I3MT-3

I3MT-3 (HMPSNE, compound 3), a pharmacological 3-MST inhibitor, exerts a bell-shaped effect on several cellular bioenergetic parameters related to oxidative phosphorylation in CT26 cells, suggesting that the 3-MST/H2S system significantly contributes to the maintenance of cell proliferation, migration and cellular bioenergetics.^[2]

The activity of 3-MST in presence of various concentrations of HMPSNE was also tested in CT26 cells homogenates. CT26 cells were centrifuged 5 min at 400× g in order to resuspend the pellet in lysis buffer 150 mM NaCl and 50 mM Tris-HCl, pH 8 containing 1% NP40 and Protease and Phosphatase Inhibitor Cocktail added just before use. Samples were sonicated with ultrasound probe three times a cycle of 5 s on and 5 s off at 70% amplitude and kept on ice for 30 min. The protein concentrations were measured as described above. HMPSNE dilutions were prepared as above and incubated 24 h at 37 °C with 100 µg/well of proteins from CT26 homogenates in black 96-well plate. After treating the homogenate with HMPSNE, 3-mercaptopyruvate was added for a final concentration of 500 µM and AzMC for a final concentration of 10 µM. Fluorescence measurement and analysis were performed.