Different structures of berberine and five other protoberberine alkaloids that affect P-glycoprotein-mediated efflux capacity

Abstract

Berberine, berberrubine, thalifendine, demethyleneberberine, jatrorrhizine, and columbamine are six natural protoberberine alkaloid (PA) compounds that display extensive pharmacological properties and share the same protoberberine molecular skeleton with only slight substitution differences. The oral delivery of most PAs is hindered by their poor bioavailability, which is largely caused by P-glycoprotein (P-gp)-mediated drug efflux. Meanwhile, P-gp undergoes large-scale conformational changes (from an inward-facing to an outward-facing state) when transporting substrates, and these changes might strongly affect the P-gp-binding specificity. To confirm whether these six compounds are substrates of P-gp, to investigate the differences in efflux capacity caused by their trivial structural differences and to reveal the key to increasing their binding affinity to P-gp, we conducted a series of in vivo, in vitro, and in silico assays. Here, we first confirmed that all six compounds were substrates of P-gp by comparing the drug concentrations in wild-type and P-gp-knockout mice in vivo. The efflux capacity (net efflux) ranked as berberrubine > berberine > columbamine ~ jatrorrhizine > thalifendine > demethyleneberberine based on in vitro transport studies in Caco-2 monolayers. Using molecular dynamics simulation and molecular docking techniques, we determined the transport pathways of the six compounds and their binding affinities to P-gp. The results suggested that at the early binding stage, different hydrophobic and electrostatic interactions collectively differentiate the binding affinities of the compounds to P-gp, whereas electrostatic interactions are the main determinant at the late release stage. In addition to hydrophobic interactions, hydrogen bonds play an important role in discriminating the binding affinities.

Keywords: P-glycoprotein; berberine; binding affinity; efflux; protoberberine alkaloid.

Scheme 1

Chemical structures BBR, BER, COL, JAT, THA, and DEM

Fig. 1

The plasma and tissue concentrations of BBR and its metabolites in WT and P-gp-KO mice. Concentrations of BBR and its metabolites in the plasma (a) and liver (b) were detected at 2 and 6 h after the oral administration of 150 mg/kg BBR (n = 5/group/time point). Data are mean ± SD. Significance was analyzed by non-paired t test. P values <0.05 were considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Transport of BBR and five other compounds in cultured Caco-2 cells. Caco-2 cell monolayers were incubated at 37 °C in HBSS, and test compounds were added to the apical or basolateral side. Four sequential samples were taken at different time points (30, 60, 90, and 120 min). The samples were withdrawn and the test compounds were determined by LC/MS/MS (n = 3). Time course for the AP-BL (a, b) and BL-AP (c, d) cumulative amounts and apparent permeability are shown. Data are mean ± SD

Fig. 3

Predicting the binding of the six compounds to P-gp through the transport pathway. The complete transition pathway between the inward-facing and outward-facing conformations of human P-gp was modeled, and the six compounds were docked into a total of 100 intermediate P-gp conformations in which the docking box center was shifted from 0 to 31.6 Å at intervals of ~0.4 Å along the Z-axis; docking scores are averaged every Z ~ 1.5 Å (b). The complete transport pathway for all six compounds from the binding site to the extracellular region along with the corresponding binding affinities to P-gp. The binding affinities (docking scores) are illustrated in a

Fig. 4

The binding modes of the six compounds interacting with P-gp at the lowest docking scores. All six compounds exhibit an inward-facing state binding mode and generally interact with P-gp through hydrophobic-driven binding modes. BBR, BER, COL, JAT, and THA binding to P-gp (a, c); DEM binding to P-gp (b, d). All residues within 4 Å of the compounds are shown in c and d

Fig. 5

The binding docking results of the six compounds to P-gp in the release stage (Z ~ 27Å). Membranes are shown as gray squares, and the binding modes of the six compounds are shown (a). Amino acids within 4 Å of the compounds are shown as yellow sticks (b). Interactions of BBR, BER, COL, JAT, THA, and DEM with P-gp (c)

Fig. 6

Hydrogen bond networks of six compounds with P-gp in the release stage. The hydroxyl group position influences the binding affinity of BER and THA (a) and of COL and JAT (b). The correlation between hydroxyl group number and binding affinities by comparing BBR with THA (c), BBR with DEM (d), and DEM with COL (e). For clarity, the hydrogen bonds of DEM are shown in d but not in e