Verinurad (formerly known as RDEA-3170; RDEA3170) is a novel, highly potent and specific URAT1 (Urate transporter 1) inhibitor with anti-hyperuricemic effects. It inhibits URAT1 with an IC50 of 25 nM and is currently under clinical trials for treating gout. High affinity inhibition of uric acid transport requires URAT1 residues Cys-32, Ser-35, Phe-365 and Ile-481. Unlike other available uricosuric agents, the requirement for Cys-32 is unique to verinurad. Two of these residues, Ser-35 and Phe-365, are also important for urate transport kinetics. A URAT1 binding assay using radiolabeled verinurad revealed that distinct URAT1 inhibitors benzbromarone, sulfinpyrazone and probenecid all inhibit verinurad binding via a competitive mechanism. However, mutations made within the predicted transporter substrate channel differentially altered the potency for individual URAT1 inhibitors. Overall, the results suggest that URAT1 inhibitors bind to a common site in the core of the transporter and sterically hinder the transit of uric acid through the substrate channel, albeit with vastly different potencies and with differential interactions with specific URAT1 amino acids.

Physicochemical Properties

Molecular Formula	C20H16N2O2S
Molecular Weight	348.42
Exact Mass	348.093
Elemental Analysis	C, 68.95; H, 4.63; N, 8.04; O, 9.18; S, 9.20
CAS #	1352792-74-5
Related CAS #	1352792-74-5
PubChem CID	54767229
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Boiling Point	566.7±50.0 °C at 760 mmHg
Flash Point	296.5±30.1 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.692
LogP	3.77
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	25
Complexity	541
Defined Atom Stereocenter Count	0
SMILES	CC(C)(SC1=C(C2=C3C=CC=CC3=C(C#N)C=C2)C=NC=C1)C(O)=O
InChi Key	YYBOLPLTQDKXPM-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H16N2O2S/c1-20(2,19(23)24)25-18-9-10-22-12-17(18)16-8-7-13(11-21)14-5-3-4-6-15(14)16/h3-10,12H,1-2H3,(H,23,24)
Chemical Name	2-((3-(4-cyanonaphthalen-1-yl)pyridin-4-yl)thio)-2-methylpropanoic acid
Synonyms	RDEA-3170; RDEA3170; RDEA 3170
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	IC50: 25 nM (URAT1)[1] Verinurad (RDEA3170) targets human URAT1 (IC50 = 25 nM); for the increase of fractional excretion of uric acid (FEUA) in humans, the half-maximal effective plasma concentration is 22 nM [1]
ln Vitro	With an IC50 of 25 nM, verinurad exhibits strong efficacy and dose-dependent inhibition of human URAT1's transport activity [1]. 1. Verinurad (RDEA3170) exhibits high potency and specificity for human URAT1, with no significant inhibitory effect on the transport activity of human OAT4 and OAT1 when tested in cell-based transport assays. In the assay, cells expressing URAT1 were incubated with ^{14}C-uric acid, and cells expressing OAT4 or OAT1 were incubated with carboxyfluorescein in the presence of different concentrations of verinurad [1] 2. High-affinity inhibition of uric acid transport by Verinurad (RDEA3170) is dependent on specific residues of URAT1, namely Cys-32, Ser-35, Phe-365 and Ile-481; the requirement for Cys-32 is unique to verinurad compared with other uricosuric agents, and Ser-35 and Phe-365 are also crucial for urate transport kinetics [1] 3. A binding assay using ^{3}H-verinurad showed that ^{3}H-verinurad binds specifically and saturably to membranes from cells transfected with human URAT1, but not to membranes from cells transfected with empty vector. Unlabeled verinurad, benzbromarone, sulfinpyrazone and probenecid all inhibit the binding of ^{3}H-verinurad to URAT1 in a dose-dependent manner, indicating a competitive inhibition mechanism [1] 4. Mutations in the predicted substrate channel of URAT1 (e.g., F241Y, F449Y, R477K) differentially alter the potency of individual URAT1 inhibitors (verinurad, benzbromarone, sulfinpyrazone, probenecid) for URAT1; for example, the hURAT1-F449Y mutant has the same affinity for verinurad but an 11-fold lower affinity for benzbromarone compared with wild-type human URAT1 [1] 5. Chimeric point mutation experiments of rat and human URAT1 (at positions 35, 365 and 481) demonstrated that Ser-35, Phe-365 and Ile-481 of human URAT1 cooperate to enhance the affinity of verinurad for URAT1; the double point mutant chimera rat URAT1 (r-N35S/F365Y) showed altered urate transport kinetics (K m) compared with wild-type rat URAT1 [1]
ln Vivo	1. In healthy human volunteers, a single 40 mg dose of Verinurad (RDEA3170) reduced baseline serum uric acid (sUA) levels by up to 60% for a sustained time period [1] 2. Verinurad (RDEA3170) increased the fractional excretion of uric acid (FEUA) in humans in a dose-dependent manner, with a half-maximal effective plasma concentration of 22 nM [1]
Enzyme Assay	1. URAT1 binding assay with radiolabeled verinurad: Membranes were prepared from cells transfected with human URAT1 or empty vector. The membranes were incubated with different concentrations of ^{3}H-verinurad (in the absence or presence of unlabeled inhibitors) for a specific period. After incubation, the membrane-bound radioactivity was measured to determine the specific binding of ^{3}H-verinurad to URAT1. For competitive inhibition experiments, 10 nM of ^{3}H-verinurad was incubated with URAT1 membranes together with different concentrations of unlabeled verinurad, benzbromarone, sulfinpyrazone or probenecid, and the bound radioactivity was quantified [1] 2. Dose-response binding assay for URAT1 mutants: Membranes were prepared from cells transfected with hURAT1 mutants (e.g., F449Y). The membranes were incubated with 10 nM of ^{3}H-verinurad and different concentrations of unlabeled verinurad or benzbromarone, and the bound radioactivity was measured to analyze the affinity of inhibitors for the mutant URAT1 [1] 3. Eadie-Hofstee linearization analysis for binding kinetics: Membranes containing human URAT1 were incubated with different concentrations of ^{3}H-verinurad in the absence or presence of fixed concentrations of unlabeled inhibitors (20 nM verinurad, 50 nM benzbromarone, 25 µM sulfinpyrazone, 200 µM probenecid). The specifically bound ^{3}H-verinurad was analyzed by Eadie-Hofstee linearization to determine the binding mechanism [1]
Cell Assay	1. URAT1/OAT transport activity assay: HEK293 cells were transfected with plasmids encoding human URAT1, OAT4 or OAT1. Transfected cells were incubated with ^{14}C-uric acid (for URAT1) or carboxyfluorescein (for OAT4/OAT1) in the presence of serial concentrations of Verinurad (RDEA3170). After incubation, the intracellular radioactivity (for URAT1) or fluorescence intensity (for OAT4/OAT1) was measured to evaluate the transport activity of the transporters, and dose-response curves were generated to calculate the inhibitory potency of verinurad [1] 2. URAT1 mutant transport activity assay: HEK293 cells were transfected with plasmids encoding wild-type human URAT1, rat URAT1, or chimeric point mutants (e.g., hURAT1-F365Y, rURAT1-Y365F, rURAT1-N35S/F365Y). The transport activity of these mutants was assessed using the same ^{14}C-uric acid incubation method as the wild-type URAT1 assay, and the K m (half maximal transport velocity) of urate transport was calculated for each construct [1] 3. Inhibitor potency assay for URAT1 mutants: HEK293 cells expressing hURAT1 mutants (F241Y, F449Y, R477K) were treated with different concentrations of verinurad, benzbromarone, sulfinpyrazone or probenecid. The transport activity of the mutants was measured via ^{14}C-uric acid uptake, and the potency of each inhibitor for the mutants was determined and compared with wild-type hURAT1 [1]
References	[1]. Discovery and characterization of verinurad, a potent and specific inhibitor of URAT1 for the treatment of hyperuricemia and gout. Sci Rep. 2017 Apr 6;7(1):665.
Additional Infomation	Verinurad has been used in trials studying the basic science and treatment of Gout, Gout and Hyperuricemia, and Gout and Asymptomatic Hyperuricemia. 1. Verinurad (RDEA3170) is a potent and specific inhibitor of URAT1, which is currently under evaluation for the therapy of hyperuricemia and gout [1] 2. All URAT1 inhibitors (including verinurad, benzbromarone, sulfinpyrazone and probenecid) bind to a common site in the core of the URAT1 transporter and sterically hinder the transit of uric acid through the substrate channel, but they exhibit vastly different potencies and differential interactions with specific URAT1 amino acids [1] 3. The requirement for the URAT1 residue Cys-32 for high-affinity inhibition is a unique characteristic of Verinurad (RDEA3170) compared with other available uricosuric agents [1]

Solubility Data

Solubility (In Vitro)

DMSO:≥ 30mg/mL Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: 2.5 mg/mL (7.18 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.18 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.18 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.8701 mL	14.3505 mL	28.7010 mL
	5 mM	0.5740 mL	2.8701 mL	5.7402 mL
	10 mM	0.2870 mL	1.4350 mL	2.8701 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.