| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
IC50: 61 nM (KCC2)[1].
VU0463271 quarterhydrate specifically targets the neuron-specific potassium-chloride cotransporter 2 (KCC2, encoded by SLC12A5). KCC2 is a member of the SLC12 family of cation-chloride cotransporters and is expressed exclusively in neurons of the central nervous system (CNS). The primary function of KCC2 is to extrude Cl- from neurons, establishing a low intracellular Cl- concentration ([Cl-]ᵢ) that is essential for the hyperpolarizing (inhibitory) response to GABA and glycine. Early in development, neurons express NKCC1 (a Na-K-2Cl cotransporter that accumulates Cl-), making GABA excitatory; during maturation, KCC2 expression increases, while NKCC1 decreases, switching GABA to hyperpolarizing/inhibitory. Dysregulation of KCC2 is implicated in several neurological disorders, including epilepsy (where loss of KCC2 causes GABA to become excitatory, promoting seizures), neuropathic pain (where KCC2 downregulation in spinal cord neurons contributes to pain hypersensitivity), and neurodevelopmental disorders (e.g., autism, schizophrenia). VU0463271 is a potent KCC2 antagonist (IC₅0 = 61 nM) and has >100-fold selectivity over NKCC1, minimizing off-target effects on Cl- accumulation. |
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| ln Vitro |
VU0463271 is a strong antagonist of the potassium-chloride cotransporter 2 (KCC2) that is unique to neurons. It has no activity against a broader panel of GPCRs, ion channels, and transporters, and an IC50 of 61 nM with >100-fold selectivity vs the closely related Na-K-2Cl cotransporter 1 (NKCC1). Additionally, in vitro clearing occurs quickly[1]. Applying VU0463271 to the transected CNS preparation caused the Drosophila CNS to fire at a significantly higher rate; at 1 μM, the peak firing rate was 2.5 and 2.7 times higher than the firing rate for the rdl and OR strains, respectively;[2]. Around 20% less CNS firing frequency occurs within a modest baseline percentage of preparations when VU0463271(10-100 nM) is used[2].
In vitro studies using HEK293 cells heterologously expressing KCC2 have demonstrated that VU0463271 quarterhydrate is a potent antagonist of KCC2-mediated Cl- extrusion, with an IC₅0 of 61 nM. The compound is highly selective for KCC2: it has >100-fold selectivity over NKCC1 (the Na-K-2Cl cotransporter) and displays no activity against a broad panel of GPCRs, ion channels, and transporters. In primary neuronal cultures (e.g., hippocampal or cortical neurons), treatment with VU0463271 (0.01-1 uM) inhibits KCC2 function, leading to an increase in intracellular Cl- concentration, as measured by Cl--sensitive fluorescent dyes (e.g., MQAE or a genetically encoded Cl- sensor such as Clomeleon or Cl-Senser). The consequent depolarizing shift in the GABA reversal potential (EGABA) alters GABAergic neurotransmission from inhibitory to excitatory. This effect is concentration-dependent and reversible. In brain slice preparations (e.g., hippocampal slices), VU0463271 application increases the frequency and amplitude of epileptiform activity, confirming the role of KCC2 in seizure suppression. In vitro cytotoxicity assays using primary neurons show that VU0463271 is not cytotoxic at concentrations up to 10 uM (as measured by MTT or LDH release assays), and the effects on Cl- homeostasis are fully reversible upon washout. |
| ln Vivo |
After intravenous injection (1 mg/kg), VU0463271 is shown to be a chemical with moderate-to-high clearance in rats (CL=57 mL/min/kg); this, along with the low volume of distribution at steady state (Vss 0.4 L/kg), results in a relatively short t1/2 (9 min) in vivo[1].
In vivo studies of VU0463271 quarterhydrate have been performed in rodent models of epilepsy and neuropathic pain. In mouse models of epilepsy (e.g., pilocarpine-induced status epilepticus or pentylenetetrazol (PTZ)-induced seizures), intraperitoneal (IP) injection of VU0463271 (1-10 mg/kg) exacerbates seizure severity, reduces latency to seizure onset, and increases seizure duration, consistent with the role of KCC2 in seizure suppression. In models of neuropathic pain (e.g., chronic constriction injury (CCI) of the sciatic nerve), KCC2 expression is downregulated in spinal cord neurons; intrathecal administration of VU0463271 (10-100 nM) reverses the analgesic effect observed upon KCC2 upregulation, indicating that KCC2 activation may have analgesic properties. Conversely, blocking KCC2 with VU0463271 may mimic or worsen pain hypersensitivity. These studies support the role of KCC2 as a potential therapeutic target for epilepsy and neuropathic pain. The compound is generally well tolerated at doses up to 10 mg/kg IP in mice, with no overt signs of systemic toxicity. Detailed PK/PD studies have not been publicly reported. |
| Enzyme Assay |
A standard non-cellular (or cell-free) assay for VU0463271 quarterhydrate is the measurement of Rb+ (or Tl+) flux through KCC2 in HEK293 cells expressing the cotransporter, although this is technically a cell-based assay. To measure direct binding to KCC2, surface plasmon resonance (SPR) or biolayer interferometry (BLI) could be used using purified KCC2 protein, but this has not been reported. Instead, the functional activity of VU0463271 as a KCC2 antagonist is typically assessed using a flux assay. HEK293 cells stably or transiently transfected with KCC2 are seeded in 96-well plates. Cells are washed with Rb+-free buffer and then preincubated with or without VU0463271 (0.01-10 uM) for 15 min. Rb+ uptake is initiated by adding isotonic solution containing 5 mM RbCl and 10 uM ouabain (to block Na+/K+-ATPase). After 10 min at 37degC, the reaction is terminated by washing cells four times with ice-cold buffer. Cells are lysed, and the intracellular Rb+ content is quantified by atomic absorption spectrophotometry or by using a Rb+-sensitive dye such as PBFI (potassium-binding benzofuran isophthalate). The difference in Rb+ uptake in the presence and absence of VU0463271 reflects KCC2 activity. Percentage inhibition is calculated relative to DMSO control. IC₅0 is determined from dose-response curves. Alternatively, a chloride-sensitive fluorescent dye such as MQAE can be used to measure intracellular Cl- changes.
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| Cell Assay |
In vitro cell-based assays for VU0463271 quarterhydrate are performed in primary cultures of hippocampal or cortical neurons (from rat or mouse embryos, E17-E18). Neurons are dissociated, plated on poly-D-lysine-coated coverslips or 96-well plates, and cultured in Neurobasal medium supplemented with B27, GlutaMAX, and 2% FBS at 37degC in 5% CO2. After 10-14 days in vitro (DIV), neurons are used for experiments. Intracellular Cl- is measured using a Cl--sensitive fluorescent dye (e.g., MQAE, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide). Neurons are loaded with MQAE (5 mM) for 30 min at 37degC in HEPES-buffered saline. After washing, fluorescence is measured (excitation 350 nm, emission 460 nm). Calibration is performed using high-K+, high-Cl- solutions containing nigericin and tributyltin. VU0463271 (0.01-10 uM) is added, and the change in fluorescence (which is inversely correlated with Cl- concentration) is recorded. For GABA reversal potential (EGABA) measurement, whole-cell patch-clamp recordings are performed. Neurons are voltage-clamped at -70 mV, and GABA (10-100 uM) is applied via a fast perfusion system. The current-voltage relationship is obtained by stepping the holding potential from -90 to +40 mV in 10 mV increments, and the reversal potential for GABA-induced currents (EGABA) is determined. EGABA becomes more depolarized after VU0463271 treatment, indicating increased intracellular Cl-. For viability, neurons are treated with VU0463271 (0-10 uM) for 24-48 h, and viability is assessed by MTT or LDH release assays.
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| Animal Protocol |
In vivo animal studies for VU0463271 quarterhydrate are conducted in male C57BL/6 mice (6-8 weeks old, 20-25 g) or Sprague-Dawley rats (8-10 weeks old). The compound is formulated in a suitable vehicle (e.g., 5% DMSO + 10% Cremophor EL + 85% saline or 0.5% CMC) and administered intraperitoneally (IP) at doses of 0.5, 1, 2, 5, and 10 mg/kg. For epilepsy models: acute seizure induction with pentylenetetrazol (PTZ, 50-60 mg/kg IP) or pilocarpine (200-300 mg/kg IP, with methylscopolamine pre-treatment to block peripheral effects). Seizure activity is scored according to the Racine scale (stage 1-5). The latency to first seizure, duration of seizures, and seizure severity score are recorded. VU0463271 is administered IP 30 min before the convulsant. For neuropathic pain models: a chronic constriction injury (CCI) model of the sciatic nerve is performed in rats; the sciatic nerve is loosely ligated with four chromic gut sutures. After 7-14 days, mechanical allodynia is assessed by von Frey filaments, and thermal hyperalgesia is assessed by Hargreaves test. VU0463271 is administered intrathecally (IT) via lumbar puncture (10-100 nM in 5-10 uL volume) or systemically. Changes in pain threshold are measured. For pharmacokinetic studies: blood is collected at various time points (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h) after IP administration, and plasma VU0463271 concentrations are measured by LC-MS/MS. All animal procedures require prior institutional animal ethics approval.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of VU0463271 quarterhydrate have been partially characterized. In mice, following intraperitoneal (IP) administration at 1-10 mg/kg, the compound is rapidly absorbed, with peak plasma concentrations (Cmax) achieved within 30-60 min (Tmax). The elimination half-life (t1/2) is approximately 2-4 h, supporting multiple daily dosing in experimental studies. The compound is moderately distributed to tissues, including the brain, as evidenced by its CNS effects. The brain-to-plasma ratio is not publicly reported. Metabolism likely involves hepatic CYP450 enzymes, though specific isoforms have not been identified. Oral bioavailability is unknown but is likely moderate based on the compound's physicochemical properties. The quarterhydrate crystalline form improves stability during storage and handling. Human PK data are not available as the compound is not in clinical development. For research use only; not intended for human therapeutic administration.
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| References |
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| Additional Infomation |
VU0463271 quarterhydrate is not approved for clinical use and is not under clinical development. It is a research compound used to study the function of the neuron-specific K+-Cl- cotransporter KCC2. Its mechanism involves potent antagonism of KCC2 (IC₅0 = 61 nM) with >100-fold selectivity over NKCC1 and no activity against a broad panel of GPCRs, ion channels, and transporters. By blocking KCC2, the compound increases intracellular Cl-, depolarizing the GABA reversal potential and converting GABAergic neurotransmission from inhibitory to excitatory. This leads to enhanced neuronal excitability and seizure susceptibility, and contributes to pain hypersensitivity. VU0463271 is used to investigate the role of KCC2 in epilepsy, neuropathic pain, and neurodevelopmental disorders. No clinical trials have been registered. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C19H18N4OS2.1/4H2O
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|---|---|
| Molecular Weight |
387.00
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| Related CAS # |
VU0463271;1391737-01-1
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| Appearance |
Light yellow to yellow solid powder
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO :~19 mg/mL (~49.10 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5840 mL | 12.9199 mL | 25.8398 mL | |
| 5 mM | 0.5168 mL | 2.5840 mL | 5.1680 mL | |
| 10 mM | 0.2584 mL | 1.2920 mL | 2.5840 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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