Inhibition of Influenza A Virus (H1N1) Fusion by Benzenesulfonamide Derivatives Targeting Viral Hemagglutinin
Figure 4
SDS-PAGE of trypsin sensitivity assay showing RO5464466 protected BHA from trypsin digestion in pH-dependent and dose-dependent manner.
(A) 6 µg BHA was incubated with compounds of different concentrations for 15 minute at 31°C prior to acidification to pH 5.0 with 0.25 M citrate (pH 4.2). The mixture was neutralized to a final pH of 7.5 and treated with 2 µg of trypsin for 30 minutes at 37°C. The extent of trypsin cleavage on BHA was analyzed on a 10% SDS-PAGE gel. MW was shown on the right in thousands. Untreated BHA, trypsin alone, BHA trypsin digestion without a prior acidification step were used as controls and included in this figure. (B) Dose-dependent protection of BHA by RO5464466. (C) pH-dependent protection of BHA by RO5464466. After incubated with 10 µM of RO5464466, BHA was acidified to different final pH shown on the top of the gel before neutralization and trypsin digestion. As a negative control, DMSO-treated BHA was adjusted to two pH values (5.0 and 5.2) before neutralization and trypsin digestion. (D) RO5464466 protected BHA from trypsin digestion not by directly inhibiting trypsin enzymatic activity. RO5464466 was added into the reaction either before or after acidification of BHA.