# Experimental and Simulation Identification of Xanthohumol as an Inhibitor and Substrate of ABCB1
## 4 Discussion  
XN is a bioactive preynlated flavonoid compound uniquely distributed in the female infloresesences of _Humulus lupulus_ L., a well-known bittering agent in the brewing industry. In recent years, XN has received much attention for its biological effects, especially in the anticancer area. A large amount of research has shown that XN can prevent and treat cancers. The related mechanisms underlying this anticancer activity have been revealed, including chemopretive activity by inhibition of the initiation and development of carcinogenesis, and therapeutic activity by proliferation inhibition, apoptosis induction, migration inhibition, as well as angiogenesis inhibition [4].  
Most recently, studies have shown that in MCF-7/ADR cells XN could reverse DOX resistance. The underlying mechanisms of this are related to the signal transduction and activation of transcription 3 (STAT3) and epidermal growth factor receptor (EGFR) suppression [7], as well as MCF-7/ADR cell stemness reduction [8]. ABCB1 plays a vital role in drug resistance by stimulating drug efflux. In this study, we investigated, for the first time, whether XN could modulate ABCB1 efflux functions. Our present results showed that XN decreased ABCB1 transporting functions, as evidenced by the increased intracellular accumulation of ABCB1 substrates, DOX and Rho123. Subsequent molecular experiments showed that XN was a substrate of ABCB1, which suggests that XN mediated-reversal of DOX resistance might be related to ABCB1 activity modulation. The finding that XN was a substrate and inhibitor of ABCB1, together with the previous finding that XN was also an inhibitor and substrate of transporter ABCG2 [9], strongly supports its efficiency in the regulation of the efflux of chemical drugs and exogenous compounds via affecting ABC transporters and its potential use for the reversal of anticancer drug resistance. Since ABCB1 is also expressed in the intestine, inhibition of ABCB1 transport function might enhance the intestinal absorption of drugs.  
Figure 7: Ligand Root Mean Square Fluctuation (RMSF). (**A**) DOX atom reference; (**B**) XN atom reference; (**C**) Ligand RMSF is used to characterize changes in the ligand atom positions. The graph shows the ligand’s fluctuations broken down by atom, corresponding to the 2D structure. Ligand RMSF was measured on the ligand heavy atoms after first being aligned on the protein backbone. The average RMSF for DOX ligand was 2.58 Å. The average RMSF for the XN ligand was 1.85 Å.  
Results of molecular modeling and simulation showed the detailed interactions between the ligands (XN and DOX) and ABCB1 and explained the molecular basis for the competitive inhibition of DOX efflux by XN. XN bound to ABCB1 at the central TMD site, which overlapped with the DOX binding site. In addition, XN binding affinity was stronger than that of DOX, resulting in less protein and ligand position fluctuation. These results support that XN competitively blocked DOX binding and efflux by ABCB1. However, further studies, including mutagenesis studies, are needed to experimentally confirm the XN-ABCB1 interactions predicted by molecular docking and simulation.