CNBSF (Irreversible GST Inhibitor)
Storage -20 ºC
CAS No. 3829-23-0
Chemical structure See Figure 3
Molecular weight 239.6 g/mol
Solubility Soluble in DMSO
Generally GSTs have two types of binding site, called G-site and H-site, for GSH and hydrophobic substrate, respectively. When GSTs bind to GSH, GSTs catalyze and stabilize thiolate anion of GSH. Once hydrophobic substrates binds to GSTs, GSTs transfer them to GSH to form GST-conjugates. GST-conjugates are quickly exported to extracellular space by multidrug resistance-associated protein (MRP) transporters. Through the above processes, GSTs detoxify toxic compounds. On the other hand, GSTs also neutralize pharmaceutical drugs via its detoxifying processes.
As many studies suggested expression level of GSTs are significantly increased in cancer cells, GSTs are considered as anticancer-resistant enzymes in cancer cells. Inhibition of GST activities is one of the promising strategy to improve drug efficiency in cancer cells. Some GST inhibitors have been developed so far, however, conventional inhibitors including ethacryic acid (EA), a representative GST inhibitor, were based on the competition with GSH. In general, competitive inhibitors are usually reversible and not sufficient in cells because of high concentration around mM order of GSH in cells.
Although several irreversible inhibitors were also discovered and show potent inhibition in vitro, these compounds have low membrane-permeability and are not good at live cell experiments. To overcome conventional problems, highly membrane-permeable and irreversible inhibitors are desired.
2-chloro-5-nitrobenzensulfonyl fluoride (CNBSF) is a novel type of irreversible GST inhibitor reported by Dr. Hiroshi Abe, Nagoya University, and co-workers (Ref.1). CNBSF is membrane-permeability and capable of entering into cytosol. Once CNBSF incorporated into cell, GSTs catch CNBSF in H-site as a hydrophobic xenobiotics. Subsequently GSTs convert CNBSF to GSH-conjugated CNBSF, called GS-5NBSF. In the case of GSTP1, a member of pi-type GST, Tyr108 residue in GSTP1 quickly reacts with sulfonyl fluoride group of GS5NBSF, fluoride anion was leaved, and form covalent bound between GST and substrate. In the result of above scheme, GSTs covalently bound inhibitor-complex are inactivated.
Reconstitution and Storage
Storage (solution): Store powder at -20oC.
After reconstitution in DMSO, aliquot and store at -20 °C. Avoid repeated freeze-thaw cycles.
For in vitro experiments : 0.1-1 mM
Recombinant human GSTP1-1 (4 ?g/ml) in 10 mM phosphate buffer was treated 1 mM CNBSF or ethacrynic acid (EA) for 2 hours at 37oC. After incubation, without or with buffer change by ultrafiltration to remove excess inhibitors, GST activities were measured by DNs-Rh (1 ?M). EA shows reversible inhibition of GSTP1-1 because GST activity was dramatically recovered by buffer change “Wash (+)”. On the other hand, CNBSF kept inhibition of GST activity after buffer change.
*DNs-Rh is also available from Funakoshi, catalog code #FDV-0030 (See below “Related products”)
Inhibition of GST under live cell condition
NCI-H522 cells were treated with trypsin to be suspended and incubated with 1 mM CNBSF or ethacryic acid (EA) for 15 min at 37oC. After wash cells by PBS, DNs-Rh was added to be final concentration 2.5 ?M and cells were incubated for 1 hour at 37oC. Cells were washed by PBS to remove excess reagents and applied into flow cytometory (Ex. 490 nm/ Em. 520 nm). Cellular GST activity was dramatically inhibited by CNBSF. Inhibition activity of CNBSD was much higher than that of EA.
Identification of covalent binding site of CNBSF on GSTP1-1
Chemically synthesized GS-5NBSF was added to recombinant human GSTP1-1 to form inhibitor-GST complex. After enzymatic digestion by trypsin, peptide fragments were analyzed by LC-MS/MS. Tyr108 on GSTP1-1 was identified as GS-5NBSF binding site.
Appendix: Proposal reaction mechanism of CNBSF to GSTs
- GSH and CNBSF are bound to G-site and H-site in GST, respectively. Tyr 7 of GSTP1-1 stabilized thiolate anion of GSH and the thiolate anion of GSH attacked to CNBSF.
- Chloro anion was left and GS-5NBSF is formed.
- Subsequently, alkoxide anion of Tyr 108 attacked to sulfonyl fluoride group
- Ternary complex is formed
- Shishido et al., Chembiochem., 20, 900-905 (2019) A covalent inhibitor for Glutathione S-Transferase Pi (GSTP1-1) in human cells.