DHFR Enzyme Inhibition assay

The inhibition of DHFR with TMP derivatives was investigated in a 96-well plate (flat-bottom wells, Nunclon Delta Cell Culture Treated Clear Polystyrene catalog no. 167008) using a dihydrofolate reductase assay kit (Sigma-Aldrich, product code CS0340). E. coli dihydrofolate reductase was prepared using an In Vitro protein synthesis kit (New England BioLabs; PURExpress In Vitro Protein Synthesis; NEB#E6800). The DHFR enzyme was diluted before using in the inhibition assay by adding 23 μL of DHFR in 1 mL of water. The assay was performed using the protocol supplied by Sigma-Aldrich; however, the procedure was adjusted for a reaction volume of 200 μL. Briefly, the compounds were provided at a stock concentration of 12 mM in 100% DMSO. The 1× buffer mixture for assay was composed of DHF and NADPH. The well containing 100 μM TMP served as negative control (“DHFR is completely inhibited”) and the well without TMP or the untreated well served as positive control (“DHFR is not inhibited”). A dilution series of compounds in water was carried out to give the 3× concentrated range from 120 to 0.018 μM. Ten microliters of each dilution was added to a 96-well plate containing 180 μL of buffer mixture. Then, 10 μL of diluted DHFR was added into each well, giving the final compound concentration range from 6 to 0.0009 μM. The samples were immediately read by the plate reader. The reaction progress was monitored by the decrease in absorbance at 340 nm, which was read every 60 s for 40 min using a POLARstar Omega plate reader, with enzymatic inhibition calculated using the equation DHRF activity % = (Abs_sample – Abs_negative/Abs_untreated – Abs_negative) × 100. The data were analyzed by GraphPad Prism 7.00 software to determine IC₅₀ values.