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nNOS/NOS1AP interaction[1]
The interaction between neuronal nitric oxide synthase (nNOS) and nitric oxide synthase 1 adaptor protein (NOS1AP, also known as CAPON). nNOS is an enzyme that produces nitric oxide (NO) in neurons, which is involved in synaptic plasticity, neurotoxicity, and pain signaling. NOS1AP binds to nNOS and modulates its activity and subcellular localization. Disruption of the nNOS-NOS1AP interaction has been shown to reduce pain responses and have anxiolytic effects. (Rac)-ZLc-002 is an inhibitor of this protein-protein interaction. |
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| ln Vitro |
(Rac)-ZLc-002 (10 μM) attenuates the co-immunoprecipitation of nNOS and NOS1AP in primary cultured cortical neurons. In vitro binding assays using AlphaScreen, (Rac)-ZLc-002 was unable to break the nNOS-NOS1AP protein-protein interaction. inert in tests conducted without cells [1]. In HEK293T cells that co-express full-length nNOS, (Rac)-ZLc-002 (10 μM) can decrease the co-immunoprecipitation of full-length NOS1AP but not PSD95-PDZ2 [1]. In order to lower tumor cell viability, (Rac)-ZLc-002 (0-50 μM; 72 h) works in concert with Paclitaxel[1].
In vitro, ZLc-002 (10 microM) attenuates the co-immunoprecipitation of nNOS and NOS1AP in primary cultured cortical neurons. However, in a cell-free AlphaScreen in vitro binding assay, (Rac)-ZLc-002 fails to disrupt the nNOS-NOS1AP protein-protein interaction, suggesting that its mechanism may involve additional factors or require cellular context. Treatment with ZLc-002 alone (0-50 microM; 72 hours) has no effect on tumor cell viability, but it shows a synergistic effect with Paclitaxel to reduce viability. |
| ln Vivo |
In mouse models, (Rac)-ZLc-002 (10 mg/kg; ip; once or daily for 8 days) suppresses Paclitaxel-induced neuropathic pain and lessens mechanical and cold ectopic pain (HY-B0015) [1]. In the spinal dorsal horn of rats, (Rac)-ZLc-002 (4 and 10 mg/kg; ip; once) decreases formalin-induced nociceptive behavior and formalin-like immunoreactivity [1]. In ICR mice, the anxiety behavior caused by chronic mild stress (CMS) is attenuated by (Rac)-ZLc-002 (40 mg/kg/day; iv; 7 days) [2]. The behavioral effects of glucocorticoids in mice were reversed when (Rac)-ZLc-002 (10 μM; 1 μl; 30 min after Corticosterone, HY-B1618) was injected into the hippocampus for 7 days [2].
In mouse models, (Rac)-ZLc-002 (10 mg/kg; intraperitoneal; once daily for 8 days) suppresses Paclitaxel-induced neuropathic pain and lessens mechanical and cold allodynia. In rats, ZLc-002 (4 and 10 mg/kg; intraperitoneal; once) reduces formalin-evoked nociceptive behavior and Fos-like immunoreactivity in the spinal dorsal horn. ZLc-002 (40 mg/kg/day; intravenous; 7 days) attenuates chronic mild stress (CMS)-induced anxiogenic behavior in ICR mice. The compound also shows efficacy in corticosterone-induced anxiety models. |
| Enzyme Assay |
Since the compound fails to disrupt nNOS-NOS1AP in a cell-free AlphaScreen assay, non-cell binding data is limited. For standard protein-protein interaction assays, recombinant nNOS and NOS1AP proteins are purified. AlphaScreen (amplified luminescent proximity homogeneous assay) is performed: donor and acceptor beads conjugated to the respective interacting proteins are incubated with varying concentrations of test compound. If the interaction is disrupted, the signal decreases. (Rac)-ZLc-002 did not show activity in this cell-free system, indicating its mechanism may involve additional factors.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: 4T1 cells Tested Concentrations: 0-50 µM Incubation Duration: 72 h Experimental Results: Treatment alone had no effect on the viability. demonstrated synergistic effect with Paclitaxel. Cellular co-immunoprecipitation (Co-IP) assays are performed in primary cultured cortical neurons or HEK293T cells co-expressing full-length nNOS and NOS1AP. Cells are treated with (Rac)-ZLc-002 (10 microM) for 2-24 hours. Cells are lysed, and nNOS is immunoprecipitated using an anti-nNOS antibody. The immunoprecipitate is analyzed by Western blot using an anti-NOS1AP antibody to detect the amount of NOS1AP bound to nNOS. A reduction in co-immunoprecipitated NOS1AP indicates successful inhibition of the interaction. ZLc-002 (10 microM) reduces co-immunoprecipitation of full-length NOS1AP but not of PSD95-PDZ2 from HEK293T cells co-expressing full-length nNOS. |
| Animal Protocol |
Animal/Disease Models: Adult C57BL/6J male mice, paclitaxel model of neuropathic pain[1]
Doses: 10 mg/kg Route of Administration: IP, once or daily for 8 days Experimental Results: Increased postinjection mechanical paw withdrawal thresholds, mechanical paw withdrawal thresholds differed across postinjection times, and the interaction between drug treatment and injection time was significant. Elevated mechanical paw withdrawal thresholds relative to vehicle treatment from 30 min. diminished postinjection cold responsiveness, cold responsiveness did not differ reliably across postinjection times, and the interaction between drug treatment and injection time was not significant. one time/day dosing increased mechanical paw withdrawal thresholds relative to the vehicle-treated group across the observation interval. For the formalin test in rats (inflammatory pain model), male Sprague-Dawley rats are injected intraperitoneally with formalin (50 microL, 5%) into the hind paw. (Rac)-ZLc-002 (4 and 10 mg/kg) is administered intraperitoneally once, 30 minutes before formalin injection. Nociceptive behavior (licking/flinching of the injected paw) is measured for 60 minutes. For the Paclitaxel-induced neuropathic pain model, mice are treated with Paclitaxel (2 mg/kg, intraperitoneal) every other day for 4 doses to induce neuropathy. Starting after the final Paclitaxel dose, (Rac)-ZLc-002 (10 mg/kg, intraperitoneal) is administered once daily for 8 days. Mechanical allodynia is measured using von Frey filaments, and cold allodynia is measured using the acetone test. For chronic mild stress (CMS) anxiety model, ICR mice are exposed to CMS for 4 weeks, then treated with ZLc-002 (40 mg/kg/day, intravenous, 7 days). Anxiety-related behaviors are assessed using open-field test, elevated plus maze, and light-dark test. |
| ADME/Pharmacokinetics |
(Rac)-ZLc-002 is soluble in DMSO at 100 mg/mL (432.43 mM). For in vivo administration, it can be formulated in vehicles such as 10% DMSO in saline or DMSO/PEG300/Tween-80/saline. The compound has a molecular weight of 231.25, which is favorable for oral bioavailability. However, detailed PK parameters such as half-life, Cmax, AUC, and oral bioavailability are not publicly available for this research compound. Storage: Powder -20degC for 3 years; In solvent -80degC for 6 months or -20degC for 1 month.
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| Toxicity/Toxicokinetics |
Toxicity data for (Rac)-ZLc-002 is not publicly available. In animal studies at efficacious doses (4-10 mg/kg intraperitoneal, 40 mg/kg intravenous), no overt signs of toxicity (e.g., significant body weight loss, abnormal behavior, or mortality) have been reported. The compound shows no activity on tumor cell viability when used alone at concentrations up to 50 microM, suggesting low intrinsic cytotoxicity. Comprehensive safety studies, including genotoxicity and chronic toxicity, have not been published for this research tool compound.
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| References |
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| Additional Infomation |
(Rac)-ZLc-002 is a research-grade chemical used to study the nNOS-NOS1AP protein-protein interaction in pain and anxiety disorders. The development of selective disruptors of the nNOS-NOS1AP interaction represents a novel therapeutic strategy for treating neuropathic pain and anxiety without the side effects associated with direct NO inhibition or nNOS enzymatic inhibitors. nNOS-NOS1AP coupling has been implicated in stress-induced anxiety and neuropathic pain. Key references include the discovery literature. This product is for research use only and is not FDA-approved for human therapy.
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| Molecular Formula |
C10H17NO5
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|---|---|
| Molecular Weight |
231.25
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| Related CAS # |
(S)-ZLc002;308277-46-5;ZLc-002;1446971-41-0
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| Appearance |
Off-white to light 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 :~100 mg/mL (~432.43 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 | 4.3243 mL | 21.6216 mL | 43.2432 mL | |
| 5 mM | 0.8649 mL | 4.3243 mL | 8.6486 mL | |
| 10 mM | 0.4324 mL | 2.1622 mL | 4.3243 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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