GTPase Activity Assay

The Effect of 1 on the GTPase activity of recombinant FtsZ was measured in 96-well microplates using a CytoPhos phosphate assay Biochem Kit (Cytoskeleton, USA) according to an optimized protocol and the manufacturer’s instructions. FtsZ (6 μM) was preincubated with vehicle (1% DMSO) or different concentrations of each test compound in 20 mM Tris buffer (pH 7.4, containing 0.01% Triton X-100 to avoid compound aggregation) for 10 min at 25°C. Then 5 mM of MgCl₂ and 200 mM of KCl were added. Reactions were started with the addition of 500 mM GTP and incubated at 37°C. After 30 min, the reactions were quenched by adding 100 mL of Cytophos reagent for 10 min. Inorganic phosphate was quantified by measuring the absorbance at 650 nm with a microplate reader (Bio-Rad laboratory Ltd., UK).