ATB107

Kinase experiment:

The concentration of mIGPS is determined. The substrate CdRP is chemically synthesized, with a yield of 30 mM. Ten microliters of 30 mM CdRP and 10 μL of 1.24 μM IGPS are added to 480 μL of 5 mM Tris/HCl (pH 7.0), and incubated at 37°C for 20 min. The enzyme activity is measured with a spectrophotometer by following the increase in absorbance of the solution at 280 nm. ATB107 is added to the assay mixture to obtain concentrations of 10-4 M, 7.5×10-5 M, 5×10-5 M, 2.5×10-5 M, and 10-5 M, respectively[1].

Cell experiment:

The tetrazolium dye reduction assay [3-[4,5-dim-ethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)] is used to determine the effect of ATB107 on cell survival and growth. At first, the THP-1 macrophage cells are inoculated at 8×104 cells/mL) into 96-well plates and incubated at 37°C in a 5% CO2/95% air atmosphere for 24 h. ATB107 is added to give concentrations of 50,100, 150 and 200 μg/mL. After incubation of cells treated with ATB107 for 12 h, 20 μL (5 g/L) of MTT solution is added to each well; this is followed by incubation for another 4 h to allow the formation of formazan crystals. Finally, 10% SDS is added to dissolve the formazan crystals, and the plates are read on a microplate reader at 570 nm. Controls are included in which only culture media are added to wells containing cells[1].

References:

[1]. Shen H, et al. A novel inhibitor of indole-3-glycerol phosphate synthase with activity against multidrug-resistant Mycobacterium tuberculosis. FEBS J. 2009 Jan;276(1):144-54.