Alkylglycerone phosphate synthase (AGPS). The compound is a covalent inhibitor targeting AGPS by binding to its active site, involving interactions with the FAD cofactor and key residues such as Asp303, Ser527, His616, and His617. [1]

- Binding Affinity (ThermoFAD): In a ThermoFAD assay measuring protein thermal shift (ΔTm), compound 2i showed the highest binding affinity among the series, with a ΔTm of +5.0 °C. [1]
Its resolved single enantiomers showed ΔTm of +3.0 °C for the first eluted enantiomer and +6.0 °C for the second eluted enantiomer. [1]
- Enzymatic Inhibition (Radioactivity Assay): In a radioactivity‑based assay using palmitoyl‑DHAP and [1‑14C]hexadecanol as substrates, 2i (180 μM) inhibited AGPS enzymatic activity to an extent comparable to or slightly better than the reference compound 1. [1]
Due to substrate hydrophobicity causing micelle formation, accurate Km and Ki/IC50 could not be determined. [1]
- Ether Lipid Reduction (231MFP cells): In 231MFP breast cancer cells treated with 500 μM 2i for 24 h, LC‑MS/MS lipidomic analysis revealed that the levels of various ether phospholipids (e.g., ether forms of MAG, PA, PI, PC, PS, LPC, LPE, LPA) were decreased by 50‑70%. [1]
- Cell Migration Inhibition (231MFP cells): In a Transwell migration assay, pre‑treatment of 231MFP cells with 500 μM 2i for 6 h reduced cell migration rate by 78% compared to DMSO control. [1]
- Cell Viability Effect (231MFP cells): Treatment of 231MFP cells with 500 μM 2i for 24 h and 48 h decreased cell viability by 44% and 57%, respectively, relative to DMSO control. [1]
- Proliferation Effect (PC‑3, MDA‑MB‑231, Met5A cells): In PC‑3 prostate and MDA‑MB‑231 breast cancer cells, 2i (50, 100, 250 μM) caused a concentration‑dependent reduction in proliferation after 24 h, with significant inhibition at 250 μM, especially in MDA‑MB‑231 cells (which express higher AGPS mRNA). [1]
In non‑tumorigenic Met5A cells, 2i showed negligible effects even at 250 μM, suggesting cancer‑cell selectivity. [1]
- EMT Marker Modulation (PC‑3, MDA‑MB‑231 cells): Treatment of PC‑3 and MDA‑MB‑231 cells with 50 or 100 μM 2i for 24 h increased E‑cadherin mRNA levels and decreased Snail and MMP2 mRNA levels, as measured by RT‑PCR. [1]
These effects were similar to or greater than those of compound 1, while the inactive analog 2s had no effect. [1]
- AGPS Knockdown Combination (MDA‑MB‑231 cells): In MDA‑MB‑231 cells transfected with AGPS siRNA and then treated with 100 μM 2i for 24 h, the changes in E‑cadherin, Snail, and MMP2 mRNA levels were similar to those observed with either siRNA alone or 2i alone, with no additive effect, confirming that the EMT modulation by 2i is specifically dependent on AGPS inhibition. [1]

- ThermoFAD Binding Assay: To evaluate compound binding to AGPS, ThermoFAD experiments were performed. AGPS (5 μM) was incubated with 180 μM test compound in buffer (50 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol) in a final volume of 20 μL. [1]
A temperature gradient from 25 °C to 70 °C was applied, and fluorescence was monitored at 0.2 °C intervals (excitation 485±30 nm, emission 625±30 nm) for 5 s. The unfolding temperature (Tm) was determined from the increase in FAD fluorescence upon release from the protein. ΔTm represents the shift in Tm upon compound addition, reflecting binding affinity. For 2i, ΔTm was +5.0 °C. [1]
- Radioactive Enzymatic Activity Assay: AGPS enzymatic activity was measured using a radioactive substrate. The reaction mixture contained AGPS protein, 100 μM palmitoyl‑DHAP, 96 μM [1‑14C]hexadecanol, and 180 μM inhibitor. [1]
Formation of [1‑14C]hexadecyl‑DHAP was monitored over time. The catalytically inactive AGPS mutant T578F served as a negative control. Inhibition was assessed by comparing product formation curves. 2i inhibited AGPS activity with an efficacy similar to or slightly better than compound 1. [1]
Due to the hydrophobicity of substrates, which tend to form micelles and precipitate under assay conditions, accurate Km and Ki/IC50 values could not be determined. [1]

- Lipidomic Analysis (231MFP cells): 231MFP cells were cultured in serum‑free medium for 24 h to minimize serum‑derived metabolites, then treated with 500 μM 2i or DMSO for 24 h. [1]
Cells (1×10⁶) were harvested, washed with PBS, and flash‑frozen. Lipids were extracted with 4 mL of chloroform:methanol:phosphate‑buffered saline (2:1:1, pH 7.4) containing internal standards (C12:0 dodecylglycerol and pentadecanoic acid). [1]
The organic layer was collected, dried under N₂, and dissolved in 120 μL chloroform. An aliquot (10 μL) was analyzed by SRM‑based LC‑MS/MS using an Agilent 6430 QQQ with ESI source and a Luna C5 reverse‑phase column. [1]
Relative levels of ether lipids were calculated by comparing peak areas to DMSO control. Treatment with 2i reduced various ether phospholipids by 50‑70%. [1]
- Cell Migration Assay (231MFP cells): Transwell chambers (pre‑coated with collagen) were used. 231MFP cells were pre‑treated with 500 μM 2i for 24 h, then trypsinized and resuspended in serum‑free medium. [1]
Cells (2×10⁴) were seeded into the upper chamber, and serum‑containing medium was placed in the lower chamber as chemoattractant. After 6 h, non‑migrated cells were removed from the upper surface, and migrated cells on the lower surface were fixed, stained, and counted. Migration rate was expressed as percentage of DMSO control. 2i reduced migration by 78%. [1]
- Cell Viability Assay (231MFP cells): 231MFP cells were seeded in 96‑well plates and treated with 500 μM 2i for 24 or 48 h. [1]
MTS reagent was added, and absorbance at 490 nm was measured. Viability was calculated as percentage of DMSO control. Reductions of 44% and 57% were observed at 24 and 48 h, respectively. [1]
- Proliferation Assay (PC‑3, MDA‑MB‑231, Met5A cells): Cells (20×10³/well) were seeded in 96‑well plates. After 24 h, medium containing 2i (50, 100, 250 μM) or DMSO was added and incubated for another 24 h. [1]
MTS reagent was then added, and absorbance at 490 nm was measured. Viability was expressed as percentage of DMSO control. Three independent experiments (total 8 measurements) were performed. [1]
- RNA Extraction and RT‑qPCR (PC‑3, MDA‑MB‑231 cells): Cells were treated with 50 or 100 μM 2i for 24 h. [1]
Total RNA was isolated using a commercial kit and reverse transcribed. qPCR was performed with specific primers for E‑cadherin, Snail, MMP2, and the housekeeping gene L32 using a SYBR Green master mix. [1]
Relative expression was calculated using the 2⁻ΔCt method and normalized to L32. 2i increased E‑cadherin and decreased Snail and MMP2 mRNA levels. [1]
- siRNA Knockdown Experiment (MDA‑MB‑231 cells): Cells were transfected with AGPS siRNAs (AGPS#1 and AGPS#2, total 100 pmol) or control siRNA using a lipid‑based transfection reagent. [1]
After 24 h, medium was replaced with fresh medium containing 100 μM 2i or an equal volume of DMSO, and cells were incubated for another 24 h. RNA was extracted and analyzed by RT‑qPCR for AGPS, E‑cadherin, Snail, and MMP2. [1]
The combination of AGPS knockdown and 2i treatment produced changes similar to either treatment alone, confirming the specificity of 2i for AGPS. [1]

Effects on non-tumor cells: In non-tumor human epithelial cells Met5A, treatment with 2i at a concentration as high as 250 μM for 24 hours had negligible effect on cell proliferation, indicating that it has a certain selectivity for cancer cells. [1]

[1]. Development of alkyl glycerone phosphate synthase inhibitors: Structure-activity relationship and effects on ether lipids and epithelial-mesenchymal transition in cancer cells. Eur J Med Chem. 2019 Feb 1;163:722-735.

Background and Mechanism: AGPS is a peroxisome flavin enzyme that catalyzes a key step in the biosynthesis of ether lipids, namely, the exchange of fatty acyl chains of acyl-DHAP with fatty alcohols through a non-redox mechanism using FAD as a cofactor. [1]
AGPS is upregulated in a variety of aggressive cancers (breast cancer, melanoma, prostate cancer), and its knockdown can reduce ether lipid levels and oncogenic signaling molecules, thereby inhibiting tumor growth. [1]
Compound 2i was developed by optimizing the structure of the first AGPS inhibitor 1, introducing a 2,6-difluoro substituent on the benzene ring to enhance the hydrophobic interaction within the substrate binding channel, thereby improving binding capacity and inhibitory efficacy. [1]
-EMT Regulation: By inhibiting AGPS, 2i upregulates E-cadherin and downregulates Snail and MMP2, thereby inhibiting epithelial-mesenchymal transition (EMT), a key process of tumor invasion and metastasis. [1]
These effects have been confirmed in PC-3 and MDA-MB-231 cells and are similar to the phenotype of AGPS gene knockdown, suggesting that these effects are specifically mediated by AGPS inhibition. [1]
-Cancer cell selectivity: The 2i cells exhibited stronger antiproliferative activity in AGPS-overexpressing cancer cells (e.g., MDA-MB-231) than in non-tumor Met5A cells, suggesting its potential for selective cancer therapy. [1]

C18H17F2N3O2

345.3

345.128

C, 62.60; H, 4.96; F, 11.00; N, 12.17; O, 9.27

2316782-88-2

155559442

Off-white to light brown solid powder

2.1

3

4

5

25

495

0

C(C1=CC=C2NC(=O)NC2=C1)NC(=O)CC(C1C(=CC=CC=1F)F)C

VDLFVZUKOKPJRH-UHFFFAOYSA-N

InChI=1S/C18H17F2N3O2/c1-10(17-12(19)3-2-4-13(17)20)7-16(24)21-9-11-5-6-14-15(8-11)23-18(25)22-14/h2-6,8,10H,7,9H2,1H3,(H,21,24)(H2,22,23,25)

3-(2,6-difluorophenyl)-N-[(2-oxo-1,3-dihydrobenzimidazol-5-yl)methyl]butanamide

AGPS-IN-1; 2316782-88-2; AGPS-IN-2i; 3-(2,6-Difluorophenyl)-N-((2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)methyl)butanamide; AGPS-IN-2i?;

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: ~125 mg/mL (362 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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