Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

Abstract

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC₅₀], <0.09 to 0.71 nM) and RSV-B (IC₅₀, 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC₅₀, 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections.

Keywords: L protein; PC786; RNA polymerases; bronchial epithelial cell; clinical isolate; inhalation; polymerase; respiratory syncytial virus.

FIG 1

Antiviral effects of PC786. (A) Chemical structure of PC786. (B) Western blotting image demonstrating the inhibitory effects of PC786 on RSV protein expression in HEp-2 cells infected with RSV A2 for 3 days. *, nonspecific band; F1, disulfide-linked F1 subunit cleaved from RSV F0 protein precursor. NT, no PC786 treatment (0.5% DMSO only). (C) Inhibitory effects of RSV A Memphis 37- and RSV B strain 1262-induced CPE in HEp-2 cells. (D and E) PCR viral loads in the supernatants from HEp-2 cells infected with Memphis 37 (D) and strain 1262 (E) in the presence of PC786. The assay was conducted in duplicate, and individual dots show data from individual wells.

FIG 2

Kinetics of antiviral activity of PC786. (A) Antiviral activity of PC786 evaluated in a time-of-addition assay. PC786 was added as a single drug exposure at 0 (just after virus inoculation), 24, 48, or 72 h after infection with RSV A2, and the resulting CPE was evaluated at 144 h postinfection, showing persistence of action. (B) GS5806 data. (C to E) PC786 (C), AZ-27 (D), or ribavirin (E) was used to dose BEAS-2B cells just after virus infection (called “nonwashout”) or added for 2 h and then removed and incubated for a further 24 h before inoculation (called “washout”). Antiviral activity was assessed by detection of RSV F protein on the cell surface using enzyme-linked immunoassay. The y axis shows percent inhibition (mean ± SEM) versus signals from vehicle-treated virus-infected controls. (F) Time course of virus titer in apical wash from air-liquid interface-cultured differentiated human bronchial epithelial cells. After infection, apical wash with PBS was collected daily up to 7 days. After each apical wash collection, PC786 was administered for 1 h apically and then removed. The limit of quantification (LOQ) is 1.5 log PFU/ml, and virus titer lower than LOQ is shown as <1.5.

FIG 3

Effects of intranasal PC786 on RSV A Long-infected cotton rats. Cotton rats were inoculated intranasally with RSV A Long (1.0 × 10⁵ PFU/cotton rat), and the animals were sacrificed after 4 days. PC786 was used for treatment once daily on day 0 (4 h before infection) and then on days 1, 2, and 3. Lung homogenates were evaluated for viral load (plaque assay) (A) and PCR products (relative quantification of cDNA from lungs on day 4 after primary RSV infection) of RSV NS1 gene (B), RANTES (C), and IP-10 (D), which were normalized to that of β-actin. Individual data are plotted, and geometric means for panel A and means ± SEM for panels B, C, and D are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (versus RSV-infected control) (PC786 = 0).

FIG 4

Mode of action of PC786. (A) Inhibition of PC786 on RSV RdRp activity in crude extracts from RSV L protein-P overexpressed in HEp-2 cells. (B) Inhibition of reporter fluorescent signal in RSV RdRp-driven minigenome assay. (C) Inhibitory activity of PC786 on CPE induced by control RSV A2 virus and by a PC786 escape mutant (Y1631H). (D) SNP genotype PCR analysis of viruses with wild-type and Y1631H mutant L protein. (E) SNP genotype PCR analysis of viruses with wild-type and V153A mutant M protein. (F) Minigenome analysis in HEp-2 cells transfected with wild-type and Y1631H mutant L protein plasmid.