PGM3 (Phosphoglucomutase 3). [1]

In breast cancer cells, FR054 (0.5–1 mM, 24-48 h) causes an early growth arrest, which is followed by a marked rise in cell death and the induction of apoptosis. Instead of having additional off-target effects, FR054 inhibits PGM3 [1]. N- and O-glycosylation levels in MDA-MB-231 cells are effectively impacted by FR054 (250 μM, 24 hours) treatment [1]. Endoplasmic reticulum (ER) stress and ROS-dependent apoptosis are induced by FR054 [1].

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Proliferation and Survival: FR054 treatment (250 μM to 1 mM for 48 h) dose-dependently reduced viability in seven different breast cancer cell lines. Six out of seven cancer cell lines were more sensitive than non-transformed hTERT-RPE-1 cells. At 72 h treatment with FR054, nearly 100% of TNBC cells (MDA-MB-231 and MDA-MB-468) appeared dead. FR054 (0.5 and 1 mM for 24 h) caused almost complete inhibition of colony formation in MDA-MB-231 cells. [1]
- Mechanism of Cell Death: FR054 (1 mM for 48 h) potently increased apoptosis (as measured by Annexin V-FITC/PI staining) in MDA-MB-231 cells, accompanied by caspase-3 activation and PARP cleavage. [1]
- Inhibition of Glycosylation: FR054 (1 mM) treatment reduced cellular UDP-GlcNAc levels by approximately 50% within 30 minutes. In MDA-MB-231 cells, FR054 treatment (250 μM for 24 h) significantly decreased membrane reactivity of PHA-L (detecting tri-/tetra-antennary N-glycans) and altered β1 integrin membrane localization. FR054 (1 mM) treatment led to a reduction of protein O-GlcNAc levels at 48 h. [1]
- Adhesion and Migration: FR054 (250 μM for 24 h) reduced MDA-MB-231 cell adhesion by about 60% and migration by about 50%. [1]
- UPR and ROS Induction: FR054 (1 mM) treatment for 24-48 h induced Unfolded Protein Response (UPR) markers (ATF4, CHOP, XBP1 mRNA upregulation; CHOP protein increase) in MDA-MB-231 cells. FR054 (1 mM) treatment significantly increased intracellular hydrogen peroxide and mitochondrial superoxide levels in a time-dependent manner. The addition of N-acetylcysteine (NAC) reduced ROS, decreased apoptosis, increased cell proliferation, and reduced CHOP expression and caspase-3 activation. [1]
- Mechanistic Confirmation: Pre-treatment with a NAGK inhibitor (NAGKi, 3-O-methyl-N-acetylglucosamine) blocked FR054-induced apoptosis. MDA-MB-231 cells with PGM3 knockdown (shPGM3 clones) showed significantly greater sensitivity to FR054 treatment (0.25 and 0.5 mM) compared to control clones, with reduced viability and increased apoptosis. [1]

In MDA-MB-231 xenograft mice, FR054 (1000 mg/kg, i.p.) suppresses the growth of cancer [1].

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Antitumor Efficacy: In an MDA-MB-231 xenograft mouse model, intraperitoneal (i.p.) administration of FR054 (fractionated dose: 500 mg/kg twice daily, total 1000 mg/kg/day for 5 days) resulted in significant tumor growth stasis and a significantly lower tumor volume variation compared to the control group (p = 0.008). Prolonged treatment (11 consecutive days with the fractionated dose) induced a reduction in tumor growth rate (Tumor Growth Inhibition, TGI = 41.77). A single daily dose of 1000 mg/kg did not show a significant difference compared to control. [1]

Cellular Thermal Shift Assay (CETSA): Cell extracts were heated from 49 to 70°C after incubation with FR054 or vehicle. Soluble proteins were collected and analyzed by Western blot. FR054 markedly increased the thermal stability of PGM3, indicating direct binding. The isothermal dose-response fingerprint (ITDRFCTESA) at 58°C showed FR054 enhanced PGM3 stability in a dose-dependent manner, with a more potent binding effect than the natural substrate GlcNAc-6-P. [1]
- UDP-GlcNAc Quantification by HPLC: Protein extracts from cells were used in an enzymatic assay with components including GlcNAc-6-P, UTP, and FR054. After 30 minutes at 37°C, UDP-GlcNAc was separated and quantified by HPLC. FR054 led to an approximate 50% reduction in UDP-GlcNAc levels compared to control. [1]

Cell Viability Assay[1]
Cell Types: MDA-MB-231 cells.
Tested Concentrations: 0.5-1 mM.
Incubation Duration: 48 h.
Experimental Results: decreased viability and a significant increase of the apoptosis as compared to the control clone.

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Cell Viability and Proliferation: Cells were seeded in 96-well plates. For viability, an MTT test was performed. For proliferation, cells were harvested and counted using a Burker chamber. Cell viability was also assessed by Trypan Blue exclusion. [1]
- Colony Formation Assay: After treatment, cells were re-seeded at low density in complete medium. After 12 days, cells were fixed, stained with crystal violet, and the absorbance was analyzed. [1]
- Apoptosis Assay: Cells were stained with Annexin V-FITC and propidium iodide (PI) in binding buffer. After 15 minutes at room temperature, samples were analyzed by flow cytometry. [1]
- ROS Measurement: Cells were stained with DCHF2DA for hydrogen peroxide or MitoSOX for mitochondrial superoxide for 30 minutes at 37°C and then analyzed by flow cytometry. [1]
- Flow Cytometry for N-Glycans: Cells were stained with fluorochrome-conjugated lectins ConA (for hybrid/high mannose and di-antennary glycans) or PHA-L (for tri-/tetra-antennary structures) for 1 hour on ice and analyzed by flow cytometry. [1]
- Confocal Microscopy: Cells were seeded on glass slides and treated with FR054. For β1 integrin, cells were incubated with anti-β1 integrin antibody, followed by an Alexa Fluor 488 conjugated secondary antibody. For N-glycans, cells were stained with PHA-L. Cells were fixed, stained with DAPI, and examined under a confocal microscope. [1]
- Adhesion and Migration Assays: For adhesion, treated cells were seeded into BSA-coated plates, allowed to adhere for 1 hour, and non-adherent cells were washed away. Adherent cells were then counted. For migration, treated cells were seeded into the upper compartments of Boyden chambers with a collagen VI-coated filter. Medium with serum was used as a chemoattractant in the lower compartment. After 4 hours, cells on the lower side of the filter were stained and counted. [1]
- Western Blot Analysis: Total protein lysates were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with specific primary antibodies overnight and then with secondary antibodies. Protein expression was detected and quantified by densitometry. [1]
- qPCR Analysis: RNA was extracted using Trizol, reverse-transcribed, and amplified by qPCR using SYBR Green. Relative expression levels were calculated using the 2-ΔΔCT method with β-actin as the endogenous control. [1]

Animal/Disease Models: Mice were subcutaneously (sc) injected with MDA-MB-231 cells[1].
Doses: 1000 mg/kg.
Route of Administration: IP, single or fractionated dose (twice a day 500 mg/kg/dose).
Experimental Results: Appears to have an in vivo antitumor efficacy that is higher when administered twice a day compared to single administration.

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Xenograft Model:** Female athymic nu/nu mice (about 6 weeks old, ~25 g) were subcutaneously injected with MDA-MB-231 cells (5x10^6 cells in 200 μL of a 1:1 mix of DMEM and Matrigel) on the right flank. [1]
- **Treatment and Dosing:** When tumors reached ~180 mm³, mice were randomized into groups. FR054 was dissolved in water with 10% DMSO. The compound was administered via intraperitoneal (i.p.) injection. A daily dose of 1000 mg/kg was given either as a single injection (once daily, 1000 mg/kg) or as a fractionated dose (twice daily, 500 mg/kg per dose). The treatment was carried out for 5 days or, in a separate study, for 11 consecutive days. Control mice received the vehicle (10% DMSO in water). [1]
- **Monitoring:** Tumor volume was measured daily using a digital caliper (volume = [long side × (short side)²]/2). Body weight was also monitored daily. Tumor Growth Inhibition (TGI) was calculated using the formula: TGI = [1 - (Tf/T0)A / (Tf/T0)V × 100]. [1]

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Xenograft Model: Female athymic nu/nu mice (about 6 weeks old, ~25 g) were subcutaneously injected with MDA-MB-231 cells (5x10^6 cells in 200 μL of a 1:1 mix of DMEM and Matrigel) on the right flank. [1]
- Treatment and Dosing: When tumors reached ~180 mm³, mice were randomized into groups. FR054 was dissolved in water with 10% DMSO. The compound was administered via intraperitoneal (i.p.) injection. A daily dose of 1000 mg/kg was given either as a single injection (once daily, 1000 mg/kg) or as a fractionated dose (twice daily, 500 mg/kg per dose). The treatment was carried out for 5 days or, in a separate study, for 11 consecutive days. Control mice received the vehicle (10% DMSO in water). [1]
- Monitoring: Tumor volume was measured daily using a digital caliper (volume = [long side × (short side)²]/2). Body weight was also monitored daily. Tumor Growth Inhibition (TGI) was calculated using the formula: TGI = [1 - (Tf/T0)A / (Tf/T0)V × 100]. [1]

Intracellular Concentration: In MDA-MB-231 cells treated with 1 mM FR054 for 24 hours, the total intracellular concentration of FR054 and its metabolites (FR053, the de-acetylated form; and FR051, the de-acetylated and phosphorylated active compound) was measured by LC/MS. The sum of all three forms was 925 ± 106 nM, and the concentration of the active compound FR051 was 106 ± 50 nM. [1]
- Chemical Stability: NMR analysis of ¹³C-labeled FR054 in cell culture medium revealed that after 24 hours, approximately 64% of the compound was hydrolyzed even in the absence of cells, indicating intrinsic instability. Only about 11% was taken up by cells. [1]

Cellular Toxicity: Non-transformed hTERT-RPE-1 cells were less sensitive to FR054 treatment compared to six out of seven breast cancer cell lines tested. [1]
- In Vivo Toxicity: In the MDA-MB-231 xenograft mouse model, no significant differences in body weight were observed between FR054-treated groups (both single and fractionated doses) and the control group over the 5- and 11-day treatment periods. No apparent signs of morbidity were observed based on body condition scoring. [1]

[1]. Inhibition of the Hexosamine Biosynthetic Pathway by Targeting PGM3 Causes Breast Cancer Growth Arrest and Apoptosis. Cell Death Dis. 2018 Mar 7;9(3):377.

Mechanism of Action: FR054 is a novel, cell-permeable inhibitor of the Hexosamine Biosynthetic Pathway (HBP). It acts as a competitive inhibitor of the enzyme PGM3. FR054 is a prodrug that is de-acetylated within the cell to form FR053, which is then phosphorylated by NAGK to yield the active compound, FR051. [1]
- Cellular Effects: FR054 treatment reduces intracellular UDP-GlcNAc, leading to decreased N- and O-linked protein glycosylation. This results in the activation of the Unfolded Protein Response (UPR), accumulation of intracellular reactive oxygen species (ROS), and ultimately, apoptosis. It also reduces cancer cell adhesion and migration by affecting β1 integrin membrane localization. [1]
- Potential Indication: The study suggests that FR054 has potential as a therapeutic agent for breast cancer, particularly for Triple-Negative Breast Cancer (TNBC). [1]

C14H19NO8

329.3026

329.111

C, 51.06; H, 5.82; N, 4.25; O, 38.87

10378-06-0

FR054;35954-65-5

9883925

Light yellow to orange semi-solid or waxy solid or viscous solid

-0.5

0

9

7

23

531

5

O1[C@@]2([H])[C@@]([H])(C([H])(C([H])(C1([H])C([H])([H])OC(C([H])([H])[H])=O)OC(C([H])([H])[H])=O)OC(C([H])([H])[H])=O)N=C(C([H])([H])[H])O2

WZFQZRLQMXZMJA-KSTCHIGDSA-N

InChI=1S/C14H19NO8/c1-6-15-11-13(22-9(4)18)12(21-8(3)17)10(5-19-7(2)16)23-14(11)20-6/h10-14H,5H2,1-4H3/t10-,11-,12-,13-,14+/m1/s1

[(3aR,5R,6S,7R,7aR)-6,7-diacetyloxy-2-methyl-5,6,7,7a-tetrahydro-3aH-pyrano[3,2-d][1,3]oxazol-5-yl]methyl acetate

6R-FR054; 6R-FR-054; 6R-FR 054;

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 (~303.67 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.59 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 25.0 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: ≥ 2.5 mg/mL (7.59 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.59 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.

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