Background/Purpose:  Gout is caused by a lack of efficient excretion of uric acid, resulting in hyperuricemia and the formation of crystal deposits of uric acid.  The renal uric acid transporter URAT1 is important for regulating serum uric acid levels, and URAT1 inhibitors reduce serum uric acid levels and are used as gout therapies.  Here, we define a molecular interaction of a novel URAT1 inhibitor lesinurad, under clinical evaluation in patients with gout.

Methods:   HEK-293T cells transiently expressing chimeric URAT1 mutants were used to measure cell-based uric acid transport activity in the presence of lesinurad and other URAT1 inhibitors.  A binding assay was also developed using membrane preparations from transfected cells and radiolabeled RDEA3170, a high-affinity URAT1 inhibitor also in development for gout.  Binding was measured after incubation with lesinurad and other URAT1 inhibitors.

Results:   Lesinurad inhibits the uric acid transport activity of human URAT1 (hURAT1) at a 20-fold higher potency compared to rat URAT1 (rURAT1), with IC₅₀‘s of 3.36 and 74.84 µM, respectively.  Chimeras containing portions of the rat and human genes identified a single residue, hURAT1 phenylalanine 365, located within transmembrane segment 7, as crucial for the higher affinity interaction with lesinurad.  This residue is a tyrosine in rURAT1.  In particular, the single chimeric point mutant hURAT1-Tyr365 shows a significantly reduced sensitivity for inhibition by lesinurad (IC₅₀ = 47.76 µM) compared to wild type hURAT1, while the converse point mutant rURAT1-Phe365 displays a significantly enhanced sensitivity (IC₅₀ = 6.82 µM) compared to wild type rURAT1.  Phe365 is also important for the interaction with hURAT1 to other commercially available inhibitors benzbromarone, probenecid, and sulfinpyrazone.  Lesinurad, the other URAT1 inhibitors, as well as salicylate and nicotinate which are known URAT1 inhibitors at high concentrations all bind competitively at the inhibitor binding site.  Among URAT1 orthologs, Phe365 occurs only in human, apes, and certain monkeys, whereas a tyrosine residue occurs in all other animals.  Consistent with in vitro inhibition of URAT1, lesinurad treatment results in an increased fractional excretion of uric acid and a decrease in serum uric acid.

Conclusion:   Lesinurad inhibits hURAT1 through an interaction that involves a critical residue, Phe365.  The gain-of-function phenotype of rURAT1-Phe365 suggests that Phe365 directly binds to lesinurad.  Phe365 is also important for the interaction with other URAT1 inhibitors, and this residue likely forms part of an inhibitor binding site within the transporter channel of URAT1.