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| 5mg |
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| 10mg |
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| Targets |
cis-Ned19 primarily targets the NAADP receptor, which has been identified as the two-pore channels (TPCs) located on the membranes of acidic organelles, specifically lysosomes and endolysosomal vesicles. It acts as a specific antagonist of the NAADP-mediated calcium signaling pathway. The compound's mechanism involves the inhibition of calcium release from these acidic stores by acting on the TPC complex. Studies indicate that Ned-19 has two binding sites on the NAADP receptor, suggesting a complex mode of interaction. The compound competes with NAADP for binding to its receptor, as demonstrated by its ability to displace [32P]NAADP in binding assays. This competitive binding effectively blocks NAADP from triggering calcium release, thereby antagonizing the downstream signaling events. Furthermore, cis-Ned19's primary mechanism involves binding to the NAADP receptor and preventing NAADP-induced calcium release from acidic organelles. Its specificity for the NAADP pathway is highlighted by its inability to inhibit calcium release mediated by other second messengers such as inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose (cADPR), as shown in concentration-inhibition curves. This selectivity makes it a valuable tool for specifically studying NAADP-dependent calcium mobilization without interfering with other major calcium signaling pathways.
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| ln Vitro |
In cell-based assays, cis-Ned19 is used to block NAADP-mediated calcium signaling. Its activity is typically assessed by measuring its ability to inhibit NAADP-induced calcium release in various cell types. For instance, it antagonizes NAADP-mediated calcium spiking in mouse pancreatic beta cells. In primary memory CD4+ T cells, Ned-19 has been shown to inhibit T cell receptor (TCR)-mediated calcium flux and its downstream effector functions, such as proliferation and cytokine production. The study revealed that both extracellular and intracellular calcium stores, including endoplasmic reticulum and lysosome-like acidic calcium stores, contribute to the TCR-mediated calcium flux, and Ned-19 effectively inhibited this process. Furthermore, cis-Ned19 fluorescently labels NAADP receptors with excitation and emission maxima at approximately 365 nm and 410 nm, respectively. This fluorescent property enables researchers to visualize the receptor's localization and potentially track its dynamics in live cells. The compound has been identified through virtual screening as a selective chemical probe for studying NAADP-dependent calcium mobilization pathways. Its ability to inhibit NAADP signaling in a variety of cell types, including immune cells and neurons, underscores its utility as a broad-spectrum tool for investigating the role of NAADP in diverse cellular functions.
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| ln Vivo |
In vivo activity data for cis-Ned19 are relatively limited in the published literature, as its primary application has been as a chemical probe in cell-based systems. However, some studies have explored its effects in animal models. For instance, Ned-19 has been reported to strongly inhibit tumor growth, vascularization, and lung metastases in mice, indicating a potential role for NAADP signaling in cancer progression. Additionally, inhibiting NAADP in vivo in an anti-CD3 mouse model resulted in an ameliorated disease course, suggesting that NAADP signaling may be involved in inflammatory or immune responses. These findings highlight the therapeutic potential of targeting the NAADP pathway. Despite these promising results, systematic and detailed in vivo efficacy studies for cis-Ned19 are not extensively documented, and further research is needed to fully understand its pharmacokinetics and pharmacodynamics in living organisms. The compound's utility as a research tool, however, remains central to its application, providing crucial insights into the biological significance of NAADP signaling.
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| Enzyme Assay |
Non-cell-based receptor binding assays for cis-Ned19 typically involve competitive binding experiments using radiolabeled NAADP. In these assays, the compound's ability to displace [32P]NAADP from its receptor is measured. For example, one study demonstrated that Ned-19 competes with [32P]NAADP binding, and the dissociation of Ned-19 (10 μM) and NAADP (10 nM) was determined by the recovery of [32P]NAADP binding after 5 days of incubation. The concentration-inhibition curves for Ned-19 on [32P]NAADP binding have also been generated. These experiments are typically performed using a calcium-mobilizing bioassay, such as sea urchin egg homogenate, which is a well-established system for studying calcium release. In this system, the release of calcium from intracellular stores is monitored using a calcium-reporting dye like fluo-3, and the inhibition of NAADP-mediated calcium release by cis-Ned19 is measured. The specificity of cis-Ned19 is confirmed by showing that it does not inhibit calcium release mediated by other second messengers, such as IP3 and cADPR, at their half-maximal concentrations. These cell-free assays are crucial for characterizing the compound's binding affinity, selectivity, and mechanism of action at the molecular level.
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| Cell Assay |
For in vitro cellular experiments, cells are typically treated with cis-Ned19 at various concentrations, often in the range of 1-100 µM, for a period of 30 minutes to several hours. The primary readout is the measurement of NAADP-induced calcium release using calcium-sensitive dyes such as Fura-2 or Fluo-4. For instance, in a study on memory CD4+ T cells, the effect of Ned-19 on TCR activation was investigated. Cells were treated with Ned-19, and the TCR-mediated calcium flux was measured. The results showed that Ned-19 inhibited this calcium flux and the downstream effector functions, including proliferation and cytokine production. In another example, NAADP-AM, a cell-permeable analogue of NAADP, was used to release calcium in memory CD4+ T cells, and this calcium flux was inhibited by Ned-19, confirming its role as an NAADP pathway antagonist. Additionally, fluorescence microscopy can be employed to visualize the labeling of NAADP receptors by cis-Ned19, utilizing its fluorescent properties (Ex/Em = 365/410 nm). These experiments are essential for confirming the compound's mechanism of action and for studying the functional consequences of NAADP signaling in various cellular contexts.
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| Animal Protocol |
In vivo animal studies for cis-Ned19 have been reported but are not as extensively documented as in vitro experiments. In one study, Ned-19 was shown to strongly inhibit tumor growth, vascularization, and lung metastases in mice, suggesting that NAADP signaling plays a critical role in cancer progression. In another in vivo model, inhibiting NAADP in an anti-CD3 mouse model resulted in an ameliorated disease course, indicating a role for NAADP in inflammatory responses. These studies typically involve administering the compound to animals via injection or other suitable routes, followed by monitoring disease progression, tumor growth, or other relevant physiological parameters. However, detailed protocols, such as specific dosages, routes of administration, and treatment schedules, are not always fully described in the available literature. The primary application of cis-Ned19 has been in cell-based systems to study NAADP signaling, and its use in vivo is an area of ongoing research. Further studies are needed to establish comprehensive in vivo efficacy and safety profiles for this compound.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of cis-Ned19, including absorption, distribution, metabolism, and excretion (ADME), have not been extensively characterized in published, peer-reviewed studies. As a research probe, its development and application have primarily focused on its use as a tool for studying NAADP signaling in vitro and in cell-based assays. While it is known to be a cell-permeant compound, which suggests it can cross biological membranes, detailed pharmacokinetic parameters such as bioavailability, half-life, protein binding, and metabolic pathways are not available in the public domain. This lack of comprehensive pharmacokinetic data is common for many research chemicals that are not intended for therapeutic use. The compound's primary value lies in its ability to selectively modulate NAADP signaling in experimental settings, rather than as a drug candidate with optimized drug-like properties. Consequently, its pharmacokinetic profile has not been a primary focus of investigation.
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| Toxicity/Toxicokinetics |
Toxicological data for cis-Ned19 are not available in the public literature. As a research chemical, its safety profile has not been systematically evaluated in standard toxicology studies. The available information primarily focuses on its biological activity as a NAADP antagonist and its use as a fluorescent probe. Researchers handling this compound should follow standard laboratory safety practices, including the use of appropriate personal protective equipment and working in a well-ventilated area. The lack of toxicological data underscores that cis-Ned19 is intended for research purposes only and is not approved for human use.
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| References | |
| Additional Infomation |
cis-Ned19 is a research tool for studying NAADP signaling pathways. Its identification as a chemical probe for NAADP was a significant breakthrough, reported in high-impact journals such as Nature Chemical Biology. It is used to investigate the role of NAADP in various physiological processes, including calcium signaling, autophagy, and lysosomal function. The compound's dual functionality as an antagonist and a fluorescent label makes it a unique and valuable reagent. It is important to note that cis-Ned19 is the ‘cis’ diastereomer, and its ‘trans’ counterpart (trans-Ned19) is also used in research. The compound is typically stored as a powder at -20°C and is soluble in DMSO. It is exclusively for research purposes and not for therapeutic or diagnostic use. The development of cis-Ned19 has paved the way for a deeper understanding of NAADP's role in health and disease, making it an indispensable tool in the field of calcium signaling.
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| Molecular Formula |
C30H31FN4O3
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| Molecular Weight |
514.59
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| Exact Mass |
514.238
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| CAS # |
1137264-00-6
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| PubChem CID |
1427626
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
4.657
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
38
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| Complexity |
810
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| Defined Atom Stereocenter Count |
2
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| SMILES |
O1C2=CC=CC=C2OCC1CNCCOCCOC
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| InChi Key |
FUHCEERDBRGPQZ-LSYYVWMOSA-N
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| InChi Code |
InChI=1S/C30H31FN4O3/c1-38-27-11-10-19(16-20(27)18-34-12-14-35(15-13-34)26-9-5-3-7-23(26)31)28-29-22(17-25(33-28)30(36)37)21-6-2-4-8-24(21)32-29/h2-11,16,25,28,32-33H,12-15,17-18H2,1H3,(H,36,37)/t25-,28-/m0/s1
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| Chemical Name |
(1S,3S)-1-[3-[[4-(2-fluorophenyl)piperazin-1-yl]methyl]-4-methoxyphenyl]-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 1.9433 mL | 9.7165 mL | 19.4329 mL | |
| 5 mM | 0.3887 mL | 1.9433 mL | 3.8866 mL | |
| 10 mM | 0.1943 mL | 0.9716 mL | 1.9433 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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