Mechanism of high affinity inhibition of the human urate transporter URAT1

Abstract

Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions.

Conflict of interest statement

Philip K. Tan, Traci M. Ostertag and Jeffrey N. Miner are employees of Ardea Biosciences, Inc., a member of the AstraZeneca group.

Figures

Figure 1. URAT1 inhibitors inhibit human URAT1 at substantially higher potency.

Dose response curves of benzbromarone (a), sulfinpyrazone (b), probenecid (c), and lesinurad (d) against human URAT1 (open symbols) and rat URAT1 (closed symbols). Data are from a single experiment and are representative of multiple experiments. Points represent the mean ± standard error of the mean (SEM) from triplicate samples. The potencies (half maximal inhibitor concentration values) are shown in Supplementary Table 1. For all inhibitors, the differences in potency between human URAT1 and rat URAT1 are statistically significant (P < 0.0001).

Figure 2. Chimeras of human (h) URAT1 and rat (r) URAT1 reveal that TM1, 7 and 11 of human (h) URAT1 confer high affinity responses to inhibitors.

(a) Diagram of human URAT1 wild type (hURAT1 WT, white box) with the predicted secondary structures showing the approximate location of the twelve transmembrane (TM) segments (numbered), the large first extracellular loop (EC1) and the large third intracellular loop (IC3), and diagrams of individual human-to-rat (h–r) chimeras, where small regions of hURAT1 were replaced with rat URAT1 (rURAT1, black boxes). (b–e) Potencies against hURAT1 and the human-to-rat (h–r) chimeras shown in (a) for benzbromarone (b), sulfinpyrazone (c), probenecid (d) and lesinurad (e). Dose-response curves for each construct were performed as in Fig. 1, and results are the mean ± SEM from at least three experiments. Asterisks indicate a significant difference in the mean value from hURAT1 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 3. Chimeras of human (h) URAT1 and rat (r) URAT1 reveal that TMS 1, 7 and 11 of hURAT1 confer high affinity responses to inhibitors.

(a) Diagram of rURAT1 wild type (rURAT1 WT, black box) with the predicted secondary structures showing the approximate location of the twelve transmembrane (TM) segments (numbered), the large first extracellular loop (EC1) and the large third intracellular loop (IC3), and diagrams of individual rat-to-human (r–h) chimeras where small regions of rURAT1 were replaced with human URAT1 (hURAT1, white boxes). (b–e) Potencies against rURAT1 and the rat-to human (r-h) chimeras shown in (a) for benzbromarone (b), sulfinpyrazone (c), probenecid (d), and lesinurad (e). Dose-response curves for each construct were performed as in Fig. 1, and results are the mean ± SEM from at least three experiments. Asterisks indicate a significant difference in the mean value from rURAT1 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 4. Identification of individual residues in TM1, 7 and 11 of hURAT1 that confer high affinity interaction with inhibitors: analysis of human-to-rat point mutants.

Potency (IC50) of URAT1 inhibitors benzbromarone (a), sulfinpyrazone (b), probenecid (c) and lesinurad (d) against human URAT1 wild type (hURAT1 WT) and point mutant chimeras with rat URAT1 (rURAT1) residues in TM1, 7, and 11. Dose-response curves were performed as in Fig. 1, and results are the mean ± SEM from at least three experiments. In general, hURAT1 residues Ser-35, Phe-365 and Ile-481 are important for high affinity interaction with URAT1 inhibitors. Asterisks indicate a significant difference in the mean value from hURAT1 WT (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 5. Identification of individual residues in TM1, 7 and 11 of hURAT1 that confer high affinity interaction with inhibitors: analysis of rat-to-human point mutants.

Potency (IC50) of URAT1 inhibitors benzbromarone (a), sulfinpyrazone (b), probenecid (c) and lesinurad (d) against rat URAT1 wild type (rURAT1 WT) and point mutant chimeras with human URAT1 (hURAT1) residues in TM1, 7 and 11. Dose-response curves were performed as in Fig. 1, and results are the mean ± SEM from at least three experiments. In general, hURAT1 residues Ser-35, Phe-365 and Ile-481 are important for high affinity interaction with URAT1 inhibitors. Asterisks indicate a significant difference in the mean value from rat URAT1 wild type (WT) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 6. Human URAT1 residues Ser-35, Phe-365 and Ile-481 are likely in close proximity and the sidechains project into the central transporter channel.

Pymol (Schrödinger) images of human OAT1 (hOAT1) three-dimensional model, a homolog of human URAT1 (hURAT1), highlighting Ser-35 (S35, multi-colored), Phe-365 (F365, orange), and Ile-481 (I481, purple). hOAT1 residues Asn-35 and Tyr-354 were converted to the corresponding URAT1 residues Ser-35 and Phe-365, while conserved hOAT1 residue Ile-470 corresponds to hURAT1 Ile-481. (a) End view with the cytoplasmic surface facing the viewer. (b) Side view with extracellular surface facing up. For both panels, the front part of the protein has been removed to reveal the central pore.

Figure 7. In a novel human URAT1 binding assay that requires Phe-365, inhibitors displace the binding of the probe.

(a) 3H-RDEA3170 binds to human URAT1 and not rat URAT1, and binding depends on Phe-365. Binding was assayed after incubation of 500 nM 3H-RDEA3170 with membranes prepared from cells transfected with the indicated constructs. (b) Inhibitors block binding of 3H-RDEA3170 to human URAT1. Human URAT1 membranes were incubated with 10 nM of 3H-RDEA3170 in the absence or presence of the indicated concentrations of inhibitors. Results are the mean ± SEM from samples assayed in triplicate.

Figure 8. Human URAT1 residues 35, 365, and 481 act additively to enhance affinity to URAT1 inhibitors.

Potencies of benzbromarone (a), sulfinpyrazone (b), probenecid (c), and lesinurad (d) against rat URAT1 wild type (rURAT1 WT) and individual or combination point mutants with human URAT1 (hURAT1) residues at positions 35, 365, and 481. Dose-response curves were performed as in Fig. 1, and results are the mean ± SEM from at least three experiments. Asterisks indicate a significant difference in the mean value from rURAT1 WT (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Figure 9. Human URAT1 Ser-35 and Phe-365 confer high affinity interaction with urate.

Potencies of uric acid against wild type (WT) and chimeric point mutants for human URAT1 (hURAT1, a) and rat URAT1 (rat URAT1, b) are shown. Dose-response curves were performed as in Fig. 1 using unlabelled uric acid, and results are the mean ± SEM from at least three experiments. Asterisks indicate a significant difference in the mean value of the sample from wild type (*P < 0.05, ***P < 0.001, ****P < 0.0001). For the mean IC50 difference between hURAT1 and rURAT1, P < 0.0001.