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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

Contribution of the 4'ClCh2 group to the QUAD-mutant resistance to ALS-8112-TP.(A) 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 S1 Text). QUAD Km CTP = 0.056±0.010 μM (n = 2), and QUAD Km ALS-8112-TP = 1.74±0.34 μM (n = 2). Km values for the WT enzyme were reported in Fig 4. (B) Fold discrimination for each enzyme was calculated as Km CTP analog / Km CTP. (C and D) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 2'F-CTP. WT Km 2'F-CTP = 0.12±0.014 μM (n = 2), and QUAD Km 2'F-CTP = 0.07±0.017 μM (n = 2). (E and F) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 4'ClCH2-CTP. WT Km 4'ClCH2-CTP = 16±2.7 μM (n = 2), and QUAD Km 4'ClCH2-CTP = 198±18 μM (n = 2).
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ppat.1004995.g007: Contribution of the 4'ClCh2 group to the QUAD-mutant resistance to ALS-8112-TP.(A) 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 S1 Text). QUAD Km CTP = 0.056±0.010 μM (n = 2), and QUAD Km ALS-8112-TP = 1.74±0.34 μM (n = 2). Km values for the WT enzyme were reported in Fig 4. (B) Fold discrimination for each enzyme was calculated as Km CTP analog / Km CTP. (C and D) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 2'F-CTP. WT Km 2'F-CTP = 0.12±0.014 μM (n = 2), and QUAD Km 2'F-CTP = 0.07±0.017 μM (n = 2). (E and F) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 4'ClCH2-CTP. WT Km 4'ClCH2-CTP = 16±2.7 μM (n = 2), and QUAD Km 4'ClCH2-CTP = 198±18 μM (n = 2).

Mentions: The four ALS-8112-selected amino acid mutations (QUAD: M628L, A789V, L795I, and I796V) were engineered into the RdRp region of the RSV L gene, and the recombinant L-P protein complex was produced. The wild-type enzyme and the QUAD mutant displayed a similar level of RdRp activity (Fig J in S1 Text). Using the primer extension assay described in Fig 4, the relative incorporation efficiencies between ALS-8112-TP substrate and natural CTP by RSV polymerase QUAD mutant led to a discrimination level of 61±1.1-fold (Fig 7A). Compared to the wild-type L-P enzyme (13±2.5-fold, Fig 4E), this represents a 4.6-fold increase in discrimination for ALS-8112-TP (Fig 7B). In order to understand at the molecular level which components of ALS-8112-TP contributed to the increased discrimination by the QUAD mutant, the same incorporation efficiency experiments were repeated with 2'F-CTP. Both WT RSV polymerase and the QUAD mutant efficiently recognized 2'F-CTP, which translated to low and comparable discrimination levels relative to CTP (Fig 7C and 7D). In contrast, 4'ClCH2-CTP was discriminated 288±48-fold by WT RSV polymerase, and 6,990±622-fold by the QUAD mutant (Fig 7E). This represents an overall resistance level of 24-fold (Fig 7F). Taken together, these results show that the increased discrimination of ALS-8112-TP by RSV polymerase QUAD mutant is conferred solely by the 4'ClCH2 moiety. Given the position of the last three of the four mutations within the RdRp domain, it is likely that motif B plays a critical role in the recognition by RSV polymerase of 4'-substitutions in nucleotide analogs.


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)

Contribution of the 4'ClCh2 group to the QUAD-mutant resistance to ALS-8112-TP.(A) 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 S1 Text). QUAD Km CTP = 0.056±0.010 μM (n = 2), and QUAD Km ALS-8112-TP = 1.74±0.34 μM (n = 2). Km values for the WT enzyme were reported in Fig 4. (B) Fold discrimination for each enzyme was calculated as Km CTP analog / Km CTP. (C and D) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 2'F-CTP. WT Km 2'F-CTP = 0.12±0.014 μM (n = 2), and QUAD Km 2'F-CTP = 0.07±0.017 μM (n = 2). (E and F) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 4'ClCH2-CTP. WT Km 4'ClCH2-CTP = 16±2.7 μM (n = 2), and QUAD Km 4'ClCH2-CTP = 198±18 μM (n = 2).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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ppat.1004995.g007: Contribution of the 4'ClCh2 group to the QUAD-mutant resistance to ALS-8112-TP.(A) 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 S1 Text). QUAD Km CTP = 0.056±0.010 μM (n = 2), and QUAD Km ALS-8112-TP = 1.74±0.34 μM (n = 2). Km values for the WT enzyme were reported in Fig 4. (B) Fold discrimination for each enzyme was calculated as Km CTP analog / Km CTP. (C and D) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 2'F-CTP. WT Km 2'F-CTP = 0.12±0.014 μM (n = 2), and QUAD Km 2'F-CTP = 0.07±0.017 μM (n = 2). (E and F) The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 4'ClCH2-CTP. WT Km 4'ClCH2-CTP = 16±2.7 μM (n = 2), and QUAD Km 4'ClCH2-CTP = 198±18 μM (n = 2).
Mentions: The four ALS-8112-selected amino acid mutations (QUAD: M628L, A789V, L795I, and I796V) were engineered into the RdRp region of the RSV L gene, and the recombinant L-P protein complex was produced. The wild-type enzyme and the QUAD mutant displayed a similar level of RdRp activity (Fig J in S1 Text). Using the primer extension assay described in Fig 4, the relative incorporation efficiencies between ALS-8112-TP substrate and natural CTP by RSV polymerase QUAD mutant led to a discrimination level of 61±1.1-fold (Fig 7A). Compared to the wild-type L-P enzyme (13±2.5-fold, Fig 4E), this represents a 4.6-fold increase in discrimination for ALS-8112-TP (Fig 7B). In order to understand at the molecular level which components of ALS-8112-TP contributed to the increased discrimination by the QUAD mutant, the same incorporation efficiency experiments were repeated with 2'F-CTP. Both WT RSV polymerase and the QUAD mutant efficiently recognized 2'F-CTP, which translated to low and comparable discrimination levels relative to CTP (Fig 7C and 7D). In contrast, 4'ClCH2-CTP was discriminated 288±48-fold by WT RSV polymerase, and 6,990±622-fold by the QUAD mutant (Fig 7E). This represents an overall resistance level of 24-fold (Fig 7F). Taken together, these results show that the increased discrimination of ALS-8112-TP by RSV polymerase QUAD mutant is conferred solely by the 4'ClCH2 moiety. Given the position of the last three of the four mutations within the RdRp domain, it is likely that motif B plays a critical role in the recognition by RSV polymerase of 4'-substitutions in nucleotide analogs.

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