Potent Irreversible GST Inhibitor CNBSF

CNBSF can be stored in DMSO stock solution for long-term in less than -20℃. However, CNBSF shows highly chemical reactivity in aqueous solutions. Especially in culture media such as DMEM, CNBSF quickly reacts with various components of DMEM, resulting in its decomposition. For example, a stability of CNBSF in serum-free DMEM at 37℃ is experimentally estimated, 60% degradation in 10 min and ～95% degradation in 30 min. Therefore, the use of inorganic salt buffers such as HEPES-buffered saline is recommended for live cell experiments. Hydrolysis of sulfonyl fluoride group of CNBSF is still observed gradually even in HBS (～50% hydrolysis in 30 min, ～70% hydrolysis in 60 min at 37℃ in HBS). So, working solution in aqueous solution such as HBS should be prepared just before use to avoid hydrolysis of CNBSF. Based on degradation or hydrolysis of CNBSF in buffer or medium, long-term incubation (>1 hour) of CNBSF for cells may not effectively. If the use of medium is required for your experiments, please optimize CNBSF concentration such as 1 mM.

Recombinant human or mouse cytosolic GST enzymes and reduced GSH (10 μM) were added into assay buffer (50 mM sodium phosphate (pH 7.4), 150 mM NaCl). DMSO for the control experiment or CNBSF (100 μM) was further added to them and incubated for 30 min. After then, a fluorescent GST activity assay reagent ☞ CellFluor™ GST (final 5 μM) was added and incubated for 30 min. Fluorescent intensity of each sample was measured by fluorescent plate leader (Ex 475±5 nm/Em 525±10 nm).

CNBSF effectively inhibited all cytosolic GST enzymes tested here.

Recombinant human GSTM1 or GSTA1, GSH (final 100 μM) and CNBSF (final 100 μM) were added to PBS and incubated for 30 min at 37℃. Each sample was split into two vials and one vial was applied into micro-dialysis kit (1 kDa-cut off) for 30 min in PBS. After dialysis, reduced GSH (additionally 100 μM) and ☞ CellFluor™ GST (1 μM) were further added to each sample and incubated for 30 min.

Neither GSTM1 nor GSTA1 activity was restored by dialysis after addition of CNBSF.

CHO cells were seeded in 96 well plate at 1×10⁴ cells/well and cultured in DMEM containing 10% FBS for 20 hours. Then culture media were replaced with HBS, treated with 100 μM CNBSF and ☞ CellFluor™ GST in 3 different protocols shown in the right scheme. The fluorescent intensities were measured with a fluorescent plate reader (Ex 475±5 nm/Em 525±10 nm).

In all protocols, GST activities were highly suppressed, indicating that CNBSF irreversibly inhibits intracellular GSTs.

786-O and CHO cells were seeded in 96 well plate at 1×10⁴ cells/well and cultured in DMEM containing 10% FBS for 20 hours. Then culture media were replaced with HBS, treated with 1-1000 μM CNBSF for 1 hour, and further treated with 2 μM ☞ CellFluor™ GST for 1 hour. The fluorescent intensities were measured with a fluorescence plate reader (Ex 475±5 nm/Em 525±10 nm).

Both 786-O and CHO cell lines showed similar IC₅₀ values around 4-5 μM in cell-based assay.

786-O and CHO were seeded in 96 well plate at ×10⁴ cells/well, cultured in DMEM containing 10% FBS for 20 hours. Then culture media were replaced with HBS, cells were incubated in the presence of 20 μM CNBSF or ethacrynic acid (EA) for 1 hour, and further treated with 2 μM ☞ CellFluor™ GST for 1 hour. Fluorescent intensities were measured with a fluorescent plate reader (Ex 475±5 nm/Em 525±10 nm) without medium change.

In both 786-O and CHO cells, CNBSF showed higher GST inhibitory activity than ethacrynic acid at 20 μM concentration.

786-O and Neuro2a were seeded in 96 well plate at 1×10⁴ cells/well, and cultured in DMEM containing 10% FBS for 20 hours. Then the culture media were replaced with serum- and phenol red-free DMEM (DMEM(−)). After incubation in HBS containing 100 μM CNBSF for 20 min, the HBS media were returned to DMEM(−) and cultured for 0, 2, 4, and 6 hours. The cells were then simultaneously treated with DMEM(−) containing ☞ CellFluor™ GST (2 μM) for 1 hour. The fluorescent intensities were measured with a fluorescent plate reader (Ex 475±5 nm/ Em 525±10 nm).

In 786-O cells, the inhibitory effect of GST was observed to continue for at least 6 hours, while a gradual recovery was observed over time for Neuro2a cells, indicating prompt rescue expression of GSTs.

CHO cells were seeded in 96 well plate at 1×10⁴ cells/well and cultured in DMEM containing 10% FBS for 20 hours. Then culture media were replaced with HBS and pretreated with 100 μM CNBSF for 1 hour. Cells were then washed with PBS, subsequently treated with lipid-peroxidation (LPO) initiators, H₂O₂ (0.1 mM or 0.5 mM), nitric oxide (NO) donor (SNAP; 0.1 mM), or radical initiator (APS; 1 mM) in HBS for 20 min or Cu²⁺ (CuSO₄; 1 mM) in HBS for 5 min. Cells were washed with HBS and ☞ AcroleinRED (2 μM), a cell-based detection reagent for acrolein, or TAMRA fluorescent dye as a negative control in HBS was treated for 30 min. Then, cells were washed 3 times with HBS to remove unreacted ☞ AcroleinRED or TAMRA, and fluorescent intensities were measured with a fluorescent plate reader (Ex 540±5 nm/ Em 580±10 nm).

Under the control condition, each drug slightly increased acrolein production. Pretreatment of CNBSF dramatically increased acrolein production and CNBSF with each LPO initiators synergistically promotes acrolein production. These results suggest the physiologically generated or drug-induced acrolein was suppressed by the cellular GST activities.