| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
SPAK[1]
STE20/SPS1-related proline-alanine-rich kinase (SPAK). ZT-1a is a non-ATP-competitive inhibitor of SPAK, meaning it binds to a site other than the ATP-binding pocket, which contributes to its selectivity. By inhibiting SPAK, it prevents the phosphorylation and activation of its downstream targets, the cation-chloride cotransporters (CCCs), including the Na-K-2Cl cotransporter (NKCC1) and the K-Cl cotransporter (KCC3). This modulation of CCC activity affects cellular ion balance, cell volume regulation, and neuronal excitability. |
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| ln Vitro |
ZT-1a decreases the phosphorylation of the Na-K-2Cl cotransporter (NKCC1) and increases the K-Cl cotransporter (KCC) through SPS1-associated proline/alanine-rich kinase (SPAK) [1]. ZT-1a inhibits the phosphorylation of NKCC1 p-Thr203/207/212 by 72±5.2% at 1 μM ZT-1a, and the phosphorylation of KCC site 1/2 in HEK-293 by 65-77% at 3 μM ZT-1a [1]. At 3–10 μM ZT-1a, there is a 70±3.8% inhibition on the phosphorylation of SPAK at Ser373 [1]. Ten micrograms of ZT-1a inhibits NKCC1, but increases KCC3 activity [1].
ZT-1a inhibits SPAK activity in vitro. In HEK-293 cells, ZT-1a at 1 µM inhibits the phosphorylation of NKCC1 at its SPAK-dependent sites (Thr203/207/212) by 72±5.2%. At 3 µM, it inhibits the phosphorylation of KCC sites 1/2 by 65-77%. SPAK phosphorylation at Ser373 is inhibited by 70±3.8% at 3-10 µM ZT-1a. These cellular activities confirm the compound's mechanism of action. |
| ln Vivo |
In vivo, ZT-1a (10–100 mg/kg) prevents SPAK-dependent phosphorylation of the cation-Cl-cotransporter (CCC)[1].
ZT-1a has been shown to protect grey and white matter in the ischemic brain by inhibiting SPAK-dependent phosphorylation of NKCC1 and KCC3. In vivo, ZT-1a (10-100 mg/kg) inhibits SPAK-dependent cation-chloride cotransporter (CCC) phosphorylation. This suggests that ZT-1a can effectively engage its target in living organisms and modulate downstream CCC activity, leading to neuroprotective effects. |
| Enzyme Assay |
The in vitro activity of ZT-1a is assessed using a kinase assay with purified SPAK enzyme. The enzyme is incubated with a peptide substrate and ATP in the presence of varying concentrations of ZT-1a. The level of substrate phosphorylation is measured, and the IC50 is determined from a dose-response curve. To confirm its non-competitive nature, the assay is performed at different ATP concentrations.
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| Cell Assay |
To study its cellular effects, HEK-293 cells or other relevant cell lines are treated with ZT-1a. The phosphorylation status of SPAK and its downstream targets, NKCC1 and KCC3, is analyzed by Western blot using phospho-specific antibodies. The effect of ZT-1a on cell volume and ion flux can also be measured using fluorescent dyes or electrophysiology.
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| Animal Protocol |
Animal/Disease Models: Naive mice[1]
Doses: 10, 30, 50, and 100 mg/kg Route of Administration: intraperitoneal (ip) administration Experimental Results: Inhibited SPAK-dependent cation-Cl- cotransporters (CCC) phosphorylation in vivo. The in vivo efficacy of ZT-1a is typically evaluated in rodent models of ischemic stroke or other neurological disorders. The compound is administered intraperitoneally or orally at doses ranging from 10-100 mg/kg. The extent of brain injury is assessed by measuring infarct volume and by evaluating neurological deficits using behavioral tests. The phosphorylation of SPAK and CCCs in brain tissue is also analyzed to confirm target engagement. |
| ADME/Pharmacokinetics |
ZT-1a has a molecular weight of 445.73 g/mol and a molecular formula of C22H15Cl3N2O2. It is soluble in DMSO at 89 mg/mL (199.67 mM) but is practically insoluble in water. For in vivo studies, it can be formulated as a clear solution in 5% DMSO, 40% PEG300, 5% Tween 80, and 50% ddH2O (4.45 mg/mL) or as a suspension in CMC-Na (≥5 mg/mL). It should be stored as a solid at -20°C, protected from light and moisture.
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| Toxicity/Toxicokinetics |
Specific toxicological data for ZT-1a are not available, as it is a research compound. It is not intended for human or veterinary use. Standard laboratory safety precautions, including the use of personal protective equipment (PPE), should be followed when handling the compound. Its safety profile would be established through dedicated preclinical toxicology studies if it were to be developed further.
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| References | |
| Additional Infomation |
ZT-1a is a research compound used to study the role of SPAK in various physiological and pathological processes, particularly in the central nervous system. Its ability to inhibit SPAK-dependent CCC phosphorylation makes it a valuable tool for investigating the pathophysiology of stroke, brain edema, and other disorders linked to ionic imbalance. It has shown therapeutic potential in preclinical models for the prevention and treatment of neurodegenerative and neurocognitive disorders. ZT-1a is not an approved drug and is strictly for research purposes.
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| Molecular Formula |
C22H15CL3N2O2
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|---|---|
| Molecular Weight |
445.725702524185
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| Exact Mass |
444.019
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| CAS # |
212135-62-1
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| PubChem CID |
10789506
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| Appearance |
White to off-white solid powder
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| LogP |
6.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
29
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| Complexity |
616
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=C(C(C)=CC=1C(C#N)C1C=CC(=CC=1)Cl)NC(C1C=C(C=CC=1O)Cl)=O
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| InChi Key |
RPTKRVXJNWPJLU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H15Cl3N2O2/c1-12-8-16(18(11-26)13-2-4-14(23)5-3-13)19(25)10-20(12)27-22(29)17-9-15(24)6-7-21(17)28/h2-10,18,28H,1H3,(H,27,29)
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| Chemical Name |
5-chloro-N-[5-chloro-4-[(4-chlorophenyl)-cyanomethyl]-2-methylphenyl]-2-hydroxybenzamide
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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: 100 mg/mL (224.35 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.67 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 20.8 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: ≥ 2.08 mg/mL (4.67 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 20.8 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (4.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2435 mL | 11.2176 mL | 22.4351 mL | |
| 5 mM | 0.4487 mL | 2.2435 mL | 4.4870 mL | |
| 10 mM | 0.2244 mL | 1.1218 mL | 2.2435 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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