Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1

Kazuki Fujita; Noriyoshi Isozumi; Qiunan Zhu; Masaya Matsubayashi; Tetsuya Taniguchi; Hiroshi Arakawa; Yoshiyuki Shirasaka; Eiichiro Mori; Ikumi Tamai

doi:10.1124/jpet.124.002096

Unique Binding Sites of Uricosuric Agent Dotinurad for Selective Inhibition of Renal Uric Acid Reabsorptive Transporter URAT1

J Pharmacol Exp Ther. 2024 Apr 26:JPET-AR-2023-002096. doi: 10.1124/jpet.124.002096. Online ahead of print.

Authors

Kazuki Fujita¹, Noriyoshi Isozumi², Qiunan Zhu¹, Masaya Matsubayashi³, Tetsuya Taniguchi⁴, Hiroshi Arakawa⁵, Yoshiyuki Shirasaka⁶, Eiichiro Mori², Ikumi Tamai⁷

Affiliations

¹ Kanazawa University, Japan.
² Department of Future Basic Medicine, Nara Medical University, Japan.
³ Research Laboratories 2, Fuji Yakuhin Co., Ltd., Japan.
⁴ Medical R&D Division, FUJIYAKUHIN CO., LTD., Japan.
⁵ Faculty of Pharmacy, Kanazawa University, Japan.
⁶ Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan.
⁷ Facylty of Pharmaceutical Sciences, Kanazawa University, Japan tamai@p.kanazawa-u.ac.jp.

PMID: 38670801
DOI: 10.1124/jpet.124.002096

Abstract

Dotinurad was developed as a uricosuric agent, inhibiting urate (UA) reabsorption through the UA transporter URAT1 in the kidneys. Due to its high selectivity for URAT1 among renal UA transporters, we investigated the mechanism underlying this selectivity by identifying dotinurad binding sites specific to URAT1. Dotinurad was docked to URAT1 using AutoDock4, utilizing the AlphaFold2-predicted structure. The inhibitory effects of dotinurad on wild-type and mutated URAT1 at the predicted binding sites were assessed through URAT1-mediated [¹⁴C]UA uptake in Xenopus oocytes. Nine amino acid residues in URAT1 were identified as dotinurad-binding sites. Sequence alignment with UA-transporting organic anion transporters (OATs) revealed that H142 and R487 were unique to URAT1 among renal UA-transporting OATs. For H142, IC₅₀ values of dotinurad increased to 62, 55, and 76 nM for mutated URAT1 (H142A, H142E, and H142R, respectively), compared to 19 nM for the wild-type, indicating that H142 contributes to URAT1-selective interaction with dotinurad. H142 was predicted to interact with the phenyl-hydroxyl group of dotinurad. The IC₅₀ of the hydroxyl group methylated dotinurad (F13141) was 165 μM, 8,420-fold higher than dotinurad, suggesting the interaction of H142 and the phenyl-hydroxyl group by forming a hydrogen bond. Regarding R487, URAT1-R487A exhibited a loss of activity. Interestingly, the URAT1-H142A/R487A double mutant restored UA transport activity, with the IC₅₀ value of dotinurad for the mutant (388 nM) significantly higher than that for H142A (73.5 nM). These results demonstrate that H142 and R487 of URAT1 determine its selectivity for dotinurad, a uniqueness observed only in URAT1 among UA-transporting OATs. Significance Statement Dotinurad selectively inhibits the urate reabsorption transporter URAT1 in renal urate-transporting OATs. This study demonstrates that dotinurad interacts with H142 and R487 of URAT1, located in the extracellular domain and unique among OATs when aligning amino acid sequences. Mutations in these residues reduce affinity of dotinurad for URAT1, confirming their role in conferring selective inhibition. Additionally, the interaction between dotinurad and URAT1 involving H142 was found to mediate hydrogen bonding.

Keywords: structure-activity relationships; transporters.