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Purity: ≥98%
ZL006 is an efficient inhibitor of the nNOS/PSD-95 protein-protein interaction and showed great promise in cellular experiments and animal models of ischemic stroke and pain. In vitro, ZL006 does not interact with the PDZ domains of nNOS or PSD-95, nor inhibit the nNOS-PDZ/PSD-95-PDZ interface by interacting with the β-finger of nNOS-PDZ. ZL006 presents little cytotoxicity, and a growth inhibition of BCECs is not found at low concentration of 0.001, 0.01, 0.1, 1 and 10 μg/mL. ZL006 does not inhibit the nNOS-PDZ/PSD-95-PDZ interaction, or perturb the nNOS β-finger.
| Targets |
ZL006 was proposed to target the nNOS-PDZ/PSD-95-PDZ protein-protein interaction; however, under the applied in vitro conditions, it did not interact with PDZ domains of nNOS or PSD-95, nor inhibit the nNOS-PDZ/PSD-95-PDZ interface [2]
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| ln Vitro |
ZL006 exhibits minimal cytotoxicity, and at low concentrations of 0.001, 0.01, 0.1, 1, and 10 μg/mL, there is no growth inhibition of BCECs. At a concentration of 10 μg/mL, T7-P-LPs/ZL006 exhibits significantly increased cytotoxicity. Following a 0.5-hour incubation period, ZL006 loads P-LPs and T7-P-LPs at concentrations ranging from 100 μg/mL to 600 μg/mL in BCECs[1]. ZL006 does not disrupt the nNOS β-finger or impede the nNOS-PDZ / PSD-95-PDZ interaction[2].
1. Fluorescence polarization (FP) assays showed that ZL006 did not bind to PDZ1, PDZ2, PDZ3 of PSD-95 or nNOS-PDZ, nor inhibit the interaction between TAMRA-nNOS and PDZ1/PDZ2/PDZ1-2 of PSD-95; in the 'indirect' FP assay, ZL006 (0-2900 μM) did not inhibit the interactions between nNOS-PDZ and PDZ domains from PSD-95/α1-Syntrophin (minor fluorescence artefacts were observed at high concentrations of 260-2900 μM) [2] 2. Isothermal titration calorimetry (ITC) showed that ZL006 had very little binding to extended nNOS-PDZ and PSD-95-PDZ2 [2] 3. ¹H-¹⁵N HSQC NMR spectroscopy revealed that ZL006 caused no significant chemical shift perturbations (Δδ < 0.1) on ¹⁵N-labelled nNOS-PDZ (alone or complexed with PSD95-PDZ2) or ¹⁵N-labelled PSD-95-PDZ2 (complexed with nNOS-PDZ) [2] 4. Cellular uptake experiments showed that T7 modification increased the uptake of ZL006-loaded PEGylated liposomes (T7-P-LPs/ZL006) by brain capillary endothelial cells (BCECs) compared with unmodified liposomes (P-LPs/ZL006); saturated T7 intervention reduced the cellular uptake of T7-P-LPs/ZL006 in BCECs (p < 0.001 compared with P-LPs/ZL006) [1] 5. Cytotoxicity assays showed little cytotoxicity of blank liposomes (V-P-LPs/V-T7-P-LPs) or ZL006-loaded liposomes (P-LPs/ZL006/T7-P-LPs/ZL006) on BCECs after 72 h of treatment at 37 °C [1] |
| ln Vivo |
Owing to its superior brain targeting delivery, T7-P-LPs/ZL006 shows a marked increase in drug accumulation in the brain tissue when compared to P-LPs/ZL006 and free ZL006. P-LPs/ZL006 show a notable reduction in drug build-up in the kidney and liver[1].
1. In vivo biodistribution and near-infrared fluorescence imaging showed that T7 modification significantly enhanced the transport of ZL006-loaded PEGylated liposomes across the blood-brain barrier (BBB) in ICR mice; the brain concentration of T7-P-LPs/ZL006 was significantly higher than free ZL006 and P-LPs/ZL006 at 0.5, 1 and 2 hours after intravenous injection (p < 0.05, p < 0.01, p < 0.001) [1] 2. In a rat model of middle cerebral artery occlusion (MCAO), T7-P-LPs/ZL006 exhibited reduced infarct volume and ameliorated neurological deficit at 24 h after MCAO compared with unmodified liposomes (P-LPs/ZL006) or free ZL006 (p < 0.05, p < 0.01, p < 0.001 compared with MCAO group, Vehicle group and free ZL006 group) [1] |
| Enzyme Assay |
1. Fluorescence polarization (FP) assays were performed to evaluate the binding of ZL006 to PDZ domains (PDZ1/PDZ2/PDZ3 of PSD-95, nNOS-PDZ) and its inhibitory effect on nNOS-PDZ/PSD-95-PDZ interactions; different fluorescent probes (Cy5-GluN2B, Cy5-CRIPT, Cy5-Nav1.4, TAMRA-nNOS, TAMRA-Cnskr2, Cy5-Cnskr2, Cy5-Sapk3) were used, and the concentration of probes was 5 nM (except Cy5-Nav1.4 at 50 nM); the affinity (Kd) and inhibitory activity (Ki/IC50) of ZL006 were calculated based on the fluorescence polarization values (mP) [2]
2. Isothermal titration calorimetry (ITC) was conducted to detect the binding between ZL006 and extended nNOS-PDZ/PSD-95-PDZ2; the titration curves were fitted to a one-site model to analyze the binding affinity (Kd), stoichiometry ratio (N), enthalpy change (∆H) and entropy change (T∆S) [2] 3. ¹H-¹⁵N HSQC NMR spectroscopy was used to detect chemical shift perturbations of ¹⁵N-labelled nNOS-PDZ (alone or complexed with PSD95-PDZ2) and ¹⁵N-labelled PSD-95-PDZ2 (complexed with nNOS-PDZ) in the presence of 20 equivalents of ZL006; the chemical shift perturbations (Δδ) were calculated using Δδ = [(ΔH)² + (0.15ΔN)²]¹/² to evaluate the binding interaction [2] |
| Cell Assay |
1. Brain capillary endothelial cells (BCECs) were cultured and incubated with coumarin-6-labeled P-LPs/T7-P-LPs at concentrations ranging from 5 ng/mL to 500 ng/mL for 0.25-6 h; cellular uptake was examined by fluorescent microscopy and quantified to compare the uptake efficiency of T7-modified and unmodified liposomes [1]
2. BCECs were treated with ZL006-loaded P-LPs/T7-P-LPs at concentrations ranging from 100 μg/mL to 600 μg/mL for 0.5 h, or with 200 μg/mL of T7-P-LPs/ZL006/P-LPs/ZL006/free ZL006 for 0.5 h/1 h (with saturated T7 intervention group as control); cellular uptake of ZL006 was detected to verify the targeting effect of T7 modification [1] 3. BCECs were treated with blank liposomes (V-P-LPs/V-T7-P-LPs), free ZL006, P-LPs/ZL006 and T7-P-LPs/ZL006 for 72 h at 37 °C; cell viability was measured to evaluate the cytotoxicity of ZL006 and liposomal formulations [1] |
| Animal Protocol |
1. ICR mice were intravenously injected with free ZL006, P-LPs/ZL006 or T7-P-LPs/ZL006; the biodistribution of ZL006 in brain and other tissues (heart, liver, spleen, lung, kidney) was detected at 0.5, 1 and 2 hours after injection (n = 4); near-infrared fluorescence imaging was used to evaluate the BBB penetration ability of DiR-labeled P-LPs/T7-P-LPs in the ischemic brain at 6 and 24 h (n = 2) [1] 2. Sprague-Dawley rats were used to establish a middle cerebral artery occlusion (MCAO) model of ischemic stroke; the rats were treated with free ZL006, P-LPs/ZL006, T7-P-LPs/ZL006, vehicle or saline (sham-operated group as control); at 24 h after MCAO, brain sections were stained with TTC to quantify infarct volume (n = 9), and neurological scores were evaluated to assess the neuroprotective effect of ZL006 [1] 3. ICR mice were intravenously administered with 100 mg/kg saline or T7-P-LPs once daily for 7 days; histochemistry analysis (hematoxylin eosin staining) was performed on brain, heart, liver, spleen, lung and kidney sections to evaluate tissue toxicity (bar: 20 μm) [1] |
| ADME/Pharmacokinetics |
1. After intravenous injection of T7-P-LPs/ZL006, the concentration of ZL006 in the brain of ICR mice was significantly higher than that of free ZL006 and P-LPs/ZL006 at 0.5, 1 and 2 hours;[1]
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| Toxicity/Toxicokinetics |
1. After intravenous injection of T7-P-LPs (100 mg/kg, once daily) into ICR mice for 7 days, histochemical analysis of brain, heart, liver, spleen, lung and kidney sections showed no obvious tissue damage; cytotoxicity test showed that ZL006 and its liposome preparation had very low cytotoxicity to BCECs [1]
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| References |
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| Additional Infomation |
1. ZL006 was previously considered an effective inhibitor of nNOS/PSD-95 protein-protein interaction and showed promising application prospects in cell experiments and animal models of ischemic stroke and pain; however, this study shows that under in vitro conditions, ZL006 does not interact with or inhibit the interaction of the nNOS/PSD-95 PDZ domain, which challenges the widely accepted mechanism of action[2]. 2. ZL006 is a novel neuroprotective agent for the treatment of ischemic stroke; T7-conjugated polyethylene glycol liposomes loaded with ZL006 (T7-P-LPs/ZL006) have satisfactory vesicle size and particle size distribution, and T7 modification (targeting transferrin receptor TfR) enhances the blood-brain barrier penetration of ZL006[1]. 3. T7-P-LPs/ZL006 can target the brain and has shown significant neuroprotective effects in a rat MCAO model, reducing infarct volume and improving neurological deficits; it is a potential targeted drug delivery system for ischemic stroke treatment [1].
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| Molecular Formula |
C14H11CL2NO4
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| Molecular Weight |
328.15
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| Exact Mass |
327.006
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| Elemental Analysis |
C, 51.24; H, 3.38; Cl, 21.61; N, 4.27; O, 19.50
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| CAS # |
1181226-02-7
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| Related CAS # |
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| PubChem CID |
44207238
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| Appearance |
Light yellow to khaki solid powder
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
530.4±50.0 °C at 760 mmHg
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| Flash Point |
274.6±30.1 °C
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| Vapour Pressure |
0.0±1.5 mmHg at 25°C
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| Index of Refraction |
1.728
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| LogP |
4.57
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
21
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| Complexity |
371
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1=C([H])C(=C([H])C(=C1O[H])C([H])([H])N([H])C1C([H])=C([H])C(C(=O)O[H])=C(C=1[H])O[H])Cl
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| InChi Key |
RTEYSQSXRFVKTJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H11Cl2NO4/c15-8-3-7(13(19)11(16)4-8)6-17-9-1-2-10(14(20)21)12(18)5-9/h1-5,17-19H,6H2,(H,20,21)
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| Chemical Name |
4-[(3,5-dichloro-2-hydroxyphenyl)methylamino]-2-hydroxybenzoic acid
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.62 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.62 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (7.62 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 | 3.0474 mL | 15.2369 mL | 30.4739 mL | |
| 5 mM | 0.6095 mL | 3.0474 mL | 6.0948 mL | |
| 10 mM | 0.3047 mL | 1.5237 mL | 3.0474 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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