Mechanistic insights into Zika virus NS3 helicase inhibition by Epigallocatechin-3-gallate: Supplementary Files

Deepak Kumar; Nitin Sharma; Murali Aarthy; Sanjeev Singh; Rajanish Giri

Inhibition assays were carried out in similar buffer conditions as used in activity assay. EGCG (Sigma-Aldrich) was dissolved in water at a stock concentration of 5mM. Inhibition assay was carried out in triplicates in 96 well plate. Initially, the 80nM enzyme is pre-incubated with a varying concentration of EGCG (serially diluted:12000 nM to 23.40 nM) in assay buffer at 25 o C for 10 minutes. After incubation, 1mM ATP substrate was added to the wells and incubated for 20 minutes. Finally, the reaction was stopped by adding 160 uL malachite green reagent to all the wells. Absorbance was taken at 630 nm after 5 minutes. IC50 value was calculated by fitting the data in non-linear regression mode using GraphPad Prism 7.0. Further, the inhibition kinetic parameters were calculated at different ATP concentrations as 100, 150, 250, 400, 600 and 1000 µM. Two concentrations of EGCG were chosen in 40 µl sample volume as 0.5 µM and 0.25 µM which are above and below from IC50 value. For all the reactions, 80 nM enzyme was preincubated in assay buffer with different EGCG concentrations for 10 minutes. Afterward ATP was added in varying concentrations as mentioned above and incubated for 20 minutes. Malachite reagent (160 µL) was added in each well and absorbance was taken at 630 nm after 5 minutes. All the measurements were taken in triplicates and Ki (Inhibition constant) was determined after fitting data in GraphPad Prism 7.0.