Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

Jérôme Deval ,Josh Taylor,Lucas K Smith,Rachel Fearns,Jyanwei Liu,Amy Fung,Lawrence M Blatt,Leo Beigelman,David B Smith,Hong Liu,Natalia Dyatkina,Qingling Zhang,Vladimir Serebryany,Marija Prhavc,Guangyi Wang,Martin L Moore,Julian Symons ,Sarah Stevens ,Sushmita Chanda ,Jin Zhang ,Hong Ji ,Yuen Yi C Tam ,Antitsa Stoycheva

doi:10.1371/journal.ppat.1004995

Abstract

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

Highlights

Respiratory syncytial virus (RSV) is a non-segmented, single-stranded, negative sense RNA virus and a member of the family Paramyxoviridae, which includes human metapneumovirus and parainfluenza virus type-3 (PIV-3)
Sofosbuvir and mericitabine are related compounds belonging to a class of drugs called nucleoside analogs that inhibit the RNA polymerase of hepatitis C virus (HCV), a positive-strand RNA virus, but have no effect on negative-strand RNA viruses
A multidisciplinary approach combining cellular, chemical, structural, and enzymatic methods was employed to demonstrate that the triphosphate form of ALS-8112 targets the RNA polymerase of RSV, but not of HCV

Summary

Introduction

Respiratory syncytial virus (RSV) is a non-segmented, single-stranded, negative sense RNA virus and a member of the family Paramyxoviridae, which includes human metapneumovirus and parainfluenza virus type-3 (PIV-3). RSV is a leading cause of lower respiratory disease in infants [3,4]. In 2005, an estimated 33.8 million episodes of RSV infection occurred worldwide in infants and young children with most of these occurring in otherwise healthy full-term infants. At least 3.4 million severe cases of lower respiratory tract infection (LRI) required hospitalization, and an estimated 66,000 to 199,000 deaths occurred, mostly in the developing world [3]. Risk factors for severe illness associated with RSV infection include prematurity ( 35 weeks gestation) and younger age (under 2 years) [6], pulmonary deficiencies, congenital heart disease, immunosuppression, low birth weight, large family size, exposure to passive smoke, and lack of breast feeding [7]

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