Mechanism of high affinity inhibition of the human urate transporter URAT1
Philip K Tan, Traci M Ostertag, Jeffrey N Miner, Philip K Tan, Traci M Ostertag, Jeffrey N Miner
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.
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References
- Choi H. K., Mount D. B. & Reginato A. M. Pathogenesis of gout. Ann. Intern. Med. 143, 499–516 (2005).
- Neogi T. Clinical practice. Gout. N. Engl. J. Med. 364, 443–452 (2011).
- Christen P., Peacock W. C., Christen A. E. & Wacker W. E. Urate oxidase in primate phylogenesis. Eur. J. Biochem. 12, 3–5 (1970).
- Kratzer J. T. et al.. Evolutionary history and metabolic insights of ancient mammalian uricases. Proc. Natl. Acad. Sci. USA 111, 3763–3768 (2014).
- Oda M., Satta Y., Takenaka O. & Takahata N. Loss of urate oxidase activity in hominoids and its evolutionary implications. Mol. Biol. Evol. 19, 640–653 (2002).
- Johnson R. J., Lanaspa M. A. & Gaucher E. A. Uric acid: a danger signal from the RNA world that may have a role in the epidemic of obesity, metabolic syndrome, and cardiorenal disease: evolutionary considerations. Semin. Nephrol. 31, 394–399 (2011).
- Levinson D. J. & Sorensen L. B. Renal handling of uric acid in normal and gouty subject: evidence for a 4-component system. Ann. Rheum. Dis. 39, 173–179 (1980).
- Roch-Ramel F. & Guisan B. Renal transport of urate in humans. News Physiol. Sci. 14, 80–84 (1999).
- Kydd A. S., Seth R., Buchbinder R., Edwards C. J. & Bombardier C. Uricosuric medications for chronic gout. Cochrane Database Syst. Rev. 11, CD010457 (2014).
- Richette P. Debulking the urate load to feel better. J Rheumatol. 39, 1311–1313 (2012).
- Dehghan A. et al.. Association of three genetic loci with uric acid concentration and risk of gout: a genome-wide association study. Lancet 372, 1953–1961 (2008).
- Kolz M. et al.. Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations. PLoS. Genet. 5, e1000504 (2009).
- Kottgen A. et al.. Genome-wide association analyses identify 18 new loci associated with serum urate concentrations. Nat. Genet. 45, 145–154 (2013).
- Vitart V. et al.. SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout. Nat. Genet. 40, 437–442 (2008).
- Wallace C. et al.. Genome-wide association study identifies genes for biomarkers of cardiovascular disease: serum urate and dyslipidemia. Am. J. Hum. Genet. 82, 139–149 (2008).
- Nakanishi T., Ohya K., Shimada S., Anzai N. & Tamai I. Functional cooperation of URAT1 (SLC22A12) and URATv1 (SLC2A9) in renal reabsorption of urate. Nephrol. Dial. Transplant. 28, 603–611 (2013).
- Anzai N. et al.. Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans. J. Biol. Chem. 283, 26834–26838 (2008).
- Enomoto A. et al.. Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature 417, 447–452 (2002).
- Ichida K. et al.. Clinical and molecular analysis of patients with renal hypouricemia in Japan-influence of URAT1 gene on urinary urate excretion. J. Am. Soc. Nephrol. 15, 164–173 (2004).
- Matsuo H. et al.. Mutations in glucose transporter 9 gene SLC2A9 cause renal hypouricemia. Am. J. Hum. Genet. 83, 744–751 (2008).
- Iharada M. et al.. Type 1 sodium-dependent phosphate transporter (SLC17A1 Protein) is a Cl(-)-dependent urate exporter. J. Biol. Chem. 285, 26107–26113 (2010).
- Jutabha P. et al.. Apical voltage-driven urate efflux transporter NPT4 in renal proximal tubule. Nucleosides Nucleotides Nucleic Acids 30, 1302–1311 (2011).
- Nakayama A. et al.. ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans. Nucleosides Nucleotides Nucleic Acids 30, 1091–1097 (2011).
- Miner JN et al.. Lesinurad, a novel, oral compound for gout, acts to decrease serum uric acid through inhibition of urate transporters in the kidney. Arthritis Res Ther (2016). In Press.
- Fleischmann R. et al.. Pharmacodynamic, pharmacokinetic and tolerability evaluation of concomitant administration of lesinurad and febuxostat in gout patients with hyperuricaemia. Rheumatology (Oxford) 53, 2167–2174 (2014).
- Iwanaga T., Sato M., Maeda T., Ogihara T. & Tamai I. Concentration-dependent mode of interaction of angiotensin II receptor blockers with uric acid transporter. J. Pharmacol. Exp. Ther. 320, 211–217 (2007).
- Huang Y., Lemieux M. J., Song J., Auer M. & Wang D. N. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620 (2003).
- Yu Z., Fong W. P. & Cheng C. H. Morin (3,5,7,2’,4’-pentahydroxyflavone) exhibits potent inhibitory actions on urate transport by the human urate anion transporter (hURAT1) expressed in human embryonic kidney cells. Drug Metab. Dispos. 35, 981–986 (2007).
- Koepsell H. & Endou H. The SLC22 drug transporter family. Pflugers. Arch. 447, 666–676 (2004).
- Feng B., Shu Y. & Giacomini K. M. Role of aromatic transmembrane residues of the organic anion transporter, rOAT3, in substrate recognition. Biochemistry 41, 8941–8947 (2002).
- Hong M., Zhou F. & You G. Critical amino acid residues in transmembrane domain 1 of the human organic anion transporter hOAT1. J. Biol. Chem. 279, 31478–31482 (2004).
- Hong M., Zhou F., Lee K. & You G. The putative transmembrane segment 7 of human organic anion transporter hOAT1 dictates transporter substrate binding and stability. J. Pharmacol. Exp. Ther. 320, 1209–1215 (2007).
- Perry J. L., Dembla-Rajpal N., Hall L. A. & Pritchard J. B. A three-dimensional model of human organic anion transporter 1: aromatic amino acids required for substrate transport. J. Biol. Chem. 281, 38071–38079 (2006).
- Mori K. et al.. Kidney-specific expression of a novel mouse organic cation transporter-like protein. FEBS Lett. 417, 371–374 (1997).
- Sato M. et al.. Identification and functional characterization of uric acid transporter Urat1 (Slc22a12) in rats. Biochim. Biophys. Acta 1808, 1441–1447 (2011).
- Eraly S. A. et al.. Multiple organic anion transporters contribute to net renal excretion of uric acid. Physiol Genomics 33, 180–192 (2008).
- Uetake D. et al.. Effect of fenofibrate on uric acid metabolism and urate transporter 1. Intern. Med. 49, 89–94 (2010).
- Ichida K. et al.. Urate transport via human PAH transporter hOAT1 and its gene structure. Kidney Int. 63, 143–155 (2003).
- National Center for Biotechnology Information. Verinurad. National Institutes of Health. Available at: . (Accessed August 17, 2016).
- Tan P. K., Farrar J. E., Gaucher E. A. & Miner J. N. Coevolution of URAT1 and uricase during primate evolution: implications for serum urate homeostasis and gout. Mol. Biol. Evol. 33, 2193-2200, doi: 10.1093/molbev/msw116. (2016).
- Chu X. Y. et al.. Transport of the dipeptidyl peptidase-4 inhibitor sitagliptin by human organic anion transporter 3, organic anion transporting polypeptide 4C1, and multidrug resistance P-glycoprotein. J. Pharmacol. Exp. Ther. 321, 673–683 (2007).
- Deng D. et al.. Crystal structure of the human glucose transporter GLUT1. Nature 510, 121–125 (2014).
- Deng D. et al.. Molecular basis of ligand recognition and transport by glucose transporters. Nature 526, 391–396 (2015).
- Nomura N. et al.. Structure and mechanism of the mammalian fructose transporter GLUT5. Nature 526, 397–401 (2015).
- Mueckler M. & Thorens B. The SLC2 (GLUT) family of membrane transporters. Mol. Aspects Med. 34, 121–138 (2013).
- Tsigelny I. F. et al.. Conformational changes of the multispecific transporter organic anion transporter 1 (OAT1/SLC22A6) suggests a molecular mechanism for initial stages of drug and metabolite transport. Cell Biochem. Biophys. 61, 251–259 (2011).
- Feng B., Dresser M. J., Shu Y., Johns S. J. & Giacomini K. M. Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3. Biochemistry 40, 5511–5520 (2001).
- Rizwan A. N., Krick W. & Burckhardt G. The chloride dependence of the human organic anion transporter 1 (hOAT1) is blunted by mutation of a single amino acid. J. Biol. Chem. 282, 13402–13409 (2007).
- Zhu C. et al.. Evolutionary Analysis and Classification of OATs, OCTs, OCTNs, and Other SLC22 Transporters: Structure-Function Implications and Analysis of Sequence Motifs. PLoS. One. 10, e0140569 (2015).
- Altschul S. F., Gish W., Miller W., Myers E. W. & Lipman D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
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