Fluorescent 4-Nitrobenzo-2-oxa-1,3-diazole-Coupled Bile Acids as Probe Substrates of Hepatic and Intestinal Bile Acid Transporters of the Solute Carrier Families SLC10 and SLCO

Several bile acid (BA) transporters are involved in the enterohepatic BA circulation between the liver and gut, including the hepatic Na⁺/taurocholate cotransporting polypeptide (NTCP) and the intestinal apical sodium-dependent BA transporter (ASBT). Fluorescent BA derivatives are helpful to measure and visualize BA transport <i>in vitro</i> and <i>in vivo</i>. We used 4-nitrobenzo-2-oxa-1,3-diazole (NBD) as the labeling fluorophore and synthesized a series of 3-NBD-coupled BA. While 3α-NBD-taurocholic acid, 3β-NBD-taurocholic acid, 3α-NBD-glycocholic acid, and 3β-NBD-glycocholic acid showed significant transport rates for human NTCP, mouse mNtcp, and mouse mAsbt, human ASBT only showed reliable transport activity for 3α-NBD-glycocholic acid. In general, NBD coupling to the 3α-position proved superior to the 3β-position, and the NBD-BA with glycine conjugation exhibited the highest overall transport rates. None of the synthesized NBD-BA was transported by the organic anion transporting polypeptides OATP1B1 and OATP1B3. Overall, 3α-NBD-glycocholic acid is most appropriate for fluorescence-based transport assays to evaluate NTCP and ASBT inhibitors.