Background URAT1 inhibitors, used for the treatment of gout, lower serum urate levels by blocking uric acid reabsorption in the kidney proximal tubule through inhibition of the uric acid transporter URAT1. We have identified a URAT1 inhibitor RDEA3170 that specifically binds to and inhibits URAT1 with nanomolar potency. RDEA3170 inhibits human URAT1 activity with over a 1000-fold higher potency compared to the ortholog rat uric acid transporter rRST, despite the two transporters sharing a 74% protein sequence identity.

Objectives To define the high affinity RDEA3170 interaction site on URAT1 and compare the interaction with other known URAT1 inhibitors.

Methods We systematically converted human URAT1 amino acid residues into the counterpart rat rRST residues, and vice versa, within the predicted transmembrane segments of the transporters and measured the activity of URAT1 inhibitors. Mutant transporters were expressed in transfected cultured cells for transport and binding assays. Transport assays measured the transport of ¹⁴C-labeled uric acid into living cells, while binding assays measured the binding of ³H-RDEA3170 to membranes prepared from transfected cells. Different amounts of unlabeled URAT1 inhibitors were added for potency determinations.

Results Binding of ³H-RDEA3170 to URAT1 was specific and saturable with a dissociation constant of 10 nM. Unlabeled RDEA3170 displaced ³H-RDEA3170 binding with a half maximal inhibition constant (IC₅₀) of 10 nM, while the URAT1 inhibitor benzbromarone displaced binding less potently (IC₅₀ of 100 nM). URAT1 inhibitors displaced RDEA3170 binding in a competitive manner, suggesting that they bind to a similar location on URAT1. Binding of ³H-RDEA3170 to rRST was not detected. In transport assays, RDEA3170 inhibits URAT1 with an IC₅₀ of 24 nM, which is 3 fold more potent than benzbromarone and over 100 fold more potent than the URAT1 inhibitors sulfinpyrazone and probenecid. RDEA3170 inhibits rRST transport with an IC₅₀ of 34,000 nM. Chimeras between URAT1 and rRST show that amino acids 35, 365, and 481 are important for RDEA3170 potency. Residue 365 is a phenylalanine in human URAT1 and a tyrosine in rat RST. Point mutation of phenylalanine 365 to tyrosine within human URAT1 decreases the potency of RDEA3170 by 100-fold, whereas conversion of tyrosine 365 to phenylalanine within rat RST increases the potency of RDEA3170 by 10-fold compared to wild type rRST. Similar but less dramatic results are seen with individual mutations at residues 35 and 481. Combinations of mutations at positions 35, 365, and 481 produce additive phenotypes, and the rRST 35/365/481 triple mutant (with URAT residues in these locations) shows an IC₅₀ for RDEA3170 of 100 nM, a near complete gain-of-function. Computer modeling shows that the amino acids responsible for high affinity binding are located in the core transport channel of URAT1.

Conclusions Three URAT1 residues required for high affinity RDEA3170 interaction have been identified. Other inhibitors interact with URAT1 at the same binding site but with lower affinity. Consistent with these preclinical results, a single dose of RDEA3170 in a phase 1 clinical trial increases uric acid fractional excretion and results in a greater than 60 percent mean decrease in sUA, which was sustained for 36 hours.

Disclosure of Interest J. Miner Employee of: Ardea Biosciences, P. Tan Employee of: Ardea Biosciences