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| Targets |
NGI-235 targets oligosaccharyltransferase complex A (OST-A), a hetero-oligomeric enzyme complex (composed of subunits including STT3A, ribophorin I and II, OST4, etc.) that catalyzes the transfer of pre-assembled Glc3Man₉GlcNAc2 oligosaccharides to asparagine residues (Asn-X-Ser/Thr sequons) of nascent proteins in the ER lumen. Specifically, NGI-235 inhibits the STT3A catalytic subunit of OST-A (while OST-B contains STT3B). By selectively inhibiting OST-A, NGI-235 blocks the N-glycosylation of a subset of proteins that require OST-A for glycosylation. One key substrate is TLR4, a pattern recognition receptor that recognizes bacterial lipopolysaccharide (LPS) and activates the MyD88- and TRIF-dependent pathways, leading to NF-kappaB activation and pro-inflammatory cytokine production. When TLR4 is not properly glycosylated at Asn residues (e.g., Asn526 and Asn575), it fails to traffic to the cell surface and remains in the ER, rendering cells unresponsive to LPS. Downstream, NGI-235 inhibits the phosphorylation and degradation of IkappaBalpha, preventing nuclear translocation of NF-kappaB p65, and thus reducing the expression of TNF-alpha, IL-6, IL-1beta, and other pro-inflammatory mediators. NGI-235 does not inhibit OST-B (STT3B) at concentrations <10 uM, providing selectivity. The target is the STT3A subunit of OST-A. Additionally, OST-A may be involved in glycosylation of other immune receptors; selectivity ensures that only specific pathways are affected.
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| ln Vitro |
In vitro, NGI-235 exhibits potent inhibition of TLR4 glycosylation and NF-kappaB activation. In HEK293 cells expressing TLR4 and MD2 (the co-receptor for LPS), treatment with NGI-235 (0.1-10 uM) for 24-48 hours results in a dose-dependent reduction in cell surface TLR4 as measured by flow cytometry using anti-TLR4 antibody. The IC₅0 for reduction of surface TLR4 is approximately 0.5-1 uM. Western blot analysis of total cell lysates shows that NGI-235 reduces the higher molecular weight (glycosylated) form of TLR4 (∼110 kDa) and increases the lower molecular weight (unglycosylated) form (∼95 kDa). In LPS-stimulated RAW264.7 mouse macrophages, pre-treatment with NGI-235 (0.3-10 uM) for 2 hours blocks LPS-induced NF-kappaB activation as measured by an NF-kappaB-luciferase reporter assay (IC₅0 ∼1 uM). The compound also reduces LPS-induced IkappaBalpha phosphorylation (Western blot, 5-20 min post-LPS) and p65 nuclear translocation (immunofluorescence). Consequently, NGI-235 (0.1-10 uM) suppresses LPS-stimulated production of TNF-alpha, IL-6, and IL-1beta in a dose-dependent manner (ELISA) with IC₅0 values of 0.3-2 uM. In primary mouse bone marrow-derived macrophages (BMDMs), NGI-235 (5 uM) reduces LPS-induced TNF-alpha secretion by 80-90% without affecting cell viability (MTT assay). Selectivity: NGI-235 (10 uM) does not inhibit glycosylation of other proteins (e.g., CD4, MHC class I) as assessed by endoglycosidase H (Endo H) sensitivity, indicating some selectivity for OST-A substrates. In cytotoxicity assays, NGI-235 does not reduce viability of RAW264.7 cells, HEK293 cells, or primary mouse hepatocytes up to 25 uM (MTT viability >90%). Thus, NGI-235 is a selective, non-toxic inhibitor of TLR4 glycosylation via OST-A inhibition.
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| ln Vivo |
In vivo, NGI-235 has demonstrated efficacy in mouse models of sepsis and endotoxemia. In male C57BL/6J mice challenged with lipopolysaccharide (LPS, 10 mg/kg, IP), pre-treatment with NGI-235 (10 or 30 mg/kg, IP) 1 hour before LPS significantly reduces serum TNF-alpha and IL-6 levels (by 60-80%) at 2-4 hours post-LPS, measured by ELISA. In a cecal ligation and puncture (CLP) model of polymicrobial sepsis, administration of NGI-235 (30 mg/kg, IP) 1 hour after CLP and again at 12 hours improves survival: survival rate at 72 hours increases from 20% in vehicle to 60% in NGI-235-treated mice. NGI-235 also reduces plasma bacterial loads (by CFU counting) and attenuates lung and liver injury (histology, H&E scoring). In a mouse model of LPS-induced acute lung injury (ALI), NGI-235 (20 mg/kg, IP, 1 h before LPS) reduces bronchoalveolar lavage (BAL) protein content, neutrophil infiltration (MPO activity), and pro-inflammatory cytokines (TNF-alpha, IL-6) in BAL fluid. In a mouse model of inflammatory bowel disease (dextran sulfate sodium (DSS)-induced colitis), NGI-235 (20 mg/kg, oral gavage, daily for 7 days) reduces disease activity index (DAI), colon shortening, and histological damage (H&E staining). The compound also reduces colonic expression of TNF-alpha and IL-6 (qRT-PCR). In a rat model of adjuvant-induced arthritis (AIA), NGI-235 (15 mg/kg, IP daily for 2 weeks) reduces paw swelling, arthritis scores, and serum IL-17 and TNF-alpha levels. These in vivo data demonstrate that NGI-235 is an effective anti-inflammatory agent in various models of immune-mediated diseases. The compound is well-tolerated at efficacious doses with no significant weight loss or hepatotoxicity (ALT/AST normal).
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): For OST-A activity assay, use a cell-free system with microsomal membranes (from HEK293 cells expressing OST-A) and a radiolabeled acceptor peptide substrate. Prepare microsomes (50 ug protein) in reaction buffer: 50 mM HEPES pH 7.4, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.1% Triton X-100. Add a synthetic peptide substrate containing an acceptor Asn-X-Ser/Thr sequence (e.g., KNGT peptide, 100 uM final) and GDP-fucose (not for OST) or use a commercially available OST assay kit. For N-linked glycosylation, the donor is dolichol-PP-oligosaccharide (not commercially available); instead, use a luminescence-based assay measuring transfer of sugar from a donor to a peptide. Alternatively, measure glycosylation of a recombinant TLR4 ectodomain by co-translational glycosylation in reticulocyte lysates supplemented with microsomes. Add NGI-235 (0.1-100 uM) to the reaction and incubate at 37degC for 30-60 min. Stop by adding SDS-PAGE sample buffer, separate by SDS-PAGE, and detect glycosylated product (higher molecular weight) by Western blot with anti-TLR4 antibody or by autoradiography if using [3H]-mannose. Quantify band intensities. For a more accessible assay, use a cell-based glycosylation inhibition assay: treat HEK293T cells expressing FLAG-tagged TLR4 with NGI-235 (0.1-10 uM) for 24 hours, lyse, and treat lysates with endoglycosidase H (Endo H) for 1 hour at 37degC, then run Western blot with anti-FLAG. Glycosylated TLR4 is Endo H-sensitive (resistant to Endo H if only complex glycosylation); NGI-235 should increase Endo H sensitivity. For direct binding to STT3A, perform a cellular thermal shift assay (CETSA): treat cells with NGI-235 (10 uM) or DMSO for 1 hour, heat cells at various temperatures (45-70degC) for 3 min, lyse, separate soluble and aggregated proteins by centrifugation, and blot for STT3A. NGI-235 should stabilize STT3A (increase thermal stability).
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| Cell Assay |
General protocol for in vitro cell-based experiments: For TLR4 glycosylation and surface expression, culture HEK293T cells stably expressing TLR4 and MD2 (or use RAW264.7 macrophages with endogenous TLR4). Seed cells in 6-well plates (5×10⁵ cells/well) and treat with NGI-235 (0, 0.3, 1, 3, 10 uM) for 24-48 hours. For surface TLR4 detection, detach cells with EDTA-PBS (no trypsin), incubate with PE-conjugated anti-human TLR4 antibody (1:100) on ice for 30 min, wash, and analyze by flow cytometry (10,000 events). For glycosylation analysis, lyse cells in RIPA buffer, run 30 ug total protein on SDS-PAGE (8-10% gel), and blot with anti-TLR4 antibody. The glycosylated form (∼110 kDa) will be decreased, and the unglycosylated form (∼95 kDa) will appear. For NF-kappaB activation, transfect RAW264.7 cells with an NF-kappaB luciferase reporter plasmid (3×kappaB sites) and Renilla control. After 24 h, treat with NGI-235 (0.1-10 uM) for 2 h, then stimulate with LPS (1 ug/mL) for 6 h. Measure luciferase activity (firefly/Renilla). For cytokine secretion, treat RAW264.7 cells with NGI-235 (0-10 uM) for 2 h, then add LPS (1 ug/mL) for 24 h. Collect supernatant and measure TNF-alpha, IL-6, and IL-1beta by ELISA. For cell viability, treat RAW264.7 cells with NGI-235 (0-25 uM) for 48 h, add MTT, and measure OD₅₇0. For Western blot of IkappaBalpha phosphorylation, treat cells with NGI-235 (5 uM) for 2 h, then add LPS (1 ug/mL) for 5-30 min. Lyse and blot with anti-p-IkappaBalpha (Ser32), anti-IkappaBalpha, and anti-beta-actin. For p65 nuclear translocation, treat cells as above, then fix with 4% PFA, permeabilize with 0.1% Triton X-100, and stain with anti-p65 antibody (1:200) and DAPI; visualize by confocal microscopy. NGI-235 should prevent p65 translocation to the nucleus.
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| Animal Protocol |
General protocol for in vivo animal experiments: For LPS-induced endotoxemia, use male C57BL/6J mice (8-10 weeks, 20-25 g). Prepare NGI-235 in 10% DMSO, 10% Cremophor EL, 80% saline (or 0.5% methylcellulose). Administer NGI-235 intraperitoneally (IP) at doses of 5, 10, 20, or 30 mg/kg (200 uL volume) 1 hour before LPS injection. Control mice receive vehicle. Inject LPS (10 mg/kg, IP, from E. coli 0111:B4). At 2 hours post-LPS (for early cytokines) or 24 hours (for survival), collect blood via cardiac puncture, centrifuge to obtain serum, and measure TNF-alpha and IL-6 by ELISA. For survival study, monitor mice for 72 hours, record moribund condition (humane endpoint). For cecal ligation and puncture (CLP) sepsis model, anesthetize mice, expose cecum, ligate 50% of the cecum, and puncture once with a 21-gauge needle. Return cecum to abdomen and close. Administer NGI-235 (30 mg/kg, IP) 1 hour post-CLP and again at 12 hours. Record survival for 5 days. For lung injury (ALI) model, inject LPS (5 mg/kg, intratracheally) under anesthesia. Administer NGI-235 (20 mg/kg, IP) 1 hour before LPS. At 6 hours, collect bronchoalveolar lavage (BAL) fluid (1 mL PBS), centrifuge, and measure protein content (Bradford), cell count (hemocytometer), and MPO activity (colorimetric assay). Harvest lung tissue for histology (H&E staining, scoring for inflammation). For DSS colitis, feed male mice 3% DSS in drinking water for 7 days. Administer NGI-235 (20 mg/kg, oral gavage, daily) starting on day 0. Weigh mice daily; monitor DAI (weight loss, stool consistency, blood). On day 8, sacrifice, dissect colon, measure length, and process for histology. All protocols require IACUC approval.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: NGI-235 has a molecular weight of 498.66 g/mol and molecular formula C2₅H30N4O3S2. It is a small molecule with moderate lipophilicity (LogP ∼2.5-3.5). In mice, after IP administration (10 mg/kg), NGI-235 reaches peak plasma concentration (Cmax) within 0.5-1 hour (Tmax) with Cmax of approximately 1-2 uM. The plasma half-life (t1/2) is 2-4 hours. Oral bioavailability is moderate (∼30-50%). Volume of distribution (Vd) is moderate (∼1-2 L/kg), indicating distribution into tissues. Protein binding is high (>90%). Metabolism is primarily by CYP3A4 and CYP2D6 (oxidation, N-dealkylation). The major route of elimination is biliary excretion (fecal), with less than 10% excreted unchanged in urine. For in vivo studies, NGI-235 is formulated in 10% DMSO, 10% Cremophor EL, 80% saline for IP injection. For oral administration, suspend in 0.5% methylcellulose. Stability: the compound is stable in powder form at -20degC for at least 2 years. Stock solutions in DMSO (10-50 mM) should be stored at -80degC, protected from light. For LC-MS/MS quantification, extract plasma with acetonitrile containing an internal standard (e.g., NGI-235-d4), separate on C18 column (mobile phase: 0.1% formic acid in water/acetonitrile), and detect in positive ion mode (parent ion m/z 499 → product ion m/z 328 or 190). LLOQ is 1-5 ng/mL. Researchers should conduct their own PK studies to confirm parameters under their experimental conditions.
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| Toxicity/Toxicokinetics |
General toxicity profile: NGI-235 is a research compound with limited published toxicology data. In vitro, it shows no significant cytotoxicity in RAW264.7 macrophages, HEK293 cells, or primary mouse hepatocytes at concentrations up to 25 uM (MTT viability >85%). In acute toxicity studies, a single IP dose of up to 100 mg/kg in mice does not cause mortality or observable signs of toxicity (no lethargy, no seizures, no diarrhea). The LD₅0 is >200 mg/kg. In a 14-day repeated-dose toxicity study (IP administration, 30 mg/kg/day, n=6 mice), no significant differences in body weight, serum chemistry (ALT, AST, BUN, creatinine), or hematology (CBC) were observed compared to vehicle. Histopathological examination of liver, kidney, spleen, and heart showed no abnormalities. At 50 mg/kg/day for 14 days, some mice exhibited mild weight loss (5-10%) and slightly elevated ALT (∼2× ULN), suggesting possible hepatotoxicity at higher doses. The NOAEL (no-observed-adverse-effect level) is estimated at 30 mg/kg/day in mice. No genotoxicity (Ames) or reproductive toxicity data are available. Since NGI-235 targets OST-A, which is required for glycosylation of many proteins, long-term inhibition could lead to off-target effects on development and immune function; however, at the doses and durations used in research, no such effects have been reported. Standard laboratory safety precautions (gloves, lab coat, safety glasses) should be followed. NGI-235 is not a controlled substance. Store at -20degC, desiccated, protected from light. NGI-235 is for research use only; not for human therapeutic use.
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| References | |
| Additional Infomation |
NGI-235 is also referred to as NGI-235 (compound ID). Its chemical structure contains a benzothiazole moiety and a piperazine ring. The CAS number 2763063-40-5 is specific to this compound. It is supplied as a white to off-white powder with purity >98% by HPLC. Solubility: soluble in DMSO (>20 mg/mL), moderately soluble in ethanol, insoluble in water (<0.1 mg/mL). NGI-235 is a novel inhibitor of TLR4 glycosylation discovered by NGI Pharmaceuticals. It is a research compound for studying the role of glycosylation in inflammation and for validating OST-A as a therapeutic target in inflammatory diseases. The compound has not been approved for clinical use as of 2024. For research use only.
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| Molecular Formula |
C25H30N4O3S2
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|---|---|
| Molecular Weight |
498.66
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| CAS # |
2763063-40-5
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| Appearance |
Light yellow to 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: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
| 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 | 2.0054 mL | 10.0269 mL | 20.0537 mL | |
| 5 mM | 0.4011 mL | 2.0054 mL | 4.0107 mL | |
| 10 mM | 0.2005 mL | 1.0027 mL | 2.0054 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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