Inhibition of Influenza A Virus (H1N1) Fusion by Benzenesulfonamide Derivatives Targeting Viral Hemagglutinin

Hemagglutinin (HA) of the influenza virus plays a crucial role in the early stage of the viral life cycle by binding to sialic acid on the surface of host epithelial cells and mediating fusion between virus envelope and endosome membrane for the release of viral genomes into the cytoplasm. To initiate virus fusion, endosome pH is lowered by acidification causing an irreversible conformational change of HA, which in turn results in a fusogenic HA. In this study, we describe characterization of an HA inhibitor of influenza H1N1 viruses, RO5464466. One-cycle time course study in MDCK cells showed that this compound acted at an early step of influenza virus replication. Results from HA-mediated hemolysis of chicken red blood cells and trypsin sensitivity assay of isolated HA clearly showed that RO5464466 targeted HA. In cell-based assays involving multiple rounds of virus infection and replication, RO5464466 inhibited an established influenza infection. The overall production of progeny viruses, as a result of the compound's inhibitory effect on fusion, was dramatically reduced by 8 log units when compared with a negative control. Furthermore, RO5487624, a close analogue of RO5464466, with pharmacokinetic properties suitable for in vivo efficacy studies displayed a protective effect on mice that were lethally challenged with influenza H1N1 virus. These results might benefit further characterization and development of novel anti-influenza agents by targeting viral hemagglutinin

Comparison of <i>in vitro</i> anti-influenza activities of RO5464466 and RO5487624.

a<p>: All numbers (EC₅₀, CC₅₀, and IC₅₀) are means of three independent experiments and shown in micromolar concentrations;</p><p>Some data from A/Weiss/43 have been shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#pone.0029120-Tang1" target="_blank">[29]</a>.</p>b<p>: MDCK cells were used;</p>c<p>: + means nearly 100% protection of BHA from trypsin degradation in the presence of compounds (10 µM).</p

RO5464466 inhibited HA-mediated hemolysis of chicken erythrocytes.

<p>After mixing RO5464466 with virus-containing allantoic fluid, suspension of freshly prepared chicken erythrocytes was added. The mixture was then acidified with different pH from 4.85 to 6. The suspension was incubated at 37°C for 30 minutes. The final concentration of RO5464466 was 10 µM. After a brief spin, supernatants containing released hemoglobin were transferred to a second plate for measuring OD₅₄₀. As a control, suspensions of erythrocytes alone were acidified (pH control) similarly to measure low pH-caused hemolysis. In the DMSO control group, every step was the same as described in RO5464466 group except the testing compound was omitted. All conditions were tested in duplicated wells. *, results significantly different (RO5464466 treated samples vs. DMSO controls) by Student's t test (<i>P</i><0.05).</p

Pharmacokinetic analysis of RO5487624.

<p>Mean plasma concentration-time profiles of RO5487624 after an oral dose (30 mg/kg, ◊, 100 mg/kg □, and 200 mg/kg, Δ) to CD-1 mice. Vertical lines are standard deviation of each group (n = 3). Concentration corresponding to RO5487624's EC₉₀ after protein binding adjustment is marked by a dotted line.</p

<i>In vivo</i> efficacy of RO5487624.

<p>40 LD₅₀ of A/FM/1/47 (H1N1) were used for viral challenge in all groups by intranasal inoculation. The first dosage of all treatments was given either 1 h before (A) or 3 h after (B) virus challenge (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#s2" target="_blank">Materials and Methods</a> section). Mice in untreated control group were orally administrated with PBS twice a day for 7 days. Animals (n = 10 in each group) was monitored for 14 days starting from virus challenge. Data was analyzed with Chi-square for mortality rate, * <i>P</i><0.05; ** <i>P</i><0.001, and with Kaplan-Meier for mean survival day, # <i>P</i><0.05; ## <i>P</i><0.001, respectively.</p

RO5464466 blocked the production of progeny virus and inhibited an established influenza virus infection.

<p>(A) Decrease of progeny virus production in cells treated with RO5464466 and other reference compounds. 12-well plates were seeded with MDCK cells at a density of 1.5×10⁵ cells per well. On the next day, serially diluted compounds and 150 pfu virus were added per well. 48 hours later, treatment was stopped and viral titer in cell monolayer was measured in TCID₅₀. Virus yield was shown in a log scale, a mean of two wells. (B) Dynamics of virus yield reduction in the presence of inhibitors. In this experiment, all treatments began at the same time when virus was added into culture wells but were stopped at different time points of 24, 48, and 72 hours post-infection, respectively. (C) Inhibition of plaque formation by RO5464466. Details of seeding MDCK cells into 6-well plates and infecting monolayers with influenza A/Weiss/43 (H1N1) were described in the plaque reduction assay in the section of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029120#s2" target="_blank">Materials and Methods</a>. Instead of using 3 ml of agarose-containing overlay for each well, 2.5 ml of overlay was used. At each time point (0, 12, 24, 48 hours post-infection), 2.5 ml of culture medium that contained compound of 2-fold final concentration was added on the top of the overlay. At 72 hours post-infection, cell monolayers were fixed and stained to show plaques. Representative data of three independent experiments was shown.</p

SDS-PAGE of trypsin sensitivity assay showing RO5464466 protected BHA from trypsin digestion in pH-dependent and dose-dependent manner.

<p>(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.</p