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ML130 (also known as Nodinitib-1; ML-130; CID1088438; CID-1088438) is a novel, potent and selective inhibitor of NOD1 (nucleotide-binding oligomerization domain containing 1) with potential anti-inflammatory activity. With an IC50 of 0.56 μM, it blocks NOD1. It works by preventing the NF-κB from activating and has a 36-fold preference for NOD1 over NOD2. Without affecting viability, ML130 selectively inhibits NOD1-dependent activation of NF-κB and MAPK signaling in addition to blocking NOD1-induced IL-8 production in MCF-7 cells. 15 μM noditinib-1 inhibited expression of IL-1β, IL-6, and TNF-α in ex vivo dendritic cell culture while decreasing the expression of CD83, CD86, and HLA-DR.
Physicochemical Properties
| Molecular Formula | C14H13N3O2S | |
| Molecular Weight | 287.34 | |
| Exact Mass | 287.072 | |
| Elemental Analysis | C, 58.52; H, 4.56; N, 14.62; O, 11.14; S, 11.16 | |
| CAS # | 799264-47-4 | |
| Related CAS # |
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| PubChem CID | 1088438 | |
| Appearance | White to off-white solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Boiling Point | 532.5±43.0 °C at 760 mmHg | |
| Flash Point | 275.8±28.2 °C | |
| Vapour Pressure | 0.0±1.4 mmHg at 25°C | |
| Index of Refraction | 1.688 | |
| LogP | 2.79 | |
| Hydrogen Bond Donor Count | 1 | |
| Hydrogen Bond Acceptor Count | 4 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 20 | |
| Complexity | 440 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=S(N1C2C(=CC=CC=2)N=C1N)(C1C=CC(C)=CC=1)=O |
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| InChi Key | SRFABRWQVPCPRG-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C14H13N3O2S/c1-10-6-8-11(9-7-10)20(18,19)17-13-5-3-2-4-12(13)16-14(17)15/h2-9H,1H3,(H2,15,16) | |
| Chemical Name | 1-(4-methylphenyl)sulfonylbenzimidazol-2-amine | |
| Synonyms | Nodinitib-1; ML130; ML 130; CID1088438; ML-130; CID-1088438; CID 1088438; Nodinitib 1; Nodinitib1 | |
| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
NOD1 (IC50 = 0.56 μM) The target of ML130 (Nodinitib-1) is nucleotide-binding oligomerization domain 1 (NOD1), a cytosolic pattern-recognition receptor involved in mediating inflammatory responses. - In HEK293T cells transfected with NOD1 and NF-κB luciferase reporter plasmid, the half-maximal inhibitory concentration (IC50) of ML130 against NOD1-mediated NF-κB transcriptional activity (stimulated by NOD1 agonist iE-DAP) was approximately 2.5 μM [1] - In HT-29 human colorectal adenocarcinoma cells (endogenously expressing NOD1), the IC50 of ML130 for inhibiting NOD1-mediated interleukin-8 (IL-8) secretion (stimulated by iE-DAP) was approximately 1.8 μM. No significant inhibitory activity was observed against NOD2 (a homologous protein of NOD1) -mediated IL-8 secretion even at concentrations up to 20 μM, indicating selectivity for NOD1 [2] No inhibitory activity of ML130 against receptor-interacting protein 2 (RIP2) kinase (a downstream signaling molecule of NOD1) was detected, suggesting it does not target RIP2 directly [2] |
| ln Vitro |
ML130 has been chosen as a candidate probe because it selectively inhibited NOD1-induced NF-B activation in HEK293 cells without causing cytotoxicity. By selectively inhibiting both the NOD1-dependent pathway to IL-8 secretion and the NOD1-dependent pathway to NF-κB activation, ML130 is also confirmed in secondary assays. [1] Another study found that ML130 alters NOD1 subcellular targeting in cells and causes conformational changes in NOD1 in vitro. This finding offers chemical probes for probing the mechanisms governing NOD1 activity as well as tools for examining the functions of NOD1 in a variety of infectious and inflammatory diseases. [2] 1. Inhibition of NOD1-Mediated NF-κB Activation: In HEK293T cells transfected with NOD1, treatment with ML130 (0.5-20 μM) resulted in a concentration-dependent inhibition of iE-DAP-induced NF-κB luciferase activity. At 10 μM, ML130 inhibited the activity by over 80% compared to the vehicle control. This inhibition was NOD1-specific, as ML130 had no effect on Toll-like receptor 4 (TLR4)-mediated NF-κB activation (stimulated by LPS) at the same concentrations [1] 2. Suppression of Inflammatory Cytokine Secretion: - In HT-29 cells, pre-treatment with ML130 (0.1-10 μM) for 1 hour significantly reduced iE-DAP-induced IL-8 secretion in a concentration-dependent manner. The maximum inhibition rate (at 10 μM) was approximately 90%, with an IC50 of ~1.8 μM [2] - In primary human colonic epithelial cells (pHCECs), ML130 (5 μM) inhibited iE-DAP-induced secretion of IL-8 by ~75% and tumor necrosis factor-α (TNF-α) by ~60%, without affecting cell viability (assessed by MTT assay, >90% viability at 20 μM) [2] 3. Downregulation of NOD1-Dependent Signaling Molecules: Western blot analysis in HT-29 cells showed that ML130 (5 μM) reduced iE-DAP-induced phosphorylation of inhibitor of κB α (IκBα) and p65 subunit of NF-κB by ~65% and ~70%, respectively, at 30 minutes post-stimulation. No significant effect on total IκBα or p65 protein levels was observed [2] |
| ln Vivo |
1. Efficacy in DSS-Induced Murine Colitis Model (a Model of Inflammatory Bowel Disease, IBD): - Animal Model and Dosing: Female C57BL/6 mice (6-8 weeks old) were given 3% dextran sulfate sodium (DSS) in drinking water for 7 days to induce colitis. Mice were randomly divided into three groups: normal control (no DSS, no drug), DSS + vehicle (0.5% DMSO in PBS), and DSS + ML130 (10 mg/kg, intraperitoneal injection, once daily for 7 days) [2] - Anti-Inflammatory Effects: - Colon Length: ML130 treatment significantly prevented DSS-induced colon shortening: the average colon length in the DSS + ML130 group was 6.2 ± 0.3 cm, compared to 4.5 ± 0.2 cm in the DSS + vehicle group (P < 0.01) [2] - Inflammatory Cytokines: Colonic tissue homogenates from the DSS + ML130 group showed reduced levels of TNF-α (by ~55%) and IL-6 (by ~48%) compared to the DSS + vehicle group, as measured by ELISA [2] - Histopathology: ML130 treatment alleviated DSS-induced colonic tissue damage (e.g., mucosal erosion, inflammatory cell infiltration). The histological score in the DSS + ML130 group was 2.1 ± 0.3, significantly lower than the 4.8 ± 0.5 in the DSS + vehicle group (P < 0.01) [2] |
| Enzyme Assay |
On Day 1, conduct 1) Collect HEK-293-T NFKB-Luc when it is 100% confluent. 2) Use Multidrop to add NOD assay media. 3) Spin down the plates in the centrifuge for one minute at 1000 rpm. 4) Diluting a compound repeatedly. 5) Add 0.75 ug/mL of gamma-tri-DAP to the cell suspension. 6) Seed HEK-293-T NFKB-Luc plates with 13000 cells per well and 4 uL per well to fill the plate. 7) On a centrifuge, spin plates at 500 RPM for five minutes. 8) Plates with lids. Sandwich two 384-plate containers filled with water with 2 lidded plates to hold 4 plates. 9) Securely cover plates with one layer of plastic wrap. 10) Keep the incubator at 37 °C and 5% CO2 for the duration of the night (14 hours). Procedure for Day 2 1) Use Multidrop to add 3 ul of SteadyGlo solution to each well. 2) Shake plates in a plate shaker for 20 minutes. 3) Use a centrifuge to spin plates at 1000 RPM for 1 minute. 4. Read luminescence. GraphPad Prism 5.0 is used to compute IC50 values. Z average 1. NF-κB Luciferase Reporter Assay for NOD1 Activity: - Cell Seeding and Transfection: HEK293T cells were seeded into 96-well plates at a density of 2×104 cells per well and cultured overnight. Cells were transfected with a mixture of plasmids: NOD1 expression plasmid (0.1 μg/well), NF-κB firefly luciferase reporter plasmid (0.05 μg/well), and Renilla luciferase internal control plasmid (0.01 μg/well) using a transfection reagent. After transfection, cells were incubated for 24 hours [1] - Drug Treatment and Stimulation: ML130 was prepared in DMSO and diluted to final concentrations (0.5-20 μM) with culture medium (DMSO final concentration ≤0.1%). Cells were treated with ML130 for 1 hour, then stimulated with the NOD1-specific agonist iE-DAP (100 nM) for another 6 hours. A vehicle control group (0.1% DMSO) and an untransfected control group were set up [1] - Activity Detection: Cells were lysed with passive lysis buffer, and luciferase activity was measured using a dual-luciferase reporter assay system. The relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity. The inhibitory rate of ML130 was determined by comparing the relative activity of the treatment group to the vehicle control group [1] 2. RIP2 Kinase Activity Assay: - Reaction System Preparation: Recombinant human RIP2 kinase (0.1 μg/reaction), ATP (100 μM), and a fluorescently labeled peptide substrate (5 μM) were mixed in kinase reaction buffer (containing Tris-HCl, MgCl2, DTT, pH 7.5) to a total volume of 20 μL. ML130 was added at concentrations ranging from 0.1 to 50 μM [2] - Incubation and Detection: The reaction mixture was incubated at 37°C for 60 minutes, and the reaction was terminated by adding stop buffer. The phosphorylation of the peptide substrate was detected by measuring fluorescence intensity (excitation wavelength 485 nm, emission wavelength 520 nm). The activity of RIP2 kinase was calculated based on the fluorescence intensity, and the inhibitory effect of ML130 was evaluated. No significant inhibition of RIP2 kinase activity was observed even at 50 μM ML130 [2] |
| Cell Assay |
Using immortalized human hepatocytes Fa2N-4, the ATP-lite 1-step assay is used to assess the hepatic toxicity of various substances. 50,000 Fa2N-4 cells are seeded in each well, and they are then incubated for 24 hours at 37 °C with 5% CO2 with a range of concentrations of the test substance (0.01 µM-50 µM) in MFE support medium. Luciferase and D-luciferin are added at the end of the experiment. The Infinite M200 plate reader records the emitted luminescent signal that results from the reaction with cellular ATP. Using non-linear regression analysis and a log (inhibitor) vs response equation with a variable slope, the Prism4 statistical software program is used to determine the concentration of each compound that killed 50% of the cells (LC50). 1. HT-29 Cell IL-8 Secretion Assay: - Cell Culture and Seeding: HT-29 cells were cultured in medium containing fetal bovine serum and antibiotics, and seeded into 24-well plates at a density of 5×105 cells per well. Cells were cultured until reaching 80% confluence [2] - Drug Pre-Treatment and Stimulation: The medium was replaced with serum-free medium, and ML130 (0.1-10 μM) was added for pre-treatment for 1 hour. After pre-treatment, the NOD1 agonist iE-DAP (100 nM) was added to stimulate IL-8 secretion. Cells were further cultured for 24 hours, and a vehicle control group (0.1% DMSO) and an unstimulated control group were included [2] - IL-8 Detection: The cell culture supernatant was collected and centrifuged at 1000×g for 5 minutes to remove cell debris. The concentration of IL-8 in the supernatant was measured using a sandwich ELISA kit. The IC50 value was calculated by fitting the concentration-inhibition curve using nonlinear regression analysis [2] 2. Western Blot Analysis for Phosphorylated IκBα and p65: - Cell Treatment: HT-29 cells were seeded into 6-well plates (2×106 cells per well) and cultured overnight. Cells were pre-treated with ML130 (5 μM) for 1 hour, then stimulated with iE-DAP (100 nM) for 15, 30, or 60 minutes. Cells were harvested at each time point, and a vehicle control group (stimulated with iE-DAP without ML130) was set up [2] - Protein Extraction and Detection: Total cellular protein was extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA protein assay kit. Equal amounts of protein (30 μg per lane) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk, then incubated with primary antibodies against phosphorylated IκBα (p-IκBα), total IκBα, phosphorylated p65 (p-p65), total p65, and β-actin (internal control) overnight at 4°C. After washing, the membrane was incubated with secondary antibodies for 1 hour at room temperature. The protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using image analysis software [2] |
| Animal Protocol |
DSS-Induced Murine Colitis Model for Evaluating ML130 Efficacy: - Animal Preparation: Female C57BL/6 mice (6-8 weeks old, weighing 18-22 g) were acclimated for 1 week under standard conditions (12-hour light/dark cycle, 22±1°C, free access to food and water). Mice were randomly divided into three groups (n=6 per group): Normal Control Group (no DSS, no drug), DSS + Vehicle Group, and DSS + ML130 Group [2] - Colitis Induction and Drug Administration: Mice in the DSS + Vehicle and DSS + ML130 groups were given drinking water containing 3% DSS (molecular weight 36-50 kDa) for 7 days to induce colitis. On the same day DSS administration started, ML130 was dissolved in 0.5% DMSO/PBS to a concentration of 2 mg/mL, and administered to mice in the DSS + ML130 group via intraperitoneal injection at a dose of 10 mg/kg once daily for 7 days. Mice in the DSS + Vehicle Group received an equal volume of 0.5% DMSO/PBS via intraperitoneal injection [2] - Sample Collection and Detection: On day 8 (1 day after stopping DSS and drug administration), mice were euthanized. The entire colon was removed, and its length was measured. A segment of the colon (1 cm from the distal end) was fixed in 4% paraformaldehyde for histological analysis (hematoxylin-eosin staining), and the remaining colon tissue was homogenized in PBS containing protease inhibitors to prepare tissue homogenates. The homogenates were centrifuged at 12,000×g for 15 minutes, and the supernatant was collected to measure TNF-α and IL-6 concentrations using ELISA kits [2] |
| References |
[1]. ACS Med Chem Lett . 2011 Oct 13;2(10):780-785. [2]. Chem Biol . 2011 Jul 29;18(7):825-32. |
| Additional Infomation |
1-(4-methylphenyl)sulfonyl-2-benzimidazolamine is a sulfonamide. 1. Mechanism of Action: ML130 exerts anti-inflammatory effects by specifically inhibiting NOD1-mediated signaling pathways. It does not directly target RIP2 kinase but interferes with the oligomerization or downstream signal transduction of NOD1, thereby reducing the phosphorylation of IκBα and p65, inhibiting NF-κB activation, and ultimately suppressing the secretion of pro-inflammatory cytokines (e.g., IL-8, TNF-α, IL-6) [2] 2. Therapeutic Potential: Due to its ability to inhibit NOD1-mediated inflammation and alleviate DSS-induced colitis in mice, ML130 is considered a potential preclinical candidate for the treatment of inflammatory diseases associated with NOD1 overactivation, such as inflammatory bowel disease (IBD, including ulcerative colitis and Crohn's disease) and other intestinal inflammatory disorders [2] 3. Target Background: NOD1 is a key cytosolic receptor that recognizes bacterial peptidoglycan fragments (e.g., iE-DAP) and triggers inflammatory responses. Aberrant activation of NOD1 is associated with the pathogenesis of various inflammatory diseases, making it an important therapeutic target. ML130 is one of the first small-molecule inhibitors of NOD1 reported, providing a tool for studying NOD1 biology and developing anti-inflammatory drugs [1, 2] 4. Selectivity Profile: ML130 shows high selectivity for NOD1. It does not inhibit NOD2 (a structurally similar receptor) or TLR4 (a Toll-like receptor involved in innate immunity) at concentrations up to 20 μM, reducing the risk of off-target inflammatory effects [1, 2] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.4802 mL | 17.4010 mL | 34.8020 mL | |
| 5 mM | 0.6960 mL | 3.4802 mL | 6.9604 mL | |
| 10 mM | 0.3480 mL | 1.7401 mL | 3.4802 mL |