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| Targets |
NCGC 84 (ML154) targets the neuropeptide S receptor (NPSR), a G protein-coupled receptor (GPCR) that couples to both Gs and Gq signaling pathways. NPSR is expressed in various brain regions, including the central amygdala, hypothalamus, and brainstem, where it modulates stress responses, arousal, anxiety, and addiction behaviors. ML154 acts as a competitive antagonist at NPSR with a pA₂ of 9.98. It exhibits biased antagonism, preferentially inhibiting ERK phosphorylation (β-arrestin pathway) over cAMP (Gαs pathway) and calcium mobilization (Gαq pathway). The compound is selective for NPSR over the vasopressin V1b receptor and 55 other targets at concentrations up to 10 µM. This biased signaling profile makes ML154 a valuable tool for dissecting NPSR signaling pathways.
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| ln Vitro |
In vitro, ML154 potently inhibits NPS-stimulated cellular responses in NPSR-expressing cells. It inhibits calcium mobilization with an IC₅₀ of 36.5 nM, cAMP accumulation with an IC₅₀ of 22.1 nM, and ERK phosphorylation with an IC₅₀ of 9.3 nM. In CHO cells expressing NPSR, ML154 (0.001-1 μM; 30 minutes) inhibits NPS-induced ERK phosphorylation in a concentration-dependent manner. The compound demonstrates biased antagonism, with preferential inhibition of ERK phosphorylation over other signaling pathways. ML154 does not inhibit the vasopressin V1b receptor or the endogenous purinergic receptor at concentrations up to 10 µM. These in vitro data establish ML154 as a potent and selective NPSR antagonist with a unique biased signaling profile.
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| ln Vivo |
In vivo, ML154 (1 mg/kg; intraperitoneal injection) blocks alcohol-induced ERK phosphorylation in the central amygdala of male Wistar rats (300-350 g). It decreases operant alcohol self-administration and reduces the motivation for alcohol reward as measured by progressive ratio responding. The compound is brain-penetrant, allowing it to reach central NPSR targets. ML154 does not affect basal locomotion or produce overt behavioral effects at the doses tested. These in vivo findings support a role for NPSR in alcohol-seeking behavior and validate ML154 as a tool for studying NPSR function in addiction and anxiety models. The compound has been used in preclinical studies to investigate the neurobiology of alcohol use disorder.
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| Enzyme Assay |
In vitro enzyme/receptor binding assays for ML154 typically involve competition binding studies using radiolabeled neuropeptide S. A typical protocol: membrane preparations from NPSR-expressing CHO cells are incubated with [¹²⁵I]-NPS (20-50 pM) and varying concentrations of ML154 (0.1 nM to 10 μM) in binding buffer (50 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 0.1% BSA) for 60-90 minutes at room temperature. Non-specific binding is determined in the presence of 1 μM unlabeled NPS. Bound radioactivity is separated by rapid filtration through glass fiber filters and quantified by scintillation counting. IC₅₀ values are calculated from competition curves. For functional assays, NPSR-expressing cells are pre-incubated with ML154 (0.001-1 μM) for 30 minutes, then stimulated with NPS (10-100 nM). ERK phosphorylation is measured by Western blot or AlphaScreen, cAMP by ELISA or HTRF, and calcium by FLIPR or Fura-2-based fluorometry.
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| Cell Assay |
In vitro cell-based assays for ML154 are performed using NPSR-expressing cell lines such as CHO-NPSR or HEK293-NPSR cells. A typical protocol: cells are seeded in 96-well plates at 20,000-50,000 cells/well and cultured overnight. Cells are serum-starved for 2-4 hours, then pre-incubated with ML154 at concentrations ranging from 0.001 to 1 μM for 30 minutes. Cells are stimulated with NPS (10-100 nM) for 5-15 minutes. ERK phosphorylation is measured using phospho-ERK ELISA or Western blot with anti-phospho-ERK antibodies. For calcium flux assays, cells are loaded with Fluo-4 or Fura-2 AM for 30-60 minutes, then fluorescence is measured upon NPS stimulation in the presence of ML154. For cAMP assays, cells are treated with forskolin (1 μM) and NPS, and cAMP levels are measured by competitive ELISA. Each concentration is tested in triplicate, and IC₅₀ values are calculated using nonlinear regression.
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| Animal Protocol |
In vivo animal studies for ML154 are conducted in male Wistar rats (300-350 g) or C57BL/6 mice. A typical protocol: ML154 is formulated in 10% Solutol, 10% N,N-dimethylacetamide, and 80% 10 mM PBS (pH 7.4) at a concentration of 1 mg/mL. Animals receive a single intraperitoneal injection of ML154 (1 mg/kg) or vehicle. Thirty minutes after injection, animals are administered alcohol (1.5-2.0 g/kg, intraperitoneal) or saline. After 15-30 minutes, animals are euthanized, and brain regions (central amygdala, prefrontal cortex) are dissected for ERK phosphorylation analysis by Western blot. For operant self-administration studies, rats are trained to press a lever for alcohol reinforcement. After stable baseline responding is established, ML154 is administered (1 mg/kg, IP) 30 minutes before the session, and lever presses are recorded. Progressive ratio sessions assess motivation for alcohol reward.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of ML154 have been partially characterized. The compound is brain-penetrant, achieving sufficient concentrations in the central nervous system to block NPSR-mediated signaling. Following intraperitoneal administration at 1 mg/kg in rats, ML154 reaches peak brain concentrations within 30-60 minutes, consistent with its ability to block alcohol-induced ERK phosphorylation at this time point. The compound is soluble in DMSO (62.5 mg/mL) and can be formulated in various vehicles for in vivo administration. Its plasma half-life, volume of distribution, and oral bioavailability have not been fully characterized in published studies. The compound is stable in powder form at -20°C for up to 3 years and in solution at -80°C for 6 months. Further pharmacokinetic studies would be needed to fully characterize its ADME properties.
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| Toxicity/Toxicokinetics |
Toxicological data for ML154 are limited to observations from in vivo studies. At the doses used in preclinical studies (1 mg/kg, IP), ML154 does not produce overt toxicity or behavioral abnormalities in rats. No significant effects on body weight, food intake, or general health have been reported. The compound has not undergone formal toxicology testing for regulatory purposes. Standard laboratory safety precautions should be followed when handling ML154: use of personal protective equipment (gloves, safety goggles, lab coat) and handling in a well-ventilated fume hood. The compound should be stored at -20°C for long-term stability and at 4°C for short-term storage. No genotoxicity, carcinogenicity, or reproductive toxicity data are available. Researchers should consult the safety data sheet (SDS) before handling.
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| References | |
| Additional Infomation |
Additional information for NCGC 84 (ML154): The compound has a CAS number of 1345964-89-7. Its molecular formula is C₂₉H₂₆BrN₂PS and molecular weight is 545.47 g/mol. Synonyms include ML154, NCGC84, NCGC-84, and NCGC00185684. The compound is a selective, brain-penetrant, non-peptide NPSR antagonist with a pA₂ of 9.98. It exhibits biased antagonism, preferentially inhibiting ERK phosphorylation (IC₅₀ = 9.3 nM) over cAMP (IC₅₀ = 22.1 nM) and calcium mobilization (IC₅₀ = 36.5 nM). ML154 is selective over vasopressin V1b and 55 other targets at 10 µM. The compound is for research use only and is not approved for clinical applications. No FDA approvals or investigational new drug applications exist. It is a valuable tool for studying NPSR function in addiction, anxiety, and stress-related disorders.
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| Molecular Formula |
C29H26N2PS+
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| Molecular Weight |
546.481
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| Exact Mass |
261.046
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| CAS # |
1345964-89-7
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| PubChem CID |
46930969
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| Appearance |
White to off-white solid powder
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| LogP |
2.615
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
2
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
34
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| Complexity |
668
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=C([N+]2=CC=CC=C2N1C/C=C/C3=CC=CC=C3)P(=S)(C4=CC=CC=C4)C5=CC=CC=C5.[Br-]
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| InChi Key |
VSWYSDPXUHVQCG-DTQAZKPQSA-N
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| InChi Code |
InChI=1S/C29H26N2PS/c1-24-29(32(33,26-17-7-3-8-18-26)27-19-9-4-10-20-27)31-22-12-11-21-28(31)30(24)23-13-16-25-14-5-2-6-15-25/h2-22H,23H2,1H3/q+1/b16-13+
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| Chemical Name |
1-cinnamyl-3-(diphenylphosphorothioyl)-2-methyl-1H-imidazo[1,2-a]pyridin-4-ium
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| Synonyms |
NCGC 84 NCGC84 NCGC-84 ML 154 ML154 ML-154 NCGC00185684.
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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 | 1.8299 mL | 9.1495 mL | 18.2989 mL | |
| 5 mM | 0.3660 mL | 1.8299 mL | 3.6598 mL | |
| 10 mM | 0.1830 mL | 0.9149 mL | 1.8299 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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