Molecular mechanism of drug inhibition of URAT1

Abstract

Hyperuricemia, characterized by elevated serum urate levels, is a key factor in the pathogenesis of gout. URAT1 is essential for renal urate reabsorption and has emerged as a critical therapeutic target for managing hyperuricemia. However, the precise transport mechanism and the inhibitory effects of uricosuric drugs on URAT1 remain unclear. Here, we present structures of the double-mutant rat homolog of URAT1 in complex with its substrate urate, and the clinical drugs benzbromarone, lesinurad, verinurad, and sulfinpyrazone. The urate-bound structure elucidates key residues involved in recognizing urate, while the structures bound with drugs clearly demonstrate the distinct binding mode of each drug with URAT1. These drugs stabilize URAT1's inward-facing state, blocking conformational transitions. Additionally, critical interactions essential for its conformational transition are identified. These findings provide a molecular framework for understanding the physiological function of URAT1 and for developing more efficacious therapies to treat hyperuricemia.

Fig. 1. Functional characterization and structure determination of URAT1^EM.

a Cryo-EM maps of URAT1^UA in LMNG detergent (gray). The N and C domains of URAT1^EM are shown in green and orange, respectively. b Cartoon representation of URAT1^EM in the same orientation and colors as in (A). Protein dimensions are indicated. Disulfide bonds in the extracellular domain are shown as sticks. c Cytoplasmic view of URAT1^EM with TM helix numbers labeled. d Cryo-EM density of urate (UA) binding pocket. The cryo-EM densities are shown as mesh, with urate and surrounding residues shown as sticks. e Sagittal slice through an electrostatic surface potential of URAT1^UA. The vertical distance from urate (UA) to cytoplasmic membrane plane is labeled. The urate molecular is shown as sphere. f Interaction of urate in binding pocket. The key residues are showed as sticks. g Comparative evaluation of the [¹⁴C] urate transportation activity of URAT1 mutants in relation to the URAT1^EM. Data are mean ± s.e.m. from three independent assays, each with one measurement (n = 3). Comparison of URAT1^EM with mutant constructs using an unpaired two-sided t-test showed significant differences (all P < 0.0001, denoted as ****).

Fig. 2. Inhibition mechanism of URAT1^EM by benzbromarone.

a The cryo-EM density of benzbromarone and surrounding residues is shown as mesh while the structure of which are presented as sticks. b Side view URAT1 in complex with benzbromarone. Electrostatic potential map of the cytoplasmic pocket is shown as a surface. URAT1^EM is dipicted in sky-blue cartoon, while benzbromarone is shown as spheres with carbon, oxygen, and bromine atoms colored in deep teal, red, and green, respectively. c Coronal section view of electrostatic surface of URAT1^BEN. Benzbromarone is represented as sphere. d Detailed view of the benzbromarone binding pocket. Surrounding residues are represented as sticks. Hydrogen bond is depicted as black dash line. e Comparative evaluation of the [¹⁴C] urate transportation activity of URAT1 mutants in relation to the URAT1^EM. Data are mean ± s.e.m. from three independent assays, each with one measurement (n = 3). Comparison of URAT1^EM with mutant constructs using an unpaired two-sided t-test showed significant differences (all P < 0.0001, denoted as ****). f Inhibition of ¹⁴C-urate transport by benzbromarone in URAT1^EM and its mutants. Data are presented as normalized mean ± s.e.m. from three biologically independent assays (n  =  3).

Fig. 3. Antagonism of URAT1^EM by lesinurad and its analog verinurad.

a Chemical structure and cryo-EM density of lesinurad and verinurad. b Coronal section view of electrostatic surface of URAT1^LES and URAT1^VER. Benzbromarone is represented as spheres. c cytoplasmic view of URAT1 in complex with lesinurad and verinurad. d Detailed view of the lesinurad binding pocket. Surrounding residues are represented as sticks. Hydrogen bond is depicted as black dash line. Inhibition of ¹⁴C-urate transport by lesinurad e and verinurad h in URAT1^EM and its mutants. Data are presented as normalized mean ± s.e.m. from three biologically independent assays (n  =  3). IC50 values are calculated by fitting into a nonlinear regression model. f Detailed view of the verinurad binding pocket. Surrounding residues are represented as sticks. Hydrogen bond is depicted as black dash line. g Superposition between URAT1^LES and URAT1^VER binding pocket. The key residues are shown as sticks. Lesinurad and verinurad are colored purple and yellow, respectively.

Fig. 4. Recognition of the sulfinpyrazone-binding site.

a Chemical structure of sulfinpyrazone. Cryo-EM density of sulfinpyrazone and surrounding residues is shown as mesh and the structure of sulfinpyrazone is shown as sticks. b Coronal section view of electrostatic surface of URAT1^SPZ. Sulfinpyrazone is represented as spheres. c cytoplasmic view of URAT1 in complex with sulfinpyrazone. d Detailed view of the sulfinpyrazone binding pocket. Surrounding residues are represented as sticks. Hydrogen bond is depicted as black dash line. e Inhibition of ¹⁴C-urate transport by sulfinpyrazone in URAT1^EM and its mutants. Data are presented as normalized mean ± s.e.m. from three biologically independent assays (n  =  3). IC50 values are calculated by fitting into a nonlinear regression model.