/ATP only. Negative feature is a center where Î³ and Î² phosphate group bind to the receptor and get hydrolyzed into the ADP for efflux of p-gp substrate. The aromatic feature (R74, R35, R42, R58, R87, R18 & R41) is a must match site; occupied all cluster groups fig-8(a,b,c,d,e&f) & fig-9(b). E-Pharmacophore feature of the modulators of highest docking score i.e LY335979 (-11.42), GF120918 (-11.37) and Biricodar-Vx-710 (-11.26) suggest that R41 and R49/R45 is a must match site with spatial distance of 11Å.
3.3.6 Ligand based pharmacophore: Phase module of Schrödinger was used to predict the ligand based pharmacophore feature of LY335979, GF120918 and Biricodar (vx-710). The best hypothesis is given by AHRR variant which matches to all three lmodulators. E-pharmacophore of these ligands also has given same feature A10H35R41R45 (Fig-10a & b).
Fig-10: (a) ligand based pharmacophore of highest docking scored modulator LY335979, GF120918 and Biricodar(vx-710)
3.3.7 Common e-pharmacophore: E- pharmacophores of all active modulators has been given in the figure-10. The scores of features R177, R155, R164, R118,R162, are -2.40, -145, -1.25, -1.77, -0.79, and -0.73 represent the all clusters. If a molecule satisfies the R177 and either R155 or R164 feature that could be a potential modulators. The R177 and R164 is also pharmacophore feature of Bircodar, a 3rd generation modulator preferred by clinician to enhance the bioavailability of several substrates. R177 and R164 is a most preferred site for purine ring of ATP/ADP, it could bind at either R177 or R164 site of NBD domain. This is also the case of other 3rd generation of inhibitors GF120918, LY335979 several other first generations of inhibitors nicardpine, amidarone, reserpine, verapamil, OC- OC144-093 and ketaconazole.
Fig-11 e-pharmacophore (f) common e-pharmacophore represents all modulators.
P-gp and cytochrome p450 (CYP3A4) have immense role in drug distribution and disposition. Many drugs despite having high potency have limited implications due to their poor bioavailability. P-gp has an immensely significant role in the bioavailability of cytotoxic hydrophobic and anticancerous drugs. Cell viability/cell cytotoxicity assay in presence of piperine in MDCK cell line has shown that piperine treated cell is more viable than untreated cells. It indicates that piperine is not toxic even at higher dose and has survival benefits in loss of growth media and in stress. Piperine treatment causes accumulation of fluorescent dye rhodamine as shown by fluorescence microscopy and fluorescent activated cell sorter. Hence piperine might be a lead molecule for development of next generation of p-gp inhibitors. Piperine can be also used as a vehicle for p-gp substrates of poor pharmacokinetic profile.
Molegro  simulation for volume of internal cavity within lipid bilayer has shown a huge cavity of (~628Å3) which can accommodate high molecular weight substrates. Loo et al also reported volume size of 600Çº which can accommodate two molecules simultaneously. Inhibitor/modulator binding site overlaps with ATP binding site [21-22]. All P-gp inhibitors were classified according to structural similarity and distance metrics of canvas module of Schrodinger. Docking simulation with glide-SP/XP module has shown ligands (Clarthromycin, Sirolimus, Tacrolimus Valinomycin and Erthromycin) do not dock i.e. these two molecules do not bind with NBD. Since these molecules having high molecular weight>1300 kDa may possibly be the binding channel of cytosolic domain of P-gp. Docking study of both generations of P-gp inhibitors close to the ATP binding site has shown that these share common residue at the binding site as indicated by the residues in the x-ray crystallographic structure. Some 1stgeneration inhibitors have show single mode of binding i.e. all the conformers bind in a single mode. It might be due to inflexibility in the molecule. E-pharmacophores have shown that ATP/ADP and piperine have similar mode of binding (R24 -1.73, N22-1.26, A6-1.21, D14 -1.21, R23 -0.66, A13 -0.17, A8 -0.38). Ligand based pharmacophore of LY335979, GF120918 and Biricodar(vx-710) and e pharmacophore have shown common features. The e- pharmacophore features and ligand based pharmacophore features of LY335979, GF120918 and Biricodar(vx-710) could be considered for the screening of database and generation of Pg-P inhibitors of next generation. The six features pharmacophore model is a most significant model drawn from the all active modulators. Conclusion drawn from these molecules can be used for design and synthesis of new lead molecules of next generation of p-gp modulator. Binding of piperine to the NBD share common residues like other P-gp inhibitors & the entire conformers bind in single fashion. These finding explains shoba et al observation why piperine enhances bioavailability of curcumin by 2000%.
Wet lab experiments suggest that piperine induce accumulation of P-gp substrates. Unlike other 1st generation inhibitors piperine does not induce secondary response. Higher docking and binding scores of second generation inhibitors in comparison to first generation inhibitors suggest that nucleotide binding domain is only site where these inhibitors bind. Higher scores of 2nd generation inhibitors are in accordance with invivo data reported in literature. Presence of common amino acids at the binding of piperine in comparison to ADP, ATP, and AMP-PNP confirm that piperine also binds in proximity to nucleotide binding domain. All these findings suggest that p-gp inhibitors bind in vicinity of ATP binding site. Binding of these inhibitors might partially or completely inhibit the binding of ATP and thereby its conversion to ADP for active efflux of its substrate. Considering the e-pharmacophore, ligand based pharmacophore and structural features of piperine and 1st generation of inhibitors which have shown better or equivalent docking scores than 2nd generation of inhibitors could be taken for the development for next generation of p-gp inhibitors. As a conclusion most potent P-gp inhibitors can be developed based on piperine as a base molecule.