O-GlcNAc transferase (IC50 = 2.7 μM)
O-GlcNAc transferase (OGT) (inhibitor) [1]

After treatment for 24 hours, OSMI-1 (50 μM; CHO cells) decreases viability by about 50% [1]. Treatment with OSMI-1 (10-100 μM; 24 hours; CHO cells) decreased O-GlcNAcylation (global O-linked N-acetylglucosamine) in a dose-dependent manner. OGT activity in cells is inhibited by OSMI-1 [1].

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In a radiometric capture assay using purified sOGT (short isoform of OGT) and a peptide substrate, OSMI-1 inhibited OGT activity in a dose-dependent manner. [1]
Kinetic analysis with fixed saturating concentrations of the protein acceptor GST-Nup62 showed that the Vmax changed as a function of OSMI-1 concentration, suggesting that OSMI-1 is not competitive with respect to the substrate UDP-GlcNAc. [1]

Mammals and zebrafish share remarkably comparable toxicity profiles; in fact, zebrafish are frequently used as a transitional species between conventional animal testing and cell-based evaluations. The in vivo acute toxicity of OSM1-1 was investigated using a zebrafish model. The LC50 values for OSM1-1 in the zebrafish model are 0.031 mg/mL (56 μM, 12 hours) and 0.025 mg/mL (45 μM, 24 hours) [2].

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OGT activity assay[2]
HPLC was used to preliminarily analyze the inhibition of OGT by the compounds. According to the previously described41, the reaction condition was optimized. 200 μM CKII, 300 nM OGT, 1 mM UDP-GlcNAc, the compounds (100 μM) and buffer (150 mM NaCl, 1 mM EDTA, 2.5 mM tris(hydroxypropyl)phosphine, 25 mM Tris-HCl, pH 7.4) were mixed and incubated at room temperature for 1 h. After being precipitated by methanol, the reaction mixtures were loaded onto HPLC (the reverse-phase chromatographic column was Zorbax SB-C18 StableBond analytical column, 250 mm × 4.6 mm, and a Zorbax SB-C18 analytical guard column, 4.6 mm × 12.5 mm, 5 μm) to quantify the yield of glycopeptide product. Mobile phase A consisted of 0.1% TFA in H2O, and mobile phase B consisted of 0.1% TFA in MeCN. The components were eluted using a gradient (flow rate at 1 mL/min; at 0 min elution solvent mixture A/B = 90/10; at 20 min elution solvent mixture A/B = 70/30; wavelength = 214 nm). IC50 values were calculated using GraphPad 5 (n = 3).[2]
A cell-free reaction system was used to determine the inhibition of O-GlcNAc level on a purified protein acceptor Nup62. Reaction mixtures containing 10 μM Nup62, 1 mM UDP-GlcNAc, 500 nM OGT, buffer (150 mM NaCl, 1 mM EDTA, 2.5 mM tris(hydroxypropyl)phosphine, 25 mM Tris-HCl, pH 7.4), and compounds were incubated at 37 °C for 1 h. Then, SDS-PAGE loading buffer was added and western blots were used to detect O-GlcNAc on Nup62.[2]
OGT activity assay based on a UDP-Glo assay kit was performed as previously described10. Following the manufacturer’s protocol, assays were optimized and performed in white, flat bottom 384-well assay plate. CKII peptide was used as the acceptor. Reactions contained the following components: 250 nM OGT, 125 μM CKII and 40 μM UDP-GlcNAc, and buffer (150 mM NaCl, 1 mM EDTA, 2.5 mM tris(hydroxypropyl)phosphine, 25 mM Tris-HCl, pH 7.4). Luminescence was measured in triplicate using a microplate luminometer. IC50 values were calculated using GraphPad 5.[2]

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OGT inhibition assays were performed using a radiometric capture assay. Purified sOGT was incubated with a biotinylated peptide substrate, UDP-[³H]GlcNAc, and varying concentrations of OSMI-1. The reaction mixture was spotted onto streptavidin-coated membranes, which were then washed to remove unreacted UDP-[³H]GlcNAc. The radioactivity retained on the membrane, corresponding to glycosylated peptide, was measured by scintillation counting. IC₅₀ values were calculated from the dose-response curves. [1]

Cell viability assay [1]
Cell Types: CHO Cell
Tested Concentrations: 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Viability diminished by approximately 50% after 24 hrs (hours).

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Western Blot Analysis[1]
Cell Types: CHO Cells
Tested Concentrations: 10 μM, 25 μM, 50 μM, 100 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Overall OGlcNAc acylation was diminished in a dose-dependent manner.

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Global O-GlcNAcylation: Chinese hamster ovary (CHO) cells were treated with 50 μM OSMI-1 or the control compound Ac4-5SGlcNAc for 24 hours. Cell lysates were prepared and analyzed by Western blot using the RL2 antibody, which recognizes O-GlcNAc-modified proteins. OSMI-1 treatment resulted in a significant reduction in global O-GlcNAcylation levels. This effect was also observed in several other mammalian cell lines. [1]
Nup62 Glycosylation: CHO cells were treated with 50 μM OSMI-1 for 24 hours. Lysates were analyzed by Western blot using an anti-Nup62 antibody. Treatment with OSMI-1 caused Nup62 to shift to a lower molecular weight, consistent with the loss of its O-GlcNAc residues and serving as a biomarker for OGT inhibition. [1]
OGA and OGT Levels: CHO cells were treated with 50 μM OSMI-1 for 24 hours. Lysates were analyzed by Western blot using anti-OGA and anti-OGT antibodies. OSMI-1 treatment reduced cellular OGA levels without affecting OGT levels, another marker of OGT inhibition. [1]
Lectin Blotting for Glycan Selectivity: CHO cells were treated with 50 μM OSMI-1 or Ac4-5SGlcNAc for 24 hours. Cell lysates were probed with a panel of nine biotinylated lectins (ConA, LCA, Jacalin, PHA-E, ECL, PHA-L, GSL-I, PNA, DBA) to assess changes in cell surface N- and O-glycan structures. OSMI-1 treatment showed minimal changes in bands detected by most lectins, indicating it does not grossly perturb glycan structures. In contrast, Ac4-5SGlcNAc caused dramatic changes in glycans recognized by Jacalin, PHA-E, and LCA. [1]
Cell Viability: CHO cells were treated with 50 μM OSMI-1 for 24 hours. Cell viability decreased by approximately 50% compared to untreated controls. A structurally related but inactive analog, PG34, was used to help distinguish OGT-dependent phenotypes from off-target effects. [1]

Acute toxicity assay[2]
All procedures of the animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the School of Life Science & Medicine, Dalian University of Technology and all experiments were conducted according to the relevant guidelines. Zebrafish embryos at 72-hours post-fertilization were selected for the acute toxicity assay. Zebrafish embryos were generated by natural pairwise mating and raised at 28.5 °C in embryo water. Zebrafish embryos were arrayed in 24-well plate (20 larvae per well) and incubated with 1 mL of embryo water per well containing various concentrations of L01 or OSMI-1 at 28.5 °C for 24 h. DMSO (0.1%, v/v) solution served as the control. The observation of zebrafish was made directly in the 24-well plate using an inverted dissecting microscope. The number of dead zebrafish in each concentration solution was recorded within 24 h, and the survival rate was calculated (%).

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Zebrafish Acute Toxicity Assay:** Zebrafish embryos (72 hours post-fertilization) were exposed to various concentrations of OSMI-1. Mortality was recorded at 12 and 24 hours. The LC₅₀ (lethal concentration for 50% of the population) was calculated. The LC₅₀ of OSMI-1 was 0.031 mg/mL (56 μM) at 12 hours and 0.025 mg/mL (45 μM) at 24 hours. [2]

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Zebrafish Acute Toxicity Assay: Zebrafish embryos (72 hours post-fertilization) were exposed to various concentrations of OSMI-1. Mortality was recorded at 12 and 24 hours. The LC₅₀ (lethal concentration for 50% of the population) was calculated. The LC₅₀ of OSMI-1 was 0.031 mg/mL (56 μM) at 12 hours and 0.025 mg/mL (45 μM) at 24 hours. [2]

OSMI-1 is cell-permeable and enters cells in an active state, leading to a more rapid onset of action compared to the prodrug Ac4-5SGlcNAc, which requires metabolic conversion to its active form. [1]

Treatment of CHO cells with 50 μM OSMI-1 for 24 hours resulted in an approximately 50% decrease in cell viability. [1]
A structurally related but inactive control compound, PG34, showed similar effects on cell viability, suggesting that some of the observed cytotoxicity may be due to off-target effects rather than OGT inhibition alone. [1]

[1]. A small molecule that inhibits OGT activity in cells. ACS Chem Biol. 2015 Jun 19;10(6):1392-7.

[2]. Discovery of a Low Toxicity O-GlcNAc Transferase (OGT) Inhibitor by Structure-based Virtual Screening of Natural Products. Sci Rep. 2017 Sep 26;7(1):12334.

OSMI-1 is a sulfonamide formed by the condensation of the sulfonic acid group of 2-oxo-1,2-dihydroquinoline-6-sulfonic acid with the primary amino group of (2R)-2-amino-N-(2-furanmethyl)-2-(2-methoxyphenyl)-N-(2-thiophenemethyl)acetamide. OSMI-1 is a cell-permeable O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) inhibitor. It is both an EC 2. (transferase) inhibitor and an EC 2.4.1.255 (protein O-GlcNAc transferase) inhibitor. It is a sulfonamide belonging to the quinoline, furan, thiophene, aromatic ether, and tertiary amide classes.

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OSMI-1 is a small molecule inhibitor of O-GlcNAc transferase (OGT) developed from a high-throughput screening hit followed by medicinal chemistry optimization. Its core structure is based on a quinolone-6-sulfonamide (Q6S) scaffold. [1]
OGT is an essential mammalian enzyme that regulates numerous cellular processes by attaching O-linked N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Aberrant OGT activity is linked to several cancers, making it a potential therapeutic target. [1]
OSMI-1 is validated as an on-target OGT inhibitor in cells through multiple readouts: reduction in global O-GlcNAcylation, a mass shift of the highly glycosylated protein Nup62, and a decrease in OGA (the enzyme that removes O-GlcNAc) levels. [1]
Unlike some other OGT inhibitors, OSMI-1 does not appear to dramatically alter cell surface N- or O-linked glycan structures, suggesting it has improved selectivity for OGT over other glycosyltransferases. However, some cytotoxicity may arise from off-target effects, and an inactive analog (PG34) is available to help control for this. [1]

563.118

C, 59.67; H, 4.47; N, 7.46; O, 17.03; S, 11.38

1681056-61-0

(Rac)-OSMI-1;2748153-92-4

118634407

White to light yellow solid

1.4±0.1 g/cm3

1.652

4.32

2

8

10

39

1000

1

COC1=CC=CC=C1[C@H](C(=O)N(CC2=CC=CO2)CC3=CC=CS3)NS(=O)(=O)C4=CC5=C(C=C4)NC(=O)C=C5

IYIGLWQQAMROOF-HHHXNRCGSA-N

InChI=1S/C28H25N3O6S2/c1-36-25-9-3-2-8-23(25)27(28(33)31(17-20-6-4-14-37-20)18-21-7-5-15-38-21)30-39(34,35)22-11-12-24-19(16-22)10-13-26(32)29-24/h2-16,27,30H,17-18H2,1H3,(H,29,32)/t27-/m1/s1

(R)-N-(Furan-2-ylmethyl)-2-(2-methoxyphenyl)-2-(2-oxo-1,2-dihydroquinoline-6-sulfonamido)-N-(thiophen-2-ylmethyl)acetamide

OSMI1 OSMI 1 OSMI-1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~177.42 mM)

Solubility in Formulation 1: 2.08 mg/mL (3.69 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.69 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)