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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

Competitive inhibition of RSV RNP complex by ALS-8112-TP.(A) 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 t test). (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.
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ppat.1004995.g003: Competitive inhibition of RSV RNP complex by ALS-8112-TP.(A) 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 t test). (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.

Mentions: The inhibition of RNA synthesis catalyzed by the wild-type RNP complex in the presence of ALS-8112-TP was inversely proportional to the concentration of cytidine triphosphate (CTP) in the reaction (Fig 3A). However, the concentration of adenosine triphosphate (ATP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) had no effect on the inhibition potency of ALS-8112-TP. Therefore, ALS-8112-TP is a competitive inhibitor of cytidine monophosphate (CMP) incorporation into nascent RNA product by the RSV RNP complex. To understand the effect of ALS-8112-TP on the production of RNA transcripts by crude wild-type RNP complex, products of RNA synthesis were treated with RNase H and visualized by high-resolution gel electrophoresis [23]. Using this method, individual gene transcripts were separated, and their inhibition correlated with the increase in concentration of ALS-8112-TP (Fig 3B).


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)

Competitive inhibition of RSV RNP complex by ALS-8112-TP.(A) 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 t test). (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.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004995.g003: Competitive inhibition of RSV RNP complex by ALS-8112-TP.(A) 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 t test). (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.
Mentions: The inhibition of RNA synthesis catalyzed by the wild-type RNP complex in the presence of ALS-8112-TP was inversely proportional to the concentration of cytidine triphosphate (CTP) in the reaction (Fig 3A). However, the concentration of adenosine triphosphate (ATP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) had no effect on the inhibition potency of ALS-8112-TP. Therefore, ALS-8112-TP is a competitive inhibitor of cytidine monophosphate (CMP) incorporation into nascent RNA product by the RSV RNP complex. To understand the effect of ALS-8112-TP on the production of RNA transcripts by crude wild-type RNP complex, products of RNA synthesis were treated with RNase H and visualized by high-resolution gel electrophoresis [23]. Using this method, individual gene transcripts were separated, and their inhibition correlated with the increase in concentration of ALS-8112-TP (Fig 3B).

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