Studies Of The Interactions Between Natural Products And OATP1B1 And The Effect Of N-glycosylation On The Function And Expression Of OATP2B1

Organic anion transporting polypeptide 1B1(OATP1B1) is an important liver-specific uptake transporter, mediating transport of numerous endogenoussubstances and drugs from blood into hepatocytes. To identify and investigate the potential modulators of OATP1B1 from natural products, the effect of 21 frequently used natural compounds and extracts on OATP1B1-mediated fluorescein methotrexate transport was studied by using Chinese hamster ovary(CHO) cells stably expressing OATP1B1 in 96-well plates. This method can be used for the screening of large compound libraries. Our results showed that some flavonoids(e.g. quercetin, quercitrin, rutin, chrysanthemum flavonoids and mulberrin) and triterpenoids(e.g. glycyrrhetinic acid and glycyrrhizic acid) were inhibitors of OATP1B1 with IC50 values less than 16 μmol·L-1. Flavonoids showed much higher inhibition activity than their glycosides. Furthermore, the type and length of saccharides had a significant effect on their activity. In order to investigate the effect of these compounds on OATP1B1-mediated transport of other substrates, we established the LC-MS/MS methods for the detection of OATP1B1 drug substrates fluvastatin and rosuvastatin. Our results indicated that the effect of these natural products on the function of OATP1B1 was substrate-dependent. This study will be conducive to predicting and avoiding potential OATP1B1-mediated drug-drug and drug-food interactions and thus provide the experimental basis and guidance for clinical rational drug use.Organic anion transporting polypeptide 2B1(OATP2B1) which expresses in the placenta, liver, mammary gland, brain, and intestine, mediates transport of bromosulfophthalein, estrone-3-sulfate and dehydroepiandroserone-3-sulfate.Glycosylation is an important type of post-translational modifications. Putative N-glycosylation sites of OATPs locate in the extracellular loops 2 and 5, which is a common feature of all OATPs.To study how N-glycosylaton affects the transport activity and expression of OATP2B1, first of all four putative sites for N-glycosylation werepredicted by computational analysis. By substituting asparagine(N) with glutamine(Q),four single mutants(N146Q, N176 Q, N350 Q and N538Q) and a double mutant(N176/538Q)of OATP2B1 were constructed.These mutants were transiently expressed in HEK293 cells.Our results showed that Asn176 and Asn538 were glycosylation sites. When these two sites were mutated individually, the protein levels were significantly reduced and transport activities were decreased; when two sites were mutated at the same time, the protein level was significantly reduced as well as the loss of transport function.These results indicated that N-glycosylation is very important for the expression and function of OATP2B1.