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The enzymatic assay was employed to evaluate the inhibitory effect of N30 on the activity of IMPDH type II by following the increase in formation of nicotinamide-adenine dinucleotide (NADH), which absorbance was read at 340 nm on Enspire at 30 °C. The 200 μL assay buffer contained 1 M Tris/HCl (pH 8.8), 1 M KCl, 30 mM EDTA, 10 μL IMPDH type II (Sigma-Aldrich), 1 mM DTT and NAD (Sigma-Aldrich). The reaction was started after addition of IMP, with final concentrations was 1 mM [19].
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