Development of a fluorescence-based assay for screening of urate transporter 1 inhibitors using 6-carboxyfluorescein

The urate transporter 1 (URAT1) inhibitors were considered a very promising class of uricosuric agents for the treatment of hyperuricemia and gout. In vitro activity testing of these compounds has been conducted by radio-labeling uric acid for a long time. However, relatively few offer the convenience and speed of fluorescence-based assays. Herein, we report the development of a non-radioactive cell-based method for the screening of URAT1 inhibitors using the human embryonic kidney 293T cells stably expressing human URAT1, and 6-carboxyfluorescein (6-CFL) as a substrate. The URAT1-mediated transport of 6-CFL was time dependent and saturable (Km = 239.5 μM, Vmax = 6.2 pmol/well/min, respectively). Molecules known to interact with organic anion transporters, including benzbromarone, probenecid, and lesinurad, demonstrated concentration-dependent inhibition of 6-CFL transport by URAT1. Moreover, we screened a small subset of compounds, and identified compound 4 as a promising URAT1 inhibitor. This in vitro assay may be employed to screen for novel URAT1 inhibitors, which are effective against hyperuricemia.