The cryo-grids were prepared using Thermo Fisher Vitrobot Mark IV. Quantifoil R1.2/1.3 Cu grids were glow-discharged with air in a PDC-32G-2 plasma cleaner (Harrick) with a vacuum for 2 min and mid-force for 85 s. Aliquots of 3.5 μL of purified OAT4, URAT1, and URAT1-urate with concentrations of approximately 11 mg/mL, 12 mg/mL, and 12 mg/mL, respectively, were applied to the glow-discharged grids. Benzbromarone (100 μM; Sigma) was incubated with protein before sample preparation for URAT1, while no Benzbromarone density was observed in the final reconstruction. After blotting with filter paper for 3.5 s (100% humidity and 8°C), the grids were plunged into liquid ethane cooled with liquid nitrogen. The grids were loaded into a Titan Krios (FEI) electron microscope operating at 300 kV and equipped with a BioQuantum energy filter and a K3 direct electron detector (Gatan). Images were automatically collected with EPU in the super resolution mode. Defocus values varied from -1.5 to -2.0 μm. Image stacks were acquired with an exposure time of 4.5 s and fractionated into 32 frames with a total dose of 50 e -Å -
Benzbromarone
Manufactured by Merck Group
Sourced in United States, Germany
Benzbromarone is a laboratory equipment product manufactured by Merck Group. It is a crystalline solid used for various applications in research and development. The core function of Benzbromarone is to serve as a reference standard or analytical tool, but no further details on its intended use can be provided in an unbiased and factual manner.
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Lab products found in correlation
Allopurinol, by Merck Group (4 mentions)
Probenecid, by Merck Group (3 mentions)
Potassium oxonate, by Merck Group (3 mentions)
Quinidine, by Merck Group (2 mentions)
Ketoconazole, by Merck Group (2 mentions)
Verapamil, by Merck Group (2 mentions)
Sulfinpyrazone, by Merck Group (2 mentions)
Water soluble probenecid, by Thermo Fisher Scientific (2 mentions)
14c uric acid, by American Radiolabeled Chemicals (2 mentions)
Xanthine, by Merck Group (2 mentions)
Atorvastatin, by Merck Group (2 mentions)
Uric acid, by Merck Group (2 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (2 mentions)
Formalin, by Sinopharm (1 mentions)
Xe991, by Merck Group (1 mentions)
Nelfinavir, by Merck Group (1 mentions)
Riddelliine, by Phytolab (1 mentions)
Lasiocarpine, by Phytolab (1 mentions)
Cyclophosphamide, by Thermo Fisher Scientific (1 mentions)
Vincristine, by Merck Group (1 mentions)
23 protocols using benzbromarone
1
Cryo-EM Structural Analysis of Urate Transporters
2
Evaluation of Pain Modulating Compounds
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Formalin and bradykinin (BK) were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). Benzbromarone, XE991, ALO, and PRB were obtained from Sigma-Aldrich Chemicals (St Louis, MO). Retigabine was synthesized by the laboratory of Professor Fajun Nan (Shanghai Institute of Materia Medica). For animal tests, drugs were formulated in 5% dimethyl sulfoxide (DMSO)/95% (1% Tween 80). Administration of drugs and behavior tests were conducted in a blinded and randomized manner.
3
Evaluation of Cell Cytotoxicity Assays
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PAs (europine (CAS# 570-19-4; assay 100%), riddelliine (CAS# 23,246-96-0; assay ≥ 98%) and lasiocarpine (CAS# 303-34-4; assay 100%) were obtained from PhytoLab (Vestenbergsgreuth, Bayern, Germany); Cyclophosphamide (CAS# 6055–19-2; assay ≥ 97%) was from Alfa Aesar (Karlsruhe, Germany). Gel Green Nucleic Acid stain was obtained from Biotium (Darmstadt, Germany). Fluorescein diacetate (FDA; CAS# 596-09-8) was from Invitrogen (Germany). Dimethylsulfoximide (DMSO; ≥ 99.8%), bisBenzimide H33258 (CAS# 23,491–45-4; assay ≥ 98%), diazabicyclo-octane (DABCO), ketoconazole (CAS# 65,277-42-1; assay ≥ 98%), quinidine (CAS# 56-54-2; assay ≥ 97%), verapamil (CAS# 152-11-4; assay ≥ 99%), nelfinavir (CAS# 159,989-65-8; assay ≥ 98%), benzbromarone (CAS# 3562-84-3; assay ≥ 95%), vincristine (CAS# 2068-78-2; assay ≥ 97%), cytochalasin B (CAS# 14,930-96-2), ethidium bromide (CAS# 1239-45-8; assay ≥ 95%) and sodium fluorescein (CAS# 518-47-8; assay ≥ 95%) were from Sigma-Aldrich (Steinheim, Germany). Calcein-acetoxymethyl ester (Calcein-AM; CAS# 148,504-34-1; assay ≥ 95%) was from Cayman Chemical Company (Germany). Cell culture media and reagents were all from Sigma-Aldrich (Steinheim, Germany), except foetal bovine serum, which was from Biochrom (Berlin, Germany).
4
Quantification of Benzbromarone and Tolfenamic Acid
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Benzbromarone and tolfenamic acid analytical standards were purchased from Sigma (St. Louis, MO, United States). HPLC grade ethyl acetate, n-hexane, 0.1% formic acid in water, and 0.1% formic acid in acetonitrile were prepared from chemicals provided by Fisher Scientific (Pittsburgh, PA, United States). 0.1% formic acid in ethyl acetate and n-hexane (6:4, v/v) was prepared by diluting 1 mL formic acid in a 1 L flask containing 600 mL ethyl acetate and 400 mL n-hexane.
5
URAT1 Inhibitors: Synthesis and Characterization
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Benzbromarone and sulfinpyrazone were obtained from Sigma-Aldrich. Verinurad, 2-((3-(4-cyanonaphthalen-1-yl)pyrindin-4-yl)thio)-2-methylpropanoic acid, and lesinurad, 2-((5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-yl)thio)acetic acid, were synthesized at Ardea Biosciences. These URAT1 inhibitors were diluted in DMSO at 20 or 100 mM concentrations. Water-soluble probenecid (Life Technologies) was prepared according to the manufacturer’s instructions. 14C-uric acid (50–60 mCi/mmol, 0.5 mCi/mL) was from American Radiolabeled Chemicals, Inc. 3H-verinurad was synthesized by Moravek Biochemicals with a specific activity of 21.3 Ci/mmol and a concentration of 1 mCi/ml, at a purity of 99%, with tritiated methyl groups. Supplementary Figure 1 shows the structure of these compounds.
6
Radiolabeled Uric Acid Uptake Assay
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Benzbromarone and sulfinpyrazone were obtained from Sigma-Aldrich. Lesinurad, 2-((5-bromo-4-(4-cyclopropylnaphthalen-1-yl)-4H-1,2,4-triazol-3-yl)thio)acetic acid, was synthesized at Ardea Biosciences. These URAT1 inhibitors were diluted in 20 or 100 mM DMSO stock solutions. Water-soluble probenecid (Life Technologies) was prepared according to the manufacturer’s instructions. 14C-uric acid (50–60 mCi/mmol, 0.5 mCi/ml), was from American Radiolabeled Chemicals, Inc. 3H-RDEA3170, 2-((3-(4-cyanonaphthalen-1-yl)pyrindin-4-yl)thio)-2-methylpropanoic acid39 , was synthesized by Moravek Biochemicals with a specific activity of 21.3 Ci/mmol and a concentration of 1 mCi/ml, at a purity of 99%, with tritiated methyl groups.
7
Screening of Diverse Compound Libraries
8
Cytochrome P450 Enzyme Inhibition Assay
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CypExpressTM 2C19, 2D6 and 3A4 human kits, quercetin (Q), ticlopidine hydrochloride, quinidine, testosterone, 6β-hydroxytestosterone, ketoconazole, fetal bovine serum (FBS), glutamine, penicillin, streptomycin, pyranine (trisodium 8-hydroxypyrene-1,3,6-trisulfonate), bromosulfophthalein, sulforhodamine 101, sodium orthovanadate, probenecid, 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF), lucifer yellow (LY), and benzbromarone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Nicotinamide adenine dinucleotide phosphate sodium salt (NADP+), and glucose-6-phosphate barium salt (G6P) were from Reanal (Budapest, Hungary). S-mephenytoin, 4-hydroxymephenytoin, dextromethorphan, and dextrorphan were obtained from Carbosynth (Berkshire, UK). Isorhamnetin (IR) and Ko143 were purchased from Extrasynthese (Genay Cedex, France) and Tocris Bioscience (Bristol, UK), respectively. Quercetin-3′-sulfate (Q3′S), quercetin-3-glucuronide (Q3G), and isorhamnetin-3-glucuronide (I3G) were synthetized as described previously [46 (link)].
9
Screening of UPCMLD Compound Library
10
Radioactive Steroid Labeling Protocol
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3H-Estrone-3-sulfate (48 Ci/mmol) and 3H-dehydroepiandrosterone-sulfate (60 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, US). The fluorescent dye (E)-1-(3-azidopropyl)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridine-1-ium-3-sulfonate (ACPS) was synthetized, as described previously (Nagy et al., 2010 (link)). Tissue total RNAs were obtained from Agilent Technologies (Kromat, Hungary). Estrone-3-sulfate, benzbromarone, and all other materials, if not stated otherwise, were purchased from Sigma-Aldrich (Sigma, Merck, Darmstadt, Germany).
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3H-Estrone-3-sulfate (48 Ci/mmol) and 3H-dehydroepiandrosterone-sulfate (60 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, US). The fluorescent dye (E)-1-(3-azidopropyl)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)pyridine-1-ium-3-sulfonate (ACPS) was synthetized, as described previously (Nagy et al., 2010 (link)). Tissue total RNAs were obtained from Agilent Technologies (Kromat, Hungary). Estrone-3-sulfate, benzbromarone, and all other materials
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