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Purity: ≥98%
A-286982 is a potent inhibitor of the LFA-1/ICAM-1 interaction with IC50 of 44 nM in an LFA-1/ICAM-1 binding assay. It blocks the binding of lymphocyte function-associated antigen-1 (LFA-1) with the cell adhesion molecule ICAM-1 with the IC50 of 35 nM in a cell based assay (the LFA-1-mediated cell adhesion assays).
| Targets |
Lymphocyte Function-Associated Antigen-1 (LFA-1/αLβ2-integrin) (IC50 = 44 nM in LFA-1/ICAM-1 binding assay; IC50 = 35 nM in LFA-1-mediated cellular adhesion assay) [1]
Lymphocyte Function-Associated Antigen-1 (LFA-1) (binds to an allosteric competitive site within the inserted (I) domain of LFA-1 αL subunit, distinct from the direct ICAM-1 binding site) [2] |
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| ln Vitro |
A-286982 connects to the allosteric site of the I domain (IDAS). When allostery passes through the A-286982 binding site and transmits from the β subunit I-like domain to the α subunit ICAM binding site, it is expected that the allosteric ICAM inhibition will display the unbeatable competition that we have seen for A-286982[2].
1. A286982 is a potent nonpeptide antagonist of LFA-1/ICAM-1 interaction; it was developed by optimizing p-arylthio cinnamides targeting an additional binding pocket adjacent to the primary site identified from an anilino diaryl sulfide lead, with a p-ethenylcarbonyl linker found optimal for accessing this pocket [1] 2. A286982 exhibited IC50 values of 44 nM in an LFA-1/ICAM-1 binding assay and 35 nM in an LFA-1-mediated cellular adhesion assay, demonstrating strong inhibitory activity against the LFA-1/ICAM-1 interaction [1] 3. In competition ELISA assays, A286982 antagonized LFA-1 binding to ICAM-1-Ig fusion protein, but did not inhibit the binding of a fluorescein-labeled analog of compound 3 (a direct competitive LFA-1 antagonist) to LFA-1; it displayed allosteric competitive behavior, and high concentrations of A286982 (20 μM in LFA-1/ICAM-1 ELISA, 50 μM in LFA-1/small-molecule ELISA) resulted in increased binding of compound 2B (a fluorescein-labeled small-molecule analog) to LFA-1 [2] |
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| ln Vivo |
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| Enzyme Assay |
1. LFA-1/ICAM-1 binding assay: Purified LFA-1 protein was immobilized on assay plates, and serial dilutions of A286982 were incubated with the immobilized LFA-1, followed by addition of ICAM-1-Ig fusion protein. The amount of bound ICAM-1-Ig was detected using a secondary antibody conjugated to a detectable label (e.g., horseradish peroxidase), and absorbance was measured to calculate the IC50 value of A286982 for inhibiting LFA-1/ICAM-1 binding (IC50 = 44 nM) [1]
2. Competition ELISA for LFA-1 ligand binding: LFA-1 was captured on assay plates, and 1/5 serial dilutions of A286982 were incubated with either ICAM-1-Ig or a fluorescein-labeled small-molecule analog (compound 2B). The binding of ICAM-1-Ig or compound 2B to LFA-1 was quantified, and the inhibitory effect of A286982 was evaluated; the assay confirmed that A286982 did not compete with compound 2B for LFA-1 binding, indicating an allosteric binding mode [2] |
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| Cell Assay |
1. LFA-1-mediated cellular adhesion assay: B-lymphocytes (or relevant cell lines expressing LFA-1) were incubated with serial concentrations of A286982, then added to plates coated with ICAM-1. After incubation, non-adherent cells were washed away, and adherent cells were quantified using a colorimetric or fluorescent method. The IC50 value of A286982 for inhibiting LFA-1-mediated cellular adhesion was determined to be 35 nM [1]
2. LFA-1 binding assay in transfected cells: 293 cells were cotransfected with β2 subunit and either wild-type αL subunit (containing the I domain) or αL subunit lacking the I domain (I-less). The binding of A286982 (assessed via competition with ICAM-1-Ig) and compound 2B (fluorescein-labeled analog) to transfected cells was measured; the assay confirmed that the I domain of LFA-1 αL subunit is essential for A286982-mediated allosteric inhibition, and cells lacking the I domain showed no significant binding of ICAM-1-Ig or compound 2B [2] |
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| References |
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| Additional Infomation |
1. A286982 belongs to a new class of p-arylthiocinnamamide compounds and is being developed as an LFA-1/ICAM-1 antagonist. It was discovered through solution-phase parallel synthesis optimization of cinnamamide compounds, targeting another binding pocket of LFA-1, and fine-tuning of diaryl substituents [1]. 2. The LFA-1/ICAM-1 interaction plays a key role in the inflammatory process, making A286982 a potential drug for treating inflammatory diseases [1]. 3. A286982, as an allosteric competitive antagonist of LFA-1, binds to a site in the LFA-1 αL subunit I domain that is opposite to the direct binding site of ICAM-1 (MIDAS motif region). This allosteric binding mode distinguishes it from direct competitive antagonists (such as compound 3) that overlap with the binding site of ICAM-1 [2].
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| Molecular Formula |
C24H27N3O4S
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| Molecular Weight |
453.56
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| Exact Mass |
453.172
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| Elemental Analysis |
C, 63.56; H, 6.00; N, 9.26; O, 14.11; S, 7.07
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| CAS # |
280749-17-9
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| Related CAS # |
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| PubChem CID |
9846729
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| Appearance |
Light yellow to green yellow solid powder
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| LogP |
4.972
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
32
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| Complexity |
700
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC(C)C1=CC=CC=C1SC2=C(C=C(C=C2)C=CC(=O)N3CCN(CC3)C(=O)C)[N+](=O)[O-]
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| InChi Key |
HTGGAYLWTDOFDK-PKNBQFBNSA-N
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| InChi Code |
InChI=1S/C24H27N3O4S/c1-17(2)20-6-4-5-7-22(20)32-23-10-8-19(16-21(23)27(30)31)9-11-24(29)26-14-12-25(13-15-26)18(3)28/h4-11,16-17H,12-15H2,1-3H3/b11-9+
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| Chemical Name |
(E)-1-(4-acetylpiperazin-1-yl)-3-[3-nitro-4-(2-propan-2-ylphenyl)sulfanylphenyl]prop-2-en-1-one
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| Synonyms |
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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 |
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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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1 mg/mL (2.20 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 1 mg/mL (2.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2048 mL | 11.0239 mL | 22.0478 mL | |
| 5 mM | 0.4410 mL | 2.2048 mL | 4.4096 mL | |
| 10 mM | 0.2205 mL | 1.1024 mL | 2.2048 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.
Antagonist competition by compounds 2A, 3,A-286982, and sICAM-1 in the LFA-1/ICAM-1 and LFA-1/small-molecule ELISAs.Protein Sci.2006 Feb;15(2):290-303. |
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Effect of antagonists on ligand binding in the LFA-1/ICAM-1 and LFA-1/small-molecule ELISAs.Protein Sci.2006 Feb;15(2):290-303. |
Structures of compounds 1–4,A-286982, and 5.Protein Sci.2006 Feb;15(2):290-303. |
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