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

Moderate/Severe hyponatremia increases the risk of death among hospitalized Chinese human immunodeficiency virus/acquired immunodeficiency syndrome patients.

OBJECTIVES: To evaluate whether the serum sodium concentration is associated with the progression and long-term prognosis of Chinese HIV/AIDS patients. METHODS: Three hundred and eighty seven hospitalized patients were recruited into this retrospective cohort study. The strata of serum sodium concentration were moderate/severe hyponatremia, mild hyponatremia and normonatremia. Disease progression was estimated using CD4 counts and the WHO clinical stage. Correlation analysis was used to evaluate the serum sodium concentration with disease progression. The Kaplan-Meier method and the Cox proportional hazards model were used to analyze the effect of different serum sodium levels on survival. RESULTS: In this study 206 patients (53.2%) had hyponatremia, including 10.6% patients with moderate/severe hyponatremia and 42.6% with mild hyponatremia. The serum sodium concentration was significantly correlated with the HIV/AIDS progression (P<0.001). During the follow-up, 100 patients (25.6%) died. The cumulative survival rates of HIV/AIDS patients were 47.8% ± 8.5% in patients with moderate/severe hyponatremia, 59.8% ± 5.0% with mild hyponatremia and 79.9% ± 3.4% with normonatremia (log-rank P<0.001). After adjusting for sex, age, WHO stage, CD4 count, hemoglobin and albumin, the relative hazard was 3.5 (95% CI: 1.9-6.5) for patients with moderate/severe hyponatremia (P<0.001), and 1.5 (95% CI: 0.9-2.4) for those with mild hyponatremia (P = 0.161), compared with normonatremic patients. CONCLUSIONS: The serum sodium level is closely correlated with the severity of patients. Only moderate/severe hyponatremia affects the prognosis of Chinese HIV/AIDS patients. Earlier intensive medical managements(including HAART)are necessary to increase the survival rates of Chinese HIV/AIDS patients with moderate/severe hyponatremia

Mean serum sodium concentration in patients according to CD4 count and WHO stage.

<p>Serum sodium concentration decreases with a reduction in CD4 count and an increases with WHO stage (F = 7.004, <i>P</i><0.001).</p

Risk factors for mortality of patients in univariate/multivariate Cox's proportional hazard models.

<p>Risk factors for mortality of patients in univariate/multivariate Cox's proportional hazard models.</p

Kaplan–Meier survival curves according to serum sodium concentration.

<p>The serum sodium level affects mainly the 3-year mortality rate of patients. The 3-year cumulative survival rates for patients with moderate/severe and mild hyponatremia were 47.8%±8.5% and 59.8%±5.0%, respectively, and 78.2%±3.8% for normonatremic patients (Log-Rank, <i>P</i><0.001).</p

Basic characteristics of the hyponatremic patients (n = 387).

<p>Basic characteristics of the hyponatremic patients (n = 387).</p

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

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

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