With-No-Lysine (K) Kinase 1 (WNK1) (Ki = 0.8 nM in ATP-competitive binding assay; IC50 = 2.1 nM in recombinant WNK1 kinase activity assay) [1]
WNK2 (IC50 = 3.5 nM in recombinant WNK2 kinase activity assay) [1]
WNK3 (IC50 = 4.2 nM in recombinant WNK3 kinase activity assay) [1]
WNK4 (IC50 = 2.8 nM in recombinant WNK4 kinase activity assay) [1]
Other serine/threonine kinases (SGK1, NKCC1, NCC, ERK1/2) (IC50 > 1000 nM for all, no significant inhibition at 1 μM) [1]

The phosphorylation of the WNK1 downstream target SPAK/OSR1 in human tissue engineered hierarchy (hTEC) is decreased by WNK463 treatment (50 nM, 1 μM, 10 μM; 6 days) [1].

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WNK463 acts as a potent and selective ATP-competitive inhibitor of the WNK kinase family (WNK1/2/3/4): it potently inhibits recombinant human WNK1 kinase activity with an IC50 of 2.1 nM, WNK2 with 3.5 nM, WNK3 with 4.2 nM, and WNK4 with 2.8 nM; it shows no significant inhibition of other ion transport-related kinases (SGK1) or ion transporters (NKCC1, NCC) at concentrations up to 1 μM (inhibition <5%) [1]
In human renal proximal tubule epithelial cells (HK-2), WNK463 (1-10 nM) dose-dependently inhibits WNK1-mediated phosphorylation of SPAK (Ser383) and OSR1 (Ser325) (key downstream effectors): at 5 nM, it reduces p-SPAK levels by 80% and p-OSR1 by 75% (Western blotting), leading to a 65% decrease in Na⁺-Cl⁻ cotransporter (NCC) activity (fluorescent ion flux assay) [1]
In rat vascular smooth muscle cells (VSMCs), WNK463 (3 nM) suppresses WNK1-dependent Ca²⁺ mobilization: it reduces phenylephrine-induced Ca²⁺ influx by 70% (Fura-2 AM fluorescence imaging) and inhibits VSMC proliferation (MTT assay, 72 hours) by 55% at 5 nM, with no effect on non-proliferating VSMCs [1]
WNK463 (5 nM) also inhibits NaCl-induced phosphorylation of NKCC1 (Thr203/Thr207/Thr212) in human umbilical vein endothelial cells (HUVECs) by 60%, reducing endothelial cell migration (scratch wound assay) by 50% vs. control [1]

In conscious SHR, administration with WNK463 (1–10 mg/kg; final product; 4 hours; autohypertensive Sprague Dawley status) resulted in a dose-dependent drop in blood glucose and an increase in heart rate concurrently. Urinary sodium WNK463 is internally generated in Sprague Dawley and is bioavailable internally in urine output, with a half-life of 2.1 hours [1].

1. Recombinant WNK1/2/3/4 kinase activity assay: Prepare recombinant human WNK1 (catalytic domain, residues 382-641), WNK2 (residues 378-637), WNK3 (residues 385-644), and WNK4 (residues 380-639) proteins, dilute to a final concentration of 5 nM in kinase reaction buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA, 0.1 mM Na₃VO₄); incubate the enzyme with serial dilutions of WNK463 (10⁻¹²-10⁻⁶ M) and ATP (100 μM) at 30°C for 15 minutes; add a WNK-specific fluorescent peptide substrate (KKKLGSLVKFSFRQDYEEVV, 200 μM) and continue incubation for 45 minutes; terminate the reaction with 50 mM EDTA, measure fluorescence intensity (excitation 360 nm, emission 480 nm) using a microplate reader; fit inhibition curves to a four-parameter logistic model to calculate IC50 values [1]
2. WNK1 ATP-competitive binding assay (surface plasmon resonance, SPR): Immobilize recombinant WNK1 catalytic domain on a CM5 sensor chip via amine coupling (pH 4.0 acetate buffer); inject serial dilutions of WNK463 (10⁻¹²-10⁻⁶ M) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) containing 1 mM ATP at a flow rate of 25 μL/min; monitor resonance units (RU) for 200 seconds of association and 300 seconds of dissociation; calculate Ki values using the Cheng-Prusoff equation [1]
3. Kinase selectivity profiling assay: Incubate 40 different recombinant human serine/threonine kinases (including SGK1, ERK1/2, PKA, PKCα) with WNK463 (1 μM) and their respective peptide substrates in kinase reaction buffer; measure kinase activity using a luminescent kinase assay kit; calculate the percentage of kinase inhibition to evaluate the selectivity of WNK463 for WNK family kinases [1]

Western Blot Analysis[2]
Cell Types: Human tissue engineered cornea (hTEC)
Tested Concentrations: 50 nM, 1 μM, 10 μM
Incubation Duration: 6 days
Experimental Results: The phosphorylation of WNK1 downstream target SPAK/OSR1 was diminished in injured hTEC.

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1. HK-2 cell NCC activity assay: Culture human renal proximal tubule HK-2 cells in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) to logarithmic phase; seed cells at 2×10⁴ cells/well in 96-well black-walled plates and allow attachment for 24 hours; load cells with a Na⁺-sensitive fluorescent dye (SBFI-AM) for 30 minutes at 37°C; treat with serial dilutions of WNK463 (1-10 nM) for 1 hour, then add NaCl (100 mM) to induce Na⁺ influx; record fluorescence intensity (excitation 340/380 nm, emission 510 nm) every 10 seconds for 5 minutes; calculate NCC activity as the rate of Na⁺ influx [1]
2. VSMC Ca²⁺ mobilization assay: Culture rat vascular smooth muscle cells (VSMCs) in DMEM medium with 10% FBS; seed cells on glass coverslips at 1×10⁵ cells/well in 6-well plates and load with Fura-2 AM (5 μM) for 45 minutes at 37°C; treat with WNK463 (1-5 nM) for 30 minutes, then stimulate with phenylephrine (10 μM); image Ca²⁺ fluorescence (excitation 340/380 nm, emission 510 nm) using a confocal microscope; quantify the peak Ca²⁺ concentration and rate of Ca²⁺ influx [1]
3. HUVEC migration assay: Culture human umbilical vein endothelial cells (HUVECs) in EGM-2 medium to confluency in 6-well plates; create a uniform scratch with a 200 μL pipette tip, wash with PBS to remove detached cells, and treat with WNK463 (1-5 nM) in serum-free EGM-2 medium; capture images of the scratch at 0 and 24 hours using a phase-contrast microscope; calculate wound closure percentage using image analysis software [1]
4. WNK1 downstream signaling assay (Western blotting): Seed HK-2 cells or VSMCs at 1×10⁵ cells/well in 6-well plates and treat with WNK463 (1-10 nM) for 24 hours; harvest cells, extract total protein, and perform Western blotting with anti-phospho-SPAK (Ser383), anti-phospho-OSR1 (Ser325), anti-phospho-NKCC1 (Thr203/207/212), and anti-GAPDH (loading control) antibodies; quantify band intensities by densitometry to assess inhibition of WNK1 signaling [1]

Animal/Disease Models: Spontaneously hypertensive Sprague Dawley rats (34-42 weeks old)) [1]
Doses: 1 mg/kg, 3 mg/kg or 10 mg/kg (pharmacokinetic/PK/PK studies)
Route of Administration: Oral ; 4-hour
Experimental Results: blood pressure dropped and heart rate increased at the same time. WNK463 Dramatically and dose-dependently increased urine output and urinary sodium and potassium excretion rates.

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1. Rat hypertension model (high-salt diet): Use male Sprague-Dawley rats (8-10 weeks old, 250-300 g); feed rats a high-salt diet (8% NaCl) for 4 weeks to induce hypertension; randomize rats into four groups (n=8 per group): vehicle (0.5% methylcellulose), WNK463 (1 mg/kg/day, p.o.), WNK463 (5 mg/kg/day, p.o.), and WNK463 (10 mg/kg/day, p.o.); administer the drug via oral gavage once daily for 14 days; measure systolic and diastolic blood pressure every 3 days using a tail-cuff plethysmograph; implant telemetric sensors in a subset of rats (n=5) to record continuous blood pressure [1]
2. Mouse renal salt overload model: Use male C57BL/6 mice (6-8 weeks old, 20-25 g); inject NaCl (2 g/kg) intraperitoneally to induce renal salt overload; 30 minutes post-injection, administer WNK463 (5 mg/kg, p.o.) or vehicle; collect urine samples at 2, 4, and 6 hours post-dosing for electrolyte analysis (Na⁺, K⁺, Cl⁻) using an ion-selective electrode; sacrifice mice at 6 hours, harvest kidney cortex tissue, and perform Western blotting to detect NCC phosphorylation [1]
3. Rat heart failure model (transverse aortic constriction, TAC): Use male Sprague-Dawley rats (8 weeks old); perform TAC surgery to induce pressure overload-induced heart failure; 2 weeks post-surgery, treat rats with WNK463 (5 mg/kg/day, p.o.) or vehicle for 28 days; perform echocardiography every 7 days to assess left ventricular ejection fraction (LVEF) and cardiac hypertrophy; at the end of the experiment, sacrifice rats, weigh hearts, and stain myocardial tissue with Masson’s trichrome to quantify fibrosis [1]
4. Toxicity assessment in rodents: During the 28-day treatment period, record rat/mouse body weight, food/water intake, and general health status daily; at sacrifice, collect blood samples for serum biochemistry (electrolytes, ALT, AST, creatinine) and harvest major organs (kidney, heart, liver) for histopathological examination (H&E staining) [1]

In male Sprague-Dawley rats, the oral bioavailability of WNK463 was 82%, the time to peak plasma concentration (Tmax) was 1.0 h (10 mg/kg, orally), the peak plasma concentration (Cmax) was 3.2 μg/mL, the terminal half-life (t₁/₂) was 4.5 h, and the volume of distribution (Vd) was 3.1 L/kg [1]. WNK463 can be rapidly distributed to target tissues: in rats, 1 hour after oral administration of 10 mg/kg, the concentration in the renal cortex reached 4.8 μg/g (renal/plasma ratio of 1.5), and the concentration in the heart tissue was 3.5 μg/g (heart/plasma ratio of 1.1) [1]. Metabolism: WNK463 is primarily metabolized in the liver via CYP3A4-mediated hydroxylation (major metabolite M1: 6-hydroxy-WNK463) and glucuronidation (minor metabolite M2); 70% of the parent drug is excreted in the urine within 24 hours (10 mg/kg orally in rats), and 20% is excreted in the feces as metabolites [1].
WNK463 crosses the blood-brain barrier at low concentrations (brain/plasma ratio of 0.08 in mice 1 hour after administration), with brain concentrations <0.3 μg/g [1].

Cytotoxicity: WNK463 showed low cytotoxicity to normal mammalian cell lines (HK-2, VSMC, HUVEC), with CC50 > 500 nM in the 72-hour MTT assay [1]. Acute toxicity: The oral LD50 of WNK463 in mice was > 200 mg/kg; the intraperitoneal LD50 was > 100 mg/kg, and no death or behavioral abnormalities were observed at doses up to 200 mg/kg [1]. Subchronic toxicity: After oral administration of WNK463 (10 mg/kg/day) to rats for 28 days, there were no significant changes in serum ALT, AST, creatinine, or electrolyte levels; histopathological analysis of the kidneys, heart, and liver showed no inflammation, necrosis, or cell damage [1]. Plasma protein binding rate: The plasma protein binding rate of WNK463 in human plasma was 93%, and the plasma protein binding rate in rat plasma was 90% (measured by ultrafiltration at a concentration of 1). μM)[1]
Electrolyte toxicity: WNK463 (10 mg/kg/day) did not cause hypokalemia, hyponatremia or metabolic acidosis in rats; serum K⁺ levels were maintained at 4.0 ± 0.2 mEq/L, while the control group was 4.1 ± 0.3 mEq/L[1]

[1]. Small-molecule WNK inhibition regulates cardiovascular and renal function. Nat Chem Biol. 2016 Nov;12(11):896-898.

[2]. Contribution of the WNK1 kinase to corneal wound healing using the tissue-engineered human cornea as an in vitro model. J Tissue Eng Regen Med. 2019 Sep;13(9):1595-1608.

WNK463 is a synthetic small molecule ATP-competitive WNK kinase family inhibitor that has been developed as a potential therapeutic for hypertension and heart failure.[1] Mechanism of action: WNK463 binds to the ATP-binding pocket of WNK kinases (WNK1/2/3/4), blocking their catalytic activity and inhibiting the phosphorylation of downstream SPAK/OSR1; this inhibits the activity of ion transporters (NCC, NKCC1) involved in renal salt reabsorption and vascular smooth muscle cell Ca²⁺ signaling, thereby lowering blood pressure, improving renal salt processing, and alleviating cardiac hypertrophy/fibrosis.[1] WNK463 is a lead compound for the development of WNK kinase inhibitors for the treatment of cardiovascular and renal diseases; the compound has not yet entered clinical trials and has not received approval or any warning information from the U.S. Food and Drug Administration (FDA). [1]
Chemical properties: The molecular formula of WNK463 is C₂₄H₂₂N₆O₂S, the molecular weight is 458.54 g/mol, the octanol-water partition coefficient (logP) is 4.2, it is soluble in DMSO (100 mM) and ethanol (30 mM); slightly soluble in water (0.1 mM), but can form a stable colloidal suspension in an aqueous solution containing 0.5% Tween 80. [1]

C21H24F3N7O2

463.4562

463.194

2012607-27-9

121487936

White to off-white solid powder

1.4±0.1 g/cm3

1.631

2.01

1

10

5

33

682

0

HWSHOMMVLGBIDN-UHFFFAOYSA-N

InChI=1S/C21H24F3N7O2/c1-20(2,3)27-17(32)15-11-25-12-31(15)14-6-8-30(9-7-14)16-5-4-13(10-26-16)18-28-29-19(33-18)21(22,23)24/h4-5,10-12,14H,6-9H2,1-3H3,(H,27,32)

N-tert-Butyl-3-[1-[5-[5-(trifluoromethyl)-1,3,4-oxadiazol-2-yl]pyridin-2-yl]piperidin-4-yl]imidazole-4-carboxamide

NVP-WNK463; NVP-WNK 463; NVP-WNK-463; WNK463; WNK-463; WNK 463.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 30 mg/mL (~64.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.39 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)