| Targets |
Naamidine J exhibits a complex pharmacology by binding to and inhibiting multiple targets. Its primary identified molecular target is the chromosome segregation 1-like (CSE1L) protein, to which it binds with a dissociation constant (KD) of 5.41 microM. By binding to CSE1L, it modulates its function. Furthermore, it has been shown to inhibit the expression of the immune checkpoint protein Programmed Death-Ligand 1 (PD-L1), making it an indirect PD-L1 inhibitor. This dual CSE1L/PD-L1 inhibitory activity is central to its mechanism of action.
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| ln Vitro |
Naamidine J (5 μM, 24 h) is cytotoxic to K562 cells with an IC50 of 11.3 μM, but non-toxic to macrophages and RKO cells (IC50 > 40 μM) [1][2]. Naamidine J (10 μM, 24 h) reduces PD-L1 expression in RKO cells and PDAC cells [1][3]. Naamidine J (1-5 μM, 5 h) inhibits LPS-induced inflammatory responses in RAW264.7 macrophages [2]. Naamidine J (5 μM, 5 h) inhibits CSE1L-mediated SP1 nuclear translocation in LPS-induced macrophages [2]. Naamidine J (10 μM, 24 hours) can restore the cytotoxicity of CD8+ T cells and significantly reduce the survival rate of PDAC cells (MIA-PaCa2, BxPC-3) [3].
In vitro, Naamidine J demonstrates potent anti-inflammatory activity. It significantly reduces the production of key pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). In contrast, it enhances the expression of anti-inflammatory markers such as CD206 and Arg-1, which are associated with M2 macrophage polarization. This shift in cytokine profile indicates it promotes an anti-inflammatory state. It also inhibits PD-L1 and shows direct anti-tumor activity in various cancer cell lines, suppressing cell proliferation. |
| ln Vivo |
Naamidine J (10-20 mg/kg, intraperitoneal injection, once daily for 3 days) significantly reduced acute lung injury induced by sensitive LPS and alleviated pulmonary edema[2]. Naamidine J (30 mg/kg, intraperitoneal injection, once every three days for 28 days) significantly increased the proportion of CD8+ T cell subsets and decreased the proportion of CD8+ Th cell subsets in tumors bearing tumors with high NAT10 expression[3].
In vivo, Naamidine J's biological activity has been confirmed in animal models of acute inflammation. In mouse models, it has been shown to significantly reduce pulmonary tissue edema, inflammatory cell infiltration, and the cytokine storm induced by systemic inflammation. This demonstrates its potent systemic anti-inflammatory effect. Furthermore, due to its inhibitory effect on PD-L1, it also exhibits anti-tumor activity in vivo by potentially reactivating the host's immune system to attack cancer cells. |
| Enzyme Assay |
General in vitro cytokine assay (ELISA): Primary macrophages or a cell line such as RAW 264.7 are seeded in 96-well plates. Cells are pre-treated with Naamidine J (0.1-10 uM) for 2 hours, then stimulated with lipopolysaccharide (LPS, 1 ug/mL) for 24 hours. Cell culture supernatants are collected, and the concentrations of TNF-alpha, IL-1beta, and IL-6 are measured using specific ELISA kits. Naamidine J will reduce the levels of these cytokines in a concentration-dependent manner. Cell viability is assessed in parallel by an MTT assay to ensure the effects are not due to cytotoxicity.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: RKO cells Tested Concentrations: 10 μM Incubation Duration: 24 h Experimental Results: Had a certain inhibitory effect on PD-L1 expression. ELISA Assay[1] Cell Types: RAW264.7 cells Tested Concentrations: 1, 2 and 5 μM Incubation Duration: Pre-treatment for 3 hours, followed by co-stimulation with LPS for 2 hours Experimental Results: Exhibited concentration-dependent inhibition of TNF-α release. RT-PCR[1] Cell Types: RAW264.7 cells Tested Concentrations: 1, 2 and 5 μM Incubation Duration: Pre-treatment for 3 hours, followed by co-stimulation with LPS for 2 hours Experimental Results: Inhibited pro-inflammatory factors (TNF-α, IL-1β, IL-6), and promoted anti-inflammatory factors (CD206, Arg-1). Western Blot Analysis[1] Cell Types: RAW264.7 cells Tested Concentrations: 5 μM Incubation Duration: Pre-treatment for 3 hours, followed by co-stimulation with LPS for 2 hours Experimental Results: Resulted in the accumulation of SP1 in the cytoplasm. Enhanced the interaction between CSE1L and SP1. General in vitro PD-L1 expression assay (Western Blot): Cancer cells (e.g., A375 or H460) are treated with Naamidine J (1-10 uM) for 24-48 hours. Cells are then lysed, and protein lysates are subjected to SDS-PAGE. The levels of PD-L1 protein are detected by Western blot using a specific anti-PD-L1 antibody. Naamidine J will cause a significant decrease in PD-L1 protein expression compared to untreated control cells. Flow cytometry can be used to quantify PD-L1 on the cell surface. |
| Animal Protocol |
Animal/Disease Models: Acute lung injury model established in mice[2]
Doses: 10 and 20 mg/kg Route of Administration: Intraperitoneal injection (i.p.), once daily for 3 days Experimental Results: Significantly reduced the increase in lung organ coefficient induced by LPS and alleviate pulmonary edema. Significantly improved the pathological damage of lung tissue and reduce the infiltration of inflammatory cells. Significantly reduced the protein levels of TNF-α in serum and lung tissue, as well as the mRNA expression of various inflammatory factors. Reduced the proportion of pro-inflammatory M1-type macrophages in the lung tissue and increased the proportion of anti-inflammatory M2-type macrophages. Animal/Disease Models: PanO2 xenograft model established in four-week-old female C57BL/6 mice (18 to 20 g)[3] Doses: 30 mg/kg Route of Administration: Intraperitoneal injection (i.p.), once every three days for 28 days Experimental Results: Significantly inhibited tumor growth in the Nat10-OE group. Reversed T cell exhaustion and restores anti-tumor function. Increased the infiltration of CD8+ T cells, reduced the PD-1 with IFNγ and exhausted phenotype, and increased the PD-1-IFNγ+ cytotoxic phenotype. General in vivo animal protocol for anti-inflammatory activity: Dissolve Naamidine J in a suitable vehicle (e.g., 5% DMSO, 10% PEG300, 5% Tween 80, 80% saline). Administer to female BALB/c mice (n=6 per group) by intraperitoneal injection at 0, 5, 10, 20 mg/kg 30 minutes prior to LPS challenge (20 mg/kg, i.p.). After 6 hours, mice are sacrificed, and blood and bronchoalveolar lavage fluid (BALF) are collected. Naamidine J will reduce the levels of TNF-alpha and IL-6 in the serum and BALF, and also reduce the total cell count and neutrophil infiltration in the lungs. |
| ADME/Pharmacokinetics |
Naamidine J is a small, moderately lipophilic natural product (MW 477.51 g/mol). As a research compound, its detailed PK/ADME properties are still under characterization. It is likely to have moderate to high bioavailability and distribute into tissues. It is expected to be metabolized by the liver, but specific pathways are not yet published. For its use as a reference standard, the compound is considered chemically stable under proper storage conditions (e.g., -20degC, protected from light).
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| Toxicity/Toxicokinetics |
Preclinical studies have shown that Naamidine J is well-tolerated at therapeutic doses in mice. It is not considered a highly toxic compound. As a natural product, it is not expected to be genotoxic. Formal toxicology studies (e.g., 28-day repeat-dose toxicity) and genotoxicity assays (Ames test, micronucleus assay) would be required before it could progress to clinical development, but for a research reagent, they are not required. For impurity qualification, it is handled as a standard laboratory chemical.
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| References |
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| Additional Infomation |
Appearance: White solid. Molecular formula: C2₅H2₇N₅O₅. Storage: -20degC, 3 years; 4degC, 2 years (Powder). Protect from light. Solubility: Soluble in DMSO. Other names: CSEL1/PD-L1 inhibitor. Safety: For research use only; avoid inhalation and skin contact.
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| CAS # |
2227550-73-2
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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 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 : ~4.55 mg/mL (~9.53 mM; with sonication (<60°C))
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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.) |
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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