Efficacy of novel hemagglutinin-neuraminidase inhibitors BCX 2798 and BCX 2855 against human parainfluenza viruses in vitro and in vivo

Abstract

Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK(2) cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 micro M in HA inhibition assays and from 0.02 to 20 micro M in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 micro M. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.

FIG. 1.

Chemical structures of BCX 2798 and BCX 2855 compounds. (A) Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid); (B) interaction of amino acids of the active site of HN NDV with Neu5Ac2en; (C) BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid); (D) BCX 2855 (4-dichloromethanesulfonylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid).

FIG. 2.

Pathogenicity of rSV(hHN) in mice. (A) Mice were infected with rSV(hHN) at doses of 10⁵ (▴), 10^5.5 (•), 10⁶ (⧫), 10^6.5 (-), or 10⁷ (▪) TCID₅₀ per mouse. Weight changes were calculated through day 21 as a percentage of the mouse's weight on day 0 (before infection). Values are the averages for each group, plotted with error bars indicating the SEM. (B) Mice were infected with rSV(hHN) at doses of 10⁵ (▴), 10⁶ (⧫), or 10⁷ (▪) TCID₅₀ per mouse. Lungs were collected 1, 3, 5, 7, and 9 days after infection. Values are mean titers of virus from three animals, plotted with error bars indicating the SEM. All mice infected with 10⁷ TCID₅₀ died by day 9.

FIG. 3.

Histopathologic changes in the lungs of mice infected with rSV(hHN). (A) Mice (three per group) were infected with a dose of 10^5.5 TCID₅₀. Lungs were removed 9 days after infection, fixed, and cut into 5-μm sections that were later stained with hematoxylin and eosin. Low-power view (×10) of the stained section is shown. (B) Control mice (three per group) were given PBS instead of virus. The lungs were prepared and examined as described in panel A.

FIG. 4.

Effect of pretreatment with BCX 2798 or BCX 2855 on virus titers from lungs of mice infected with rSV(hHN). BCX 2798 (10 mg/kg per day [▴]) and BCX 2855 (50 mg/kg per day [•]) were intranasally administered to 129x1/SvJ mice for 5 days; administration began 4 h before viral infection. Three mice per group were infected with rSV(hHN) (10^6.5 TCID₅₀ per mouse). Control infected mice were treated only with PBS (▪). The averages for each group are plotted with error bars indicating the SEM.