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| Targets |
The primary target of IW-927 is the TNF receptor 1 (TNFR1), a cell-surface receptor that mediates many of the pro-inflammatory and cytotoxic effects of TNF-α. TNF-α is a pleiotropic cytokine that signals through two distinct receptors, TNFR1 and TNFR2. TNFR1 is ubiquitously expressed and is responsible for the majority of TNF-α's effects, including the activation of the NF-κB pathway (which promotes inflammation and cell survival) and the induction of apoptosis (programmed cell death). IW-927 is a potent and selective antagonist of the TNF-α/TNFR1 interaction. It specifically blocks the binding of TNF-α to TNFR1, thereby inhibiting the downstream signaling cascades. This is demonstrated by its ability to disrupt TNF-α-induced IκB phosphorylation, a key event in the activation of the NF-κB pathway, with an IC50 of 600 nM. Importantly, IW-927 does not bind to TNFR2 or CD40, indicating its high selectivity for TNFR1. This selectivity is crucial for dissecting the specific functions of TNFR1 versus TNFR2. The compound binds reversibly to TNFR1, but it has been shown to covalently modify the receptor via a photochemical reaction. This suggests that IW-927 can form a stable, long-lasting interaction with its target, which may be useful for certain applications.
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| ln Vitro |
In vitro studies have characterized IW-927 as a potent inhibitor of the TNF-α/TNFR1 interaction. Its activity is typically measured using cell-free binding assays and cell-based functional assays. In binding assays, IW-927 has been shown to block the binding of TNF-α to TNFR1 with an IC50 of 50 nM. This indicates that it is a highly potent inhibitor at the molecular level. In cell-based assays, IW-927 disrupts TNF-α-induced IκB phosphorylation with an IC50 of 600 nM. IκB is an inhibitor of the transcription factor NF-κB. When TNF-α binds to TNFR1, it triggers a signaling cascade that leads to the phosphorylation and degradation of IκB, allowing NF-κB to enter the nucleus and activate the transcription of pro-inflammatory genes. By inhibiting IκB phosphorylation, IW-927 effectively blocks the activation of the NF-κB pathway, which is a key mediator of TNF-α's pro-inflammatory effects. These in vitro studies confirm that IW-927 is a potent and selective inhibitor of TNFR1 signaling. The compound's ability to block both the binding of TNF-α to its receptor and the downstream signaling events makes it a valuable tool for studying the role of this pathway in various cellular processes.
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| ln Vivo |
In vivo activity of IW-927 has been demonstrated in animal models of TNF-α-mediated inflammation. While specific protocols are not detailed in the available literature, the compound is designed to be used in such models to study the role of TNFR1 signaling in disease. For example, it could be used in a mouse model of rheumatoid arthritis (collagen-induced arthritis) or inflammatory bowel disease (DSS-induced colitis). In these models, IW-927 would be administered to the animals, and its effect on disease severity, inflammatory markers, and immune cell infiltration would be assessed. As a potent antagonist of the TNF-α/TNFR1 interaction, IW-927 is expected to reduce inflammation and tissue damage in these models. The compound's ability to disrupt TNF-α-induced IκB phosphorylation in vitro suggests that it would also be effective in blocking NF-κB activation in vivo, leading to a reduction in the production of pro-inflammatory cytokines. However, because IW-927 is a research compound, extensive in vivo data is not as widely published as for clinically approved TNF-α inhibitors like adalimumab or infliximab. Its primary use is as a tool to validate TNFR1 as a therapeutic target.
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| Enzyme Assay |
The in vitro receptor binding assays for IW-927 are designed to measure its interaction with TNFR1. A common approach is a competitive binding assay. In this assay, TNFR1 protein (or cell membranes expressing TNFR1) is immobilized on a solid support (e.g., a 96-well plate). A labeled form of TNF-α (e.g., biotinylated or radiolabeled) is then added to the wells along with increasing concentrations of unlabeled IW-927. After incubation, the unbound components are washed away, and the amount of labeled TNF-α bound to the receptor is measured. A decrease in the binding of labeled TNF-α in the presence of IW-927 indicates that IW-927 is competing for the same binding site. The IC50 for this competition is 50 nM. To confirm that IW-927 is binding directly to TNFR1, a direct binding assay can be performed using a labeled version of IW-927. Surface Plasmon Resonance (SPR) is another technique that can be used to measure the binding affinity (Kd) of IW-927 for TNFR1, which has been reported to be 40-100 μM. This indicates a weak affinity, which is characteristic of small-molecule inhibitors that target protein-protein interactions. These assays provide a quantitative measure of IW-927's potency and its mechanism of action.
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| Cell Assay |
In vitro cell-based assays for IW-927 are used to measure its functional activity in inhibiting TNFR1 signaling. A key assay is the measurement of IκB phosphorylation. In this assay, cells expressing TNFR1 (e.g., HeLa cells or fibroblasts) are treated with TNF-α in the presence or absence of IW-927. After a short stimulation (e.g., 15-30 minutes), the cells are lysed, and the level of phosphorylated IκB is measured by Western blotting using a phospho-specific antibody. Alternatively, a cell-based ELISA can be used for a more quantitative, high-throughput measurement. The IC50 for inhibition of IκB phosphorylation by IW-927 is 600 nM. Another functional assay is the measurement of NF-κB activation. This can be done using a reporter gene assay, where cells are transfected with a plasmid containing a luciferase gene under the control of an NF-κB-responsive promoter. The cells are then treated with TNF-α in the presence or absence of IW-927, and the luciferase activity is measured. A decrease in luciferase activity indicates that IW-927 is inhibiting NF-κB activation. These cell-based assays confirm that IW-927's binding to TNFR1 translates into the inhibition of downstream signaling pathways.
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| Animal Protocol |
In vivo animal experiments for IW-927 would be designed to evaluate its efficacy in models of TNF-α-mediated disease. A typical protocol might involve a mouse model of acute inflammation, such as the LPS-induced endotoxemia model. In this model, mice are injected with lipopolysaccharide (LPS), a component of bacterial cell walls that triggers a strong inflammatory response, including the production of TNF-α. IW-927 would be administered to the mice (e.g., intraperitoneally or intravenously) either before or after the LPS challenge. The levels of pro-inflammatory cytokines (e.g., TNF-α, IL-6, IL-1β) in the blood would be measured by ELISA. The effect of IW-927 on these cytokine levels would be compared to a vehicle control group. Another model is the collagen-induced arthritis (CIA) model for rheumatoid arthritis. In this model, mice are immunized with collagen to induce an autoimmune response that leads to joint inflammation. IW-927 would be administered during the disease course, and the severity of arthritis would be assessed by clinical scoring of paw swelling and by histological analysis of joint tissue. These in vivo studies are crucial for validating the therapeutic potential of targeting the TNF-α/TNFR1 interaction with a small molecule like IW-927.
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| ADME/Pharmacokinetics |
Pharmacokinetic (PK) data for IW-927 is not provided in standard product descriptions. As a research compound, its PK profile would need to be determined experimentally. However, some basic properties can be inferred. IW-927 has a molecular weight of 441.57 g/mol and a molecular formula of C22H23N3O3S2, indicating it is a relatively large, lipophilic molecule. Its density is 1.42 g/cm³. For in vivo administration, it would need to be formulated in a suitable vehicle, such as a mixture of DMSO, PEG, and saline. Its solubility in aqueous buffers is not specified, but its lipophilic nature suggests it may have poor aqueous solubility, which could limit its oral bioavailability. For research use, the compound is typically stored as a powder at -20°C, where it is stable for up to three years. A comprehensive PK study would involve administering IW-927 to rodents via both intravenous and oral routes, collecting blood samples at various time points, and analyzing plasma concentrations using LC-MS/MS to determine key parameters such as half-life, clearance, volume of distribution, and oral bioavailability. Such data is essential for any in vivo efficacy study to ensure proper dosing.
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| Toxicity/Toxicokinetics |
Detailed toxicity data for IW-927 is not provided in standard product descriptions. However, it is reported that IW-927 shows no cytotoxicity at concentrations greater than 100 μM, suggesting a favorable safety profile in vitro. This indicates that the compound is not inherently toxic to cells at concentrations far exceeding its IC50 for TNFR1 inhibition, which is a positive attribute. This lack of general cytotoxicity suggests that its effects are specific to its target, TNFR1, and that it does not cause off-target cell damage. As a research compound, its toxicity in vivo has not been extensively characterized. The safety of IW-927 would depend on the role of TNFR1 in normal tissues and the potential for on-target toxicity. Since TNFR1 is involved in many physiological processes, including immune defense and tissue homeostasis, its chronic inhibition could have adverse effects. However, for short-term research studies, the compound is expected to be well-tolerated. All handling should be done with standard laboratory safety precautions, using appropriate personal protective equipment (PPE). Any in vivo use would require prior toxicological assessment.
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| References | |
| Additional Infomation |
IW-927 is a research compound and is not approved for any clinical use. It is a potent and selective antagonist of the TNF-α/TNFR1 interaction, making it a valuable tool for studying the biology of TNF-α and the role of TNFR1 in various diseases. TNF-α is a major therapeutic target for autoimmune and inflammatory diseases, and several biologics (monoclonal antibodies) that neutralize TNF-α, such as adalimumab and infliximab, are clinically approved and highly effective. However, these biologics are administered by injection and can be expensive. There is significant interest in developing small-molecule inhibitors that can block the TNF-α/TNFR1 interaction, as they could be administered orally and potentially have better tissue penetration. IW-927 serves as a lead compound for such efforts. Its mechanism of action, binding to TNFR1 and blocking the downstream NF-κB pathway, is well-characterized. By studying IW-927, researchers can gain insights into the structural requirements for inhibiting this protein-protein interaction and can develop more potent and drug-like analogs. IW-927 is therefore an important research tool in the field of inflammation and immunology.
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| Molecular Formula |
C22H23N3O3S2
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| Molecular Weight |
441.566322565079
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| Exact Mass |
441.118
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| CAS # |
389142-48-7
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| PubChem CID |
10433428
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| Appearance |
Yellow to orange solid powder
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
30
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| Complexity |
660
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(C2C=CC=C(C=2)OC)=CC2=C1C=C1C(N(C(N12)=S)CCCN1CCOCC1)=O
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| InChi Key |
RQGIIRZJMKNCHS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H23N3O3S2/c1-27-16-5-2-4-15(12-16)19-13-17-20(30-19)14-18-21(26)24(22(29)25(17)18)7-3-6-23-8-10-28-11-9-23/h2,4-5,12-14H,3,6-11H2,1H3
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| Chemical Name |
4-(3-methoxyphenyl)-10-(3-morpholin-4-ylpropyl)-11-sulfanylidene-5-thia-1,10-diazatricyclo[6.3.0.02,6]undeca-2(6),3,7-trien-9-one
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| Synonyms |
IW-927; IW927; IW 927;
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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) |
DMSO : ~10 mg/mL (~22.65 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 | 2.2646 mL | 11.3232 mL | 22.6465 mL | |
| 5 mM | 0.4529 mL | 2.2646 mL | 4.5293 mL | |
| 10 mM | 0.2265 mL | 1.1323 mL | 2.2646 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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