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

Deval J, Hong J, Wang G, Taylor J, Smith LK, Fung A, Stevens SK, Liu H, Jin Z, Dyatkina N, Prhavc M, Stoycheva AD, Serebryany V, Liu J, Smith DB, Tam Y, Zhang Q, Moore ML, Fearns R, Chanda SM, Blatt LM, Symons JA, Beigelman L - PLoS Pathog. (2015)

Bottom Line: 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-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 lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses.

View Article: PubMed Central - PubMed

Affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Identification of RSV polymerase as the molecular target for ALS-8112.(A) 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) 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 t test). (C) 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. (D) 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 = 3). (E) 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 t test).
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ppat.1004995.g002: Identification of RSV polymerase as the molecular target for ALS-8112.(A) 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) 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 t test). (C) 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. (D) 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 = 3). (E) 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 t test).

Mentions: RSV A2 viruses were repeatedly passaged in HEp-2 cells with or without increasing concentrations of ALS-8112. Two independent adaptations were conducted, as well as a no-drug control virus pool that was passaged at the same time. After > 35 passages, the virus grown in the presence of ALS-8112 from each ALS-8112-treated pool exhibited a > 50-fold EC50 shift in the antiviral assay when compared with the control virus pool cultured for the same duration in the absence of ALS-8112. When the complete RSV genome was sequenced, all ALS-8112-selected viruses showed four amino acid substitutions (QUAD); methionine 628 to leucine (M628L), alanine 789 to valine (A789V), leucine 795 to isoleucine (L795I), and isoleucine 796 to valine (I796V) within the CRIII of the RdRp, the RNA polymerase coding region of the RSV L gene (Fig 2A). Within the ALS-8112 drug selection pool, >95% of the viruses carried all four mutations on the same genome. Three of the four amino acid changes associated with ALS-8112 resistance are co-located within motif B. This particular sequence of the CRIII region is in close proximity to the catalytic motif C residues (810-GDNQ-813) that are responsible for nucleotide incorporation by paramyxovirus RNA polymerases. No mutations were identified in any other RSV genes. Phenotypic validation of the QUAD mutations was conducted by reverse genetics using the RSV minigenome system. In this assay, four plasmids encoding RSV N, P, M2-1, and L proteins were co-transfected into HEp-2 cells. The transient expression of each of the corresponding proteins leads to the formation of a functional RSV polymerase complex, and its RNA-dependent RNA polymerase (RdRp) activity was monitored in the cells using a luciferase-based reporter [22]. We mutated the L gene at all four positions previously identified from sequencing, and analyzed the effect of these substitutions on the inhibition potency of the drug. In this system, ALS-8112 inhibited the wild-type RSV polymerase-dependent luciferase activity with an EC50 of 0.25 ± 0.04 μM (Fig CA in S1 Text). In comparison, ALS-8112 inhibited the QUAD-mutant RSV polymerase-dependent luciferase activity with an EC50 of 9.7 ± 5.4 μM, only reaching about 60% maximum inhibition at the top concentration (Fig 2B and Fig CA in S1 Text). The presence of the four amino acid changes did not significantly affect the amount of firefly luciferase signal, which may indicate similar RSV polymerase activity between the two clones (Fig CB in S1 Text). The 39-fold shift in inhibition potency caused by the four mutations in the L gene indicates that ALS-8112 inhibits RSV replication by targeting the L protein responsible for the viral RNA transcription and replication functions. A detailed description of the characterization of the ALS-8112 resistant virus and the role of each individual mutation will be the subject of a separate study.


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

Deval J, Hong J, Wang G, Taylor J, Smith LK, Fung A, Stevens SK, Liu H, Jin Z, Dyatkina N, Prhavc M, Stoycheva AD, Serebryany V, Liu J, Smith DB, Tam Y, Zhang Q, Moore ML, Fearns R, Chanda SM, Blatt LM, Symons JA, Beigelman L - PLoS Pathog. (2015)

Identification of RSV polymerase as the molecular target for ALS-8112.(A) 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) 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 t test). (C) 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. (D) 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 = 3). (E) 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 t test).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476725&req=5

ppat.1004995.g002: Identification of RSV polymerase as the molecular target for ALS-8112.(A) 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) 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 t test). (C) 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. (D) 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 = 3). (E) 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 t test).
Mentions: RSV A2 viruses were repeatedly passaged in HEp-2 cells with or without increasing concentrations of ALS-8112. Two independent adaptations were conducted, as well as a no-drug control virus pool that was passaged at the same time. After > 35 passages, the virus grown in the presence of ALS-8112 from each ALS-8112-treated pool exhibited a > 50-fold EC50 shift in the antiviral assay when compared with the control virus pool cultured for the same duration in the absence of ALS-8112. When the complete RSV genome was sequenced, all ALS-8112-selected viruses showed four amino acid substitutions (QUAD); methionine 628 to leucine (M628L), alanine 789 to valine (A789V), leucine 795 to isoleucine (L795I), and isoleucine 796 to valine (I796V) within the CRIII of the RdRp, the RNA polymerase coding region of the RSV L gene (Fig 2A). Within the ALS-8112 drug selection pool, >95% of the viruses carried all four mutations on the same genome. Three of the four amino acid changes associated with ALS-8112 resistance are co-located within motif B. This particular sequence of the CRIII region is in close proximity to the catalytic motif C residues (810-GDNQ-813) that are responsible for nucleotide incorporation by paramyxovirus RNA polymerases. No mutations were identified in any other RSV genes. Phenotypic validation of the QUAD mutations was conducted by reverse genetics using the RSV minigenome system. In this assay, four plasmids encoding RSV N, P, M2-1, and L proteins were co-transfected into HEp-2 cells. The transient expression of each of the corresponding proteins leads to the formation of a functional RSV polymerase complex, and its RNA-dependent RNA polymerase (RdRp) activity was monitored in the cells using a luciferase-based reporter [22]. We mutated the L gene at all four positions previously identified from sequencing, and analyzed the effect of these substitutions on the inhibition potency of the drug. In this system, ALS-8112 inhibited the wild-type RSV polymerase-dependent luciferase activity with an EC50 of 0.25 ± 0.04 μM (Fig CA in S1 Text). In comparison, ALS-8112 inhibited the QUAD-mutant RSV polymerase-dependent luciferase activity with an EC50 of 9.7 ± 5.4 μM, only reaching about 60% maximum inhibition at the top concentration (Fig 2B and Fig CA in S1 Text). The presence of the four amino acid changes did not significantly affect the amount of firefly luciferase signal, which may indicate similar RSV polymerase activity between the two clones (Fig CB in S1 Text). The 39-fold shift in inhibition potency caused by the four mutations in the L gene indicates that ALS-8112 inhibits RSV replication by targeting the L protein responsible for the viral RNA transcription and replication functions. A detailed description of the characterization of the ALS-8112 resistant virus and the role of each individual mutation will be the subject of a separate study.

Bottom Line: 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-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 lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses.

View Article: PubMed Central - PubMed

Affiliation: Alios BioPharma, Inc., South San Francisco, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus