Fig. 2.

Inhibition of NS2B/3_pro in vitro by ARDP0006. An in vitro assay was developed to measure inhibition of intramolecular cleavage at NS2B-3 in the dengue minimal proteinase. (A and B) Minimal proteinase was produced in RRL in the presence of increasing concentrations of ARDP0006 (50 μM to 2 mM), and remaining precursor was plotted to determine the IC₅₀ of self-cleavage in vitro (n = 1). (C and D) A pulse–chase assay was developed to monitor the effect of the inhibitor on reaction rate. Peak precursor abundance in RRL occurred at 25 to 30 min (C, lanes 1 to 3), and inactive NS3-S135A protein was robustly translated (C, lane 4) but no cleavage products were observed. The 32-kDa species seen in lane 4 is frequently observed background in this system. After labeling and translation, addition of 100 μM ARDP0006 resulted in modest but statistically significant inhibition of cleavage (D, dashed line) relative to mock treated reactions (D, solid line). Line fitting was performed using GraphPad Prism v.7 using a log[inhibitor] vs. response variable-slope equation for determining the apparent IC₅₀ (B) and single-phase exponential decay functions to fit the data in D. The P value in D was determined by comparing the fit of individual and shared models using the extra sum-of-squares F test. n = 3 (dashed line) or 5 (solid line).