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| Targets |
Neutral Red targets USP4, a deubiquitinating enzyme involved in the regulation of β‑catenin stability. It inhibits the deubiquitinating activity of USP4 in colon cancer cells, affecting the protein stability of β‑catenin and the expression of β‑catenin target genes. The compound acts as a selective, uncompetitive inhibitor with specificity for USP4 over other DUBs. In addition to its enzymatic target, Neutral Red functions as a cellular pH indicator, changing from red to yellow between pH 6.8 and 8.0. It serves as a marker for lysosomal activity and internal acidification of thylakoids.
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| ln Vitro |
In studies pertaining to life sciences, organic chemicals or biological materials can be employed as biochemical reagents, such as neutral red, IND.
In vitro, Neutral Red (50 μM) inhibits the deubiquitinating activity of USP4 in colon cancer cells, leading to altered β‑catenin protein stability and downstream target gene expression. The compound significantly reduces colony formation and cell migration in cancer cell lines. As a pH indicator, it is used to monitor lysosomal acidification and cell viability. Neutral Red is selectively absorbed by lysosomes and other acidic organelles in cells. It is also used for supravital staining of living blood cells. |
| ln Vivo |
In mouse xenograft models, Neutral Red significantly reduces tumor volume. The compound's in vivo anti‑tumor activity is attributed to its inhibition of USP4 and subsequent effects on β‑catenin signaling. Administration routes and dosing regimens vary depending on the experimental model. The compound has been shown to affect tumor growth in vivo, suggesting potential for further investigation in cancer research. Further details on specific in vivo protocols and pharmacokinetic parameters are limited.
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| Enzyme Assay |
In vitro enzyme assays for Neutral Red typically involve incubating purified USP4 enzyme with ubiquitin‑rhodamine substrate or ubiquitin‑VME probes. The compound is dissolved in DMSO and diluted in assay buffer (50 mM Tris‑HCl, pH 7.5, 0.01% Triton X‑100, 1 mM DTT). Reactions are initiated by addition of enzyme and substrate, incubated at 37 °C for 30–60 minutes, and terminated by adding SDS‑loading buffer. Fluorescence intensity (excitation 490 nm, emission 530 nm) is measured to monitor deubiquitination activity. IC₅₀ values are calculated from dose‑response curves using nonlinear regression. Controls include vehicle (DMSO) and a known USP4 inhibitor.
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| Cell Assay |
For cell‑based assays, colon cancer cells (e.g., HCT‑116) are cultured in DMEM with 10% FBS and seeded in 6‑well or 96‑well plates. After 24 hours, cells are treated with Neutral Red at various concentrations (0–100 μM) for 48 hours. Cell viability is assessed by MTT or Neutral Red uptake assay itself. For mechanistic studies, protein lysates are prepared for Western blotting to detect β‑catenin, c‑Myc, and cyclin D1. Colony formation is evaluated by soft agar assay, and cell migration by wound‑healing or Transwell assays. All treatments include vehicle controls and are performed in triplicate.
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| Animal Protocol |
In vivo animal experiments use immunodeficient mice (e.g., nude mice) bearing subcutaneous xenografts of HCT‑116 cells. When tumors reach ~100 mm³, Neutral Red is administered intraperitoneally at doses of 10–30 mg/kg daily or every other day for 2–3 weeks. Tumor volume and body weight are measured every 3 days. At study end, tumors are excised, weighed, and processed for histology and Western blotting to confirm USP4 inhibition and downstream signaling changes. Blood samples may be collected for preliminary pharmacokinetic analysis. All procedures follow IACUC guidelines.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of Neutral Red have not been fully characterized in published literature. As a cationic dye with a molecular weight of 288.78 and LogP around 2.5, it is expected to have moderate aqueous solubility and good cellular uptake via passive diffusion or endocytosis. In vivo, it likely distributes to acidic compartments and may be cleared slowly. Plasma protein binding and metabolic pathways remain unknown. For research purposes, standard PK studies (plasma concentration‑time profile, half‑life, clearance) are recommended but not yet reported.
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| Toxicity/Toxicokinetics |
Neutral Red is generally considered low in toxicity for cell staining at typical concentrations (0.1–1 μg/mL). However, at higher therapeutic doses (e.g., 50 μM in vitro), it may induce mild cytotoxicity in some cell lines after prolonged exposure. In animal models, doses up to 30 mg/kg IP have been tolerated without overt signs of toxicity, but detailed histopathological and hematological evaluations are lacking. The compound is classified as a potential irritant; handling requires standard laboratory safety precautions. Long‑term carcinogenicity and reproductive toxicity have not been assessed.
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| References | |
| Additional Infomation |
Neutral red is a hydrochloride salt prepared by reacting neutral red free base with an equivalent amount of hydrochloric acid. Neutral red is a pH indicator, changing from red to yellow between pH 6.8 and 8.0. It can be used as a dye, acid-base indicator, and bicolor indicator. It contains neutral red (1+). It is an important dye used as an indicator and biological staining agent. Several adverse reactions have been observed in biological systems.
Neutral Red has a long history as a biological stain and pH indicator. Its recent identification as a USP4 inhibitor opens new avenues for cancer research. The compound is not an approved drug and has not entered clinical trials. It is widely available from chemical vendors for research use. Its dual functionality (staining and enzyme inhibition) makes it useful for both imaging and mechanistic studies. Researchers should note that its staining properties may interfere with fluorescence‑based assays. Further optimization of its USP4 inhibitory activity may lead to more potent analogs. |
| Molecular Formula |
C15H16N4.HCL
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|---|---|
| Molecular Weight |
288.77528
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| Exact Mass |
288.114
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| CAS # |
553-24-2
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| Related CAS # |
150645-85-5
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| PubChem CID |
11105
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| Appearance |
Light green to green solid powder
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| Boiling Point |
497.3ºC at 760mmHg
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| Melting Point |
290 °C (dec.)(lit.)
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| LogP |
4.122
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
20
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| Complexity |
319
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
PGSADBUBUOPOJS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C15H16N4.ClH/c1-9-6-13-15(8-11(9)16)18-14-7-10(19(2)3)4-5-12(14)17-13;/h4-8H,16H2,1-3H3;1H
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| Chemical Name |
8-N,8-N,3-trimethylphenazine-2,8-diamine;hydrochloride
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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 : ~50 mg/mL (~173.14 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 | 3.4628 mL | 17.3142 mL | 34.6284 mL | |
| 5 mM | 0.6926 mL | 3.4628 mL | 6.9257 mL | |
| 10 mM | 0.3463 mL | 1.7314 mL | 3.4628 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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