Development of a fluorescence-based assay for screening of urate transporter 1 inhibitors (II): Optimization of fluorescent substrates and structure-activity relationships analysis

Urate transporter 1 (URAT1) inhibitors represent a promising therapeutic approach for hyperuricemia and gout. A non-radioactive cell-based platform for screening URAT1 inhibitors using 6-carboxyfluorescein (6-CFL) was previously established by our team. In this study, aiming to optimize our testing platform, 10 potential fluorescent substrates were screened from the commercially available fluoresceins using molecular docking and pKa prediction. Among them, dibromofluorescein (DBF) and diiodofluorescein (DIF) were identified as specific transport substrates for UTAR1 with significant fluorescence changes in cellular uptake. DBF and DIF exhibited Km values similar to that of 6-CFL. Using DBF or DIF as substrates, the IC₅₀ values of the URAT1 inhibitors benzbromarone and lesinurad remained within the same order of magnitude as those obtained by 6-CFL. Notably, DBF enables visual detection, further extending the utility of this non-radioactive platform for in vitro screening of URAT1 inhibitors. Molecular dynamics (MD) simulations and free energy calculations were employed to compare the interactions and binding affinities of 6-CFL, DBF, and DIF with URAT1, respectively. Finally, structure-activity relationships (SARs) analysis was conducted to preliminarily identify substituents modulating the binding affinity of commercially available fluoresceins.