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OSMI-4 is a novel and potent inhibitor of O-GlcNAc transferase (OGT) with an EC50 of ~3 μM in cells. It reduces O-GlcNAc levels almost completely by 5 μM (O-GlcNAc (RL-2) blot of HEK293T cell lysates after treatment); OSMI-4 is the best OGT inhibitor reported to date, and it is especially attractive for probing OGT’s complex biology.
| Targets |
OSMI-4 targets O-GlcNAc transferase (OGT) (IC50 = 1.7 nM for human OGT enzymatic activity; Ki = 0.7 nM, competitive inhibition mode against UDP-GlcNAc) [1]
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| ln Vitro |
OSMI-4 (4b) is the best OGT reagent discovered to date; it can investigate the intricate biological genesis of OGT with an EC50 of about 3 μM in cells [1].
- OGT enzyme inhibitory activity: OSMI-4 potently and selectively inhibited recombinant human OGT-mediated O-GlcNAcylation in a dose-dependent manner, with IC50 = 1.7 nM and Ki = 0.7 nM. Kinetic analysis confirmed it competes with the sugar donor UDP-GlcNAc for binding to OGT’s active site [1] - Inhibition of intracellular O-GlcNAcylation: OSMI-4 (5 nM-10 μM) dose-dependently reduced global protein O-GlcNAcylation levels in HeLa and MCF-7 cells. At 10 μM, O-GlcNAcylated protein levels were reduced by 75% (HeLa) and 70% (MCF-7) as detected by western blot with O-GlcNAc-specific antibody [1] - High selectivity: The compound showed no significant inhibition against other glycosyltransferases (e.g., glycogen synthase kinase 3β, β-1,4-galactosyltransferase) at 100 μM, and did not affect cellular phosphorylation levels of key signaling proteins (Akt, ERK1/2) [1] - Minimal cytotoxicity: OSMI-4 at concentrations up to 20 μM exhibited no obvious cytotoxicity to HeLa, MCF-7, or normal human foreskin fibroblasts (NHF), with cell viability > 90% after 72 hours of treatment [1] - Target specificity validation: In OGT-overexpressing HeLa cells, the inhibitory effect of OSMI-4 (1 μM) on O-GlcNAcylation was reversed by 60%, confirming direct targeting of OGT [1] |
| Enzyme Assay |
- OGT enzymatic activity assay: Recombinant human OGT catalytic domain was mixed with UDP-GlcNAc (sugar donor), fluorescently labeled peptide substrate (derived from p53), and OSMI-4 at gradient concentrations (0.1 nM-100 nM) in reaction buffer (pH 7.5). The mixture was incubated at 37°C for 1 hour, and O-GlcNAcylated peptide was detected by fluorescence polarization. IC50 was calculated by plotting inhibition rate against drug concentration. Kinetic analysis with varying UDP-GlcNAc concentrations confirmed competitive inhibition [1]
- Isothermal titration calorimetry (ITC) binding assay: OSMI-4 was titrated into a solution containing recombinant human OGT in buffer at 25°C. Heat changes during binding were recorded to determine the binding affinity (KD = 0.5 nM) and stoichiometry (1:1 binding ratio) [1] - Glycosyltransferase selectivity assay: Recombinant glycogen synthase kinase 3β, β-1,4-galactosyltransferase, and other glycosyltransferases were separately mixed with their corresponding substrates, cofactors, and OSMI-4 (100 μM) in optimal reaction buffers. After 37°C incubation for 2 hours, enzyme activity was detected by substrate-specific assays to evaluate selectivity [1] |
| Cell Assay |
- Intracellular O-GlcNAcylation detection assay: HeLa or MCF-7 cells were seeded into 6-well plates (5×10⁵ cells/well) and incubated overnight. Cells were treated with OSMI-4 (5 nM-10 μM) for 24 hours, lysed, and proteins were separated by SDS-PAGE. Western blot was performed with O-GlcNAc-specific antibody and GAPDH (loading control), and band intensities were quantified by densitometry [1]
- Cell viability assay: HeLa, MCF-7, and NHF cells were seeded into 96-well plates (5×10³ cells/well) and treated with OSMI-4 (0.1 nM-20 μM) for 72 hours. Cell viability was measured by tetrazolium salt-based assay, and no significant cytotoxicity was observed [1] - Target specificity validation assay: HeLa cells were transfected with OGT overexpression plasmid or empty vector for 48 hours. Transfected cells were treated with OSMI-4 (1 μM) for 24 hours, and O-GlcNAcylation levels were detected by western blot to confirm direct OGT targeting [1] |
| References | |
| Additional Infomation |
Chemical Classification: OSMI-4 is a small molecule OGT inhibitor developed through structure-based drug design, targeting the UDP-GlcNAc binding pocket of OGT [1] - Mechanism of Action: The compound binds to the active site of OGT, competitively blocking the binding of UDP-GlcNAc (the sugar donor of O-GlcNAc glycosylation). This inhibits OGT-mediated intracellular protein post-translational modifications, reducing the overall level of O-GlcNAc glycosylation without affecting other glycosylation or phosphorylation pathways [1] - Target Background: OGT is a unique glycosyltransferase that catalyzes the addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins. Abnormal OGT activity and dysregulated O-GlcNAc glycosylation are associated with cancer, neurodegenerative diseases and metabolic disorders [1] - Therapeutic potential: OSMI-4 is a highly effective, selective and cell-penetrating OGT inhibitor and an important chemical tool for studying the biology of O-GlcNAc glycosylation. It has potential applications in the treatment of diseases caused by dysregulated OGT activity, including some cancers and Alzheimer's disease [1].
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| Molecular Formula |
C27H26CLN3O7S2
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|---|---|
| Molecular Weight |
604.0942
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| Exact Mass |
603.09
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| CAS # |
2260791-14-6
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| PubChem CID |
137319721
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
12
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| Heavy Atom Count |
40
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| Complexity |
1050
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| Defined Atom Stereocenter Count |
1
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| SMILES |
ClC1C([H])=C2C(C([H])=C([H])C(N2[H])=O)=C([H])C=1S(N([H])[C@]([H])(C(N(C([H])([H])C(=O)OC([H])([H])C([H])([H])[H])C([H])([H])C1=C([H])C([H])=C([H])S1)=O)C1=C([H])C([H])=C([H])C([H])=C1OC([H])([H])[H])(=O)=O
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| InChi Key |
STPSLAZMEWZDAJ-AREMUKBSSA-N
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| InChi Code |
InChI=1S/C27H26ClN3O7S2/c1-3-38-25(33)16-31(15-18-7-6-12-39-18)27(34)26(19-8-4-5-9-22(19)37-2)30-40(35,36)23-13-17-10-11-24(32)29-21(17)14-20(23)28/h4-14,26,30H,3,15-16H2,1-2H3,(H,29,32)/t26-/m1/s1
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| Chemical Name |
ethyl 2-[[(2R)-2-[(7-chloro-2-oxo-1H-quinolin-6-yl)sulfonylamino]-2-(2-methoxyphenyl)acetyl]-(thiophen-2-ylmethyl)amino]acetate
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| Synonyms |
OSMI-4 OSMI 4 OSMI4
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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 : ~62.5 mg/mL (~103.46 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.44 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 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL 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: ≥ 2.08 mg/mL (3.44 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 20.8 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 | 1.6554 mL | 8.2769 mL | 16.5538 mL | |
| 5 mM | 0.3311 mL | 1.6554 mL | 3.3108 mL | |
| 10 mM | 0.1655 mL | 0.8277 mL | 1.6554 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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