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

<div><p>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.</p></div

Identification of RSV polymerase as the molecular target for ALS-8112.

<p>(<b>A</b>) Selection of resistance mutations associated with the prolonged culture of RSV A2 in the presence of increasing concentrations of ALS-8112. All four mutations detected from full-genome sequence analysis mapped to motif B, a region of the RdRp domain (residues ~500–1100) of the RSV L protein just upstream of the CRIII region containing the catalytic motif _810-GDNQ_-813 (red) responsible for nucleotide incorporation by the RSV polymerase. (<b>B</b>) In vitro inhibition potency of ALS-8112 against the RSV minigenome luciferase-based reporter assay. HEp-2 cells were co-transfected to transiently express the RSV N, P, M2-1 and L proteins containing either the wild-type or the QUAD mutated sequence (n = 3) *P < 0.05 (Student's <i>t</i> test). (<b>C</b>) Inhibitory effect of ALS-8112-TP on the RdRp activity of the crude RSV RNP complex. The RNP complex containing the RSV L protein was extracted from virus-infected cells (RSV+), and uninfected cells were used as negative control for RdRp activity (RSV-). The enzymatic reaction was conducted in the presence of ALS-8112, ALS-8112-MP, or ALS-8112-TP. Labeled transcripts were separated from the initial radiolabeled CTP substrate by urea PAGE prior to phosphor-imaging. (<b>D</b>) Effect of increasing concentration of ALS-8112-TP on the RdRp activity of the RNP complex. The intensity of RNA product after gel electrophoresis was reported for each nucleotide concentration (n <i>= 3</i>). (<b>E</b>) In vitro inhibition potency of ALS-8112-TP against RSV wild-type (n = 4) and QUAD (n = 2) RdRp activity **P < 0.005 (Student's <i>t</i> test).</p

Competitive inhibition of RSV RNP complex by ALS-8112-TP.

<p>(<b>A</b>) Effect of low (1 μM) and high (100 μM) concentration of ATP, UTP, CTP, or GTP, on inhibition of RSV RNP by ALS-8112-TP used at a single concentration of 30 μM. (n = 2) *P < 0.05 (Student's <i>t</i> test). (<b>B</b>) Effect of increasing concentration of ALS-8112-TP on the RdRp activity of the RNP complex. RNA products were treated with RNase H prior to high-resolution electrophoresis in order to visualize individual gene transcripts. Lane 1 had no inhibitor, and ALS-8112-TP concentration ranged from 0.0003 μM (lane 2) to 300 μM (lane 8) by 10-fold increments.</p

Contribution of the 4'ClCh₂ group to the QUAD-mutant resistance to ALS-8112-TP.

<p><b>(A)</b> The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of either CTP or ALS-8112-TP. Product formation was quantified and expressed as % primer extension from the +3 position (see calculation in Fig D in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004995#ppat.1004995.s001" target="_blank">S1 Text</a>). QUAD K_{m CTP} = 0.056±0.010 μM (<i>n</i> = 2), and QUAD K_{m ALS-8112-TP} = 1.74±0.34 μM (<i>n</i> = 2). K_m values for the WT enzyme were reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004995#ppat.1004995.g004" target="_blank">Fig 4</a>. <b>(B)</b> Fold discrimination for each enzyme was calculated as K_{m CTP analog} / K_{m CTP}. <b>(C and D)</b> The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 2'F-CTP. WT K_{m 2'F-CTP} = 0.12±0.014 μM (<i>n</i> = 2), and QUAD K_{m 2'F-CTP} = 0.07±0.017 μM (<i>n</i> = 2). <b>(E and F)</b> The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 4'ClCH₂-CTP. WT K_{m 4'ClCH2-CTP} = 16±2.7 μM (<i>n</i> = 2), and QUAD K_{m 4'ClCH2-CTP} = 198±18 μM (<i>n</i> = 2).</p

Rational design of ALS-8112 as a selective RSV inhibitor.

<p><b>(A)</b> The nucleotide analog 2'-F-CTP is a substrate for both RSV and HCV polymerase, but it does not cause any inhibition by immediate chain termination. The addition of a 4'ClCH₂ group (ALS-8112-TP) makes the molecule a selective inhibitor of RSV polymerase. The addition of a 2'Me group favors recognition by HCV polymerase, and the addition of a 4'N₃ group causes dual RSV/HCV polymerase inhibition. <b>(B)</b> X-ray structure of natural CDP in the active site of HCV polymerase (PDB 4WTC, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004995#ppat.1004995.ref028" target="_blank">28</a>]). (<b>C, D</b>, and <b>E</b>) Docked binding modes of 2'F-2'Me-, 2'F-4'ClCH₂-, and 2'F-4'N₃-CDP, respectively.</p