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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

Chain termination of RNA synthesis by ALS-8112-TP.(A) SDS PAGE of recombinant RSV L-P polymerase complex. (B) Principle of the single nucleotide incorporation assay with RSV L-P polymerase: The 11-mer template contains a single G at the +4 position. In presence of radiolabled GTP (G*), the primer can be extended by one base (+1). GTP+ATP allows for a +3 extension, while the addition of CTP enables full-length RNA product synthesis (+7). (C) Wild-type recombinant RSV L-P polymerase complex (all lanes except 3 and 4) was incubated with primer/template (P/T) and GTP* (lane 1), or GTP* + 10 μM ATP (lane 2). Lanes 3 and 4: same as 1 and 2, except that the lethal N812A mutation was introduced within the L subunit. Lane 5: enzyme + GTP* + 10 μM CTP. Lane 6: enzyme + GTP* + 10 μM ALS-8112-TP. Lane 7: enzyme + GTP* + ATP + 10 μM CTP. Lane 8: enzyme + GTP* + ATP + 10 μM ALS-8112-TP. (D) RSV L-P incubated in the presence of GTP* + ATP and increasing concentrations of either ALS-8112-TP or mericitabine-TP: lane 1, 0.021 μM; lane 2, 0.062 μM; lane 3, 0.19 μM; lane 4, 0.56 μM; lane 5, 1.7 μM; and lane 6, 5.0 μM. (E) Product formation was quantified and expressed as % primer extension from the +3 position (see calculation in Fig D in S1 Text). CTP Km = 0.057±0.009 μM (n = 4), and ALS-8112-TP Km = 0.74±0.08 μM (n = 4).
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ppat.1004995.g004: Chain termination of RNA synthesis by ALS-8112-TP.(A) SDS PAGE of recombinant RSV L-P polymerase complex. (B) Principle of the single nucleotide incorporation assay with RSV L-P polymerase: The 11-mer template contains a single G at the +4 position. In presence of radiolabled GTP (G*), the primer can be extended by one base (+1). GTP+ATP allows for a +3 extension, while the addition of CTP enables full-length RNA product synthesis (+7). (C) Wild-type recombinant RSV L-P polymerase complex (all lanes except 3 and 4) was incubated with primer/template (P/T) and GTP* (lane 1), or GTP* + 10 μM ATP (lane 2). Lanes 3 and 4: same as 1 and 2, except that the lethal N812A mutation was introduced within the L subunit. Lane 5: enzyme + GTP* + 10 μM CTP. Lane 6: enzyme + GTP* + 10 μM ALS-8112-TP. Lane 7: enzyme + GTP* + ATP + 10 μM CTP. Lane 8: enzyme + GTP* + ATP + 10 μM ALS-8112-TP. (D) RSV L-P incubated in the presence of GTP* + ATP and increasing concentrations of either ALS-8112-TP or mericitabine-TP: lane 1, 0.021 μM; lane 2, 0.062 μM; lane 3, 0.19 μM; lane 4, 0.56 μM; lane 5, 1.7 μM; and lane 6, 5.0 μM. (E) Product formation was quantified and expressed as % primer extension from the +3 position (see calculation in Fig D in S1 Text). CTP Km = 0.057±0.009 μM (n = 4), and ALS-8112-TP Km = 0.74±0.08 μM (n = 4).

Mentions: Recently, it has been shown that, once co-purified, recombinant RSV L and P proteins form a dimer that recognizes synthetic RNA templates to synthesize short products (Fig 4A, [25]). In a similar assay format, the RNA polymerase activity of recombinant RSV L-P polymerase in complex with a short synthetic primer/template substrate was monitored (Fig 4B). In the presence of GTP alone or GTP+ATP, wild-type RSV L-P RNA polymerase complex specifically extended the RNA primer by 1 or 3 bases, respectively (Fig 4C, lanes 1 and 2). As a control, the L-P protein variant containing a single N812A mutation within the catalytic site of the RdRp region of L was inactive (lanes 3 and 4). Under these conditions, no signs of cytidine mis-incorporation were observed in the absence of ATP (lane 5). However, the specific incorporation of natural CMP in the presence of GTP and ATP led to the formation of +7 full-length RNA products (lane 7). In comparison, ALS-8112-MP was also incorporated into the nascent RNA at a specific +4 position opposite to a single guanosine on the template (lane 8), but not at any other positions (lane 6). After the incorporation of ALS-8112-MP, no subsequent nucleotide could be incorporated at the 3ʹ-end of the RNA primer and full-length product formation (+7 position) was not achieved (lane 8). This finding demonstrates that ALS-8112-TP inhibits the polymerase activity of the RSV L-P RNA polymerase complex by immediate termination of chain synthesis. The incorporation of ALS-8112-MP by RSV polymerase was efficient, as judged by the modest 13±2.5-fold discrimination relative to natural CTP (Fig 4D and 4E). In comparison, the specific inhibitor of HCV RNA polymerase mericitabine-TP (2'F-2'Me-CTP) was not recognized as a substrate for RSV polymerase under standard assay conditions. Even at a concentration of 300 μM, the level of incorporation of mericitabine-MP was less than 20% (Fig G in S1 Text). Compared to natural CTP, this represents a discrimination level greater than 5,000-fold. Consistently, mericitabine did not inhibit RSV replication in the subgenomic replicon assay (Table 3).


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)

Chain termination of RNA synthesis by ALS-8112-TP.(A) SDS PAGE of recombinant RSV L-P polymerase complex. (B) Principle of the single nucleotide incorporation assay with RSV L-P polymerase: The 11-mer template contains a single G at the +4 position. In presence of radiolabled GTP (G*), the primer can be extended by one base (+1). GTP+ATP allows for a +3 extension, while the addition of CTP enables full-length RNA product synthesis (+7). (C) Wild-type recombinant RSV L-P polymerase complex (all lanes except 3 and 4) was incubated with primer/template (P/T) and GTP* (lane 1), or GTP* + 10 μM ATP (lane 2). Lanes 3 and 4: same as 1 and 2, except that the lethal N812A mutation was introduced within the L subunit. Lane 5: enzyme + GTP* + 10 μM CTP. Lane 6: enzyme + GTP* + 10 μM ALS-8112-TP. Lane 7: enzyme + GTP* + ATP + 10 μM CTP. Lane 8: enzyme + GTP* + ATP + 10 μM ALS-8112-TP. (D) RSV L-P incubated in the presence of GTP* + ATP and increasing concentrations of either ALS-8112-TP or mericitabine-TP: lane 1, 0.021 μM; lane 2, 0.062 μM; lane 3, 0.19 μM; lane 4, 0.56 μM; lane 5, 1.7 μM; and lane 6, 5.0 μM. (E) Product formation was quantified and expressed as % primer extension from the +3 position (see calculation in Fig D in S1 Text). CTP Km = 0.057±0.009 μM (n = 4), and ALS-8112-TP Km = 0.74±0.08 μM (n = 4).
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Related In: Results  -  Collection

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Show All Figures
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ppat.1004995.g004: Chain termination of RNA synthesis by ALS-8112-TP.(A) SDS PAGE of recombinant RSV L-P polymerase complex. (B) Principle of the single nucleotide incorporation assay with RSV L-P polymerase: The 11-mer template contains a single G at the +4 position. In presence of radiolabled GTP (G*), the primer can be extended by one base (+1). GTP+ATP allows for a +3 extension, while the addition of CTP enables full-length RNA product synthesis (+7). (C) Wild-type recombinant RSV L-P polymerase complex (all lanes except 3 and 4) was incubated with primer/template (P/T) and GTP* (lane 1), or GTP* + 10 μM ATP (lane 2). Lanes 3 and 4: same as 1 and 2, except that the lethal N812A mutation was introduced within the L subunit. Lane 5: enzyme + GTP* + 10 μM CTP. Lane 6: enzyme + GTP* + 10 μM ALS-8112-TP. Lane 7: enzyme + GTP* + ATP + 10 μM CTP. Lane 8: enzyme + GTP* + ATP + 10 μM ALS-8112-TP. (D) RSV L-P incubated in the presence of GTP* + ATP and increasing concentrations of either ALS-8112-TP or mericitabine-TP: lane 1, 0.021 μM; lane 2, 0.062 μM; lane 3, 0.19 μM; lane 4, 0.56 μM; lane 5, 1.7 μM; and lane 6, 5.0 μM. (E) Product formation was quantified and expressed as % primer extension from the +3 position (see calculation in Fig D in S1 Text). CTP Km = 0.057±0.009 μM (n = 4), and ALS-8112-TP Km = 0.74±0.08 μM (n = 4).
Mentions: Recently, it has been shown that, once co-purified, recombinant RSV L and P proteins form a dimer that recognizes synthetic RNA templates to synthesize short products (Fig 4A, [25]). In a similar assay format, the RNA polymerase activity of recombinant RSV L-P polymerase in complex with a short synthetic primer/template substrate was monitored (Fig 4B). In the presence of GTP alone or GTP+ATP, wild-type RSV L-P RNA polymerase complex specifically extended the RNA primer by 1 or 3 bases, respectively (Fig 4C, lanes 1 and 2). As a control, the L-P protein variant containing a single N812A mutation within the catalytic site of the RdRp region of L was inactive (lanes 3 and 4). Under these conditions, no signs of cytidine mis-incorporation were observed in the absence of ATP (lane 5). However, the specific incorporation of natural CMP in the presence of GTP and ATP led to the formation of +7 full-length RNA products (lane 7). In comparison, ALS-8112-MP was also incorporated into the nascent RNA at a specific +4 position opposite to a single guanosine on the template (lane 8), but not at any other positions (lane 6). After the incorporation of ALS-8112-MP, no subsequent nucleotide could be incorporated at the 3ʹ-end of the RNA primer and full-length product formation (+7 position) was not achieved (lane 8). This finding demonstrates that ALS-8112-TP inhibits the polymerase activity of the RSV L-P RNA polymerase complex by immediate termination of chain synthesis. The incorporation of ALS-8112-MP by RSV polymerase was efficient, as judged by the modest 13±2.5-fold discrimination relative to natural CTP (Fig 4D and 4E). In comparison, the specific inhibitor of HCV RNA polymerase mericitabine-TP (2'F-2'Me-CTP) was not recognized as a substrate for RSV polymerase under standard assay conditions. Even at a concentration of 300 μM, the level of incorporation of mericitabine-MP was less than 20% (Fig G in S1 Text). Compared to natural CTP, this represents a discrimination level greater than 5,000-fold. Consistently, mericitabine did not inhibit RSV replication in the subgenomic replicon assay (Table 3).

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