Pro-GA Cell-permeable GGCT inhibitor

Pro-GA (Cell-permeable GGCT inhibitor)

Cat. No. FNK-FDV-0019
Store at -20°C
Size 2 mg
Formulation C₁₂H₁₉O₇N
Molecular weight 289.28 g/mol
Solubility Soluble in DMSO


Description

For research of glutathione metabolic cycle
Pro-GA
The cell permeable GGCT Inhibitor

GGCT (γ-Glutamylcyclotransferase, C7orf24) is a glutathione homeostasis-related enzyme, which is highly expressed in cancer cells. While GGCT expression is low in normal cells, GGCT overexpressed cells dramatically increase its proliferation. Meanwhile, cell proliferation and migration are suppressed in GGCT KO cancer cells. This knowledge suggests that GGCT accelerates cell proliferation.
Existing GGCT inhibitors, such as N-glutaryl-L-alanine (GA), do not have cell membrane permeability, and cannot be used for cell culture and animal experiment.Pro-GA is the world’s first pro-drug type of GA, which penetrates into cells with inhibition activity to GGCT.

This product has been commercialized with the support of Kyoto Pharmaceutical University.

Product Background

y-Glutamylcyclotransferase (GGCT) catalyzes the formation of 5-oxoproline and free amino acids from y-glutamyl dipeptides for maintaining glutathione homeostasis. Although the enzymatic activity of GGCT was firstly reported in 1956, the gene of GGCT had not been identified for many years. In 2008, a biochemical study revealed C7orf24, a functionally unknown and highly expressed protein in bladder cancer cells, shows GGCT activity (ref.1, 2). Many studies show GGCT is highly expressed in various cancer cells as compared with normal tissues.

Overexpression of GGCT promotes cell proliferation in NIH-3T3 with low-level GGCT expression and depletion of GGCT mRNA by RNA interference inhibits cell-growth in several types of cancer cell lines which show high expression of GGCT. A potent inhibitor of GGCT like RNAi methods will be powerful tool to investigate the physiological function of GGCT. N-Glutaryl-L-alanine (GA) is one of the potent inhibitor for GGCT activity, but GA has little membrane-permeability. Pro-GA is a novel diester-type cell-permeable GGCT inhibitor pro-drug and shows the inhibitory effects for proliferation of multiple malignant cancer cells in vitro and tumor growth in vivo.

Features

• Pro-drug type of N-glutaryl-L-alanine (GA)
• High cell membrane permeability
• Transient inhibition of GGCT activity
• Specifically suppresses proliferation of cancer cells.
• Compatible to in vitro and in vivo use
  (not for human or veterinary use)

• Molecular Formula: C12H19O7N
• Chemical Structure: see below
• Molecular Weight: 289.28
• Solubility: Soluble in DMSO

 
 
Fig.1 GA-release efficiency of Pro-GA in cells
MCF7 cells were treated with 100 μM of fluorescent NBD-conjugated Pro-GA for 0-60 min. After washing, cell lysates were prepared and analyzed by HPLC to evaluate GA amount released from Pro-GA. NBD-conjugated Pro-GA was immediately incorporated into cells within a few minutes and converted to GA in the cells.

 
Fig.2 Inhibition of proliferation in cancer cells
Cells were cultured in serum starve condition with DMSO (as control), GA or Pro-GA for the indicated days. Cell proliferation was measured by WST cell viability assay. Pro-GA specifically suppressed proliferation of cancer cells.

 
Fig.3 Stability of Pro-GA in culture medium at room temperature
Stabilities of fluorescent NBD-conjugated Pro-GA in PBS or 10% FBS/DMEM were tested by HPLC analysis. While conversion of Pro-GA to GA was slowly in PBS (t1/2>5 hours), Pro-GA was quickly converted into GA in 10% FBS/DMEM by esterase activity in FBS (t1/2~50 min). 

Please prepare Pro-GA solution in assay medium just before use.

How to use

General procedure of cell-based inhibition of GGCT by Pro-GA
 Before Use

pH of assay medium is very important to avoid non-enzymatic hydrolysis of Pro-GA. Because both acidic and basic conditions can induce hydrolysis of Pro-GA, pH should be neutral. As the assay medium, DMEM containing 25 mM HEPES (DMEM/HEPES) is strongly recommended.

1. Prepare 100 µM of Pro-GA in DMEM/HEPES just before use.

Note Pro-GA is not stable in DMEM/HEPES for long time. Please use Pro-GA in DMEM/HEPES within 5 min.

2. Exchange culture medium to the Pro-GA in DMEM/HEPES and culture cells for 1-120 hours.

Note Pro-GA is immediately penetrated into cells and converted to GA inside the cells by esterases. Regarding detail information, please find “GA-release efficiency of Pro-GA in cells” below.

Reference

1. Oakley et al., J. Biol. Chem. 283, 22031-22042, (2008)
2. Kageyama et al., Proteomics Clin. Appl., 1, 192-199 (2007)
3. Ii et al., ChemMedChem., 13, 155-163 (2018)