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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 dual RSV/HCV polymerase inhibitors.(A) Incorporation by recombinant RSV polymerase of CTP analogs with a ribose containing either an OH, F, diF, or F-Me at the 2'-position, and either an H, N3, or ClCH2 at the 4'-position. Immediate chain terminators form a product at the +4 position, while natural substrate (CTP) fully extends the primer to the +7 position. (B) Comparative analysis of IC50 values of CTP analogs tested against RSV RNP complex versus HCV polymerase. Inhibitors of RSV polymerase are circled in green, and inhibitors of HCV polymerase are circled in red.
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ppat.1004995.g005: Identification of dual RSV/HCV polymerase inhibitors.(A) Incorporation by recombinant RSV polymerase of CTP analogs with a ribose containing either an OH, F, diF, or F-Me at the 2'-position, and either an H, N3, or ClCH2 at the 4'-position. Immediate chain terminators form a product at the +4 position, while natural substrate (CTP) fully extends the primer to the +7 position. (B) Comparative analysis of IC50 values of CTP analogs tested against RSV RNP complex versus HCV polymerase. Inhibitors of RSV polymerase are circled in green, and inhibitors of HCV polymerase are circled in red.

Mentions: To understand at the molecular level why ALS-8112-TP, unlike mericitabine-TP, is a substrate for RSV polymerase, we analyzed the incorporation profile of a series of structural intermediates between ALS-8112-TP and mericitabine-TP. Nine CTP analogs were modified to contain either an OH, F, diF, or F-Me at the 2'-position, and either an H, N3, or ClCH2 at the 4'-position (Fig 5A). Some of these molecules included the triphosphate form of other clinically relevant molecules such as gemcitabine (2'diF-CTP) and balapiravir (4'N3-CTP). We found that 2'F- and 2'diF-CTP were substrates for RSV polymerase, but did not cause immediate chain termination (Fig 5A). In contrast, 2'F-4'ClCH2- (ALS-8112-TP), 4'N3-, 2'F-4'N3- and 2'diF-4'N3-CTP were both substrates and chain terminators of recombinant RSV polymerase. Finally, 4'ClCH2-CTP seemed less efficiently recognized by the enzyme, while 2'diF-4'ClCH2- and 2'F-2'Me-CTP (mericitabine-TP) were almost completely inactive. Balapiravir-TP (4'N3-CTP) is a known inhibitor of HCV polymerase [26], which suggested that other CTP analogs could have dual RSV/HCV inhibition properties. This was further investigated by measuring the IC50 values of all compounds against HCV polymerase and RSV RNP complex. As expected, 2'F- and 2'diF-CTP did not efficiently inhibit either of the two polymerases, most likely because they did not cause immediate chain termination (Fig 5B) [27]. In contrast, 4'N3-, 2'F-4'N3-, 2'diF-4'N3-, and 4'ClCH2-CTP were all dual RSV/HCV polymerase inhibitors. These results were in agreement with the dual RSV/HCV antiviral effect of balapiravir in subgenomic replicons (Table 3). From this series of molecules, 2'F-4'ClCH2- (ALS-8112-TP) was the only CTP analog to efficiently inhibit RSV but not HCV polymerase. We conclude that, at the structural level, the selectivity of ALS-8112 towards RSV is provided by a combination of the 2'F- and the 4'ClCH2 moieties on the ribose group (Fig 6A and Fig H in S1 Text). On the other hand, the presence of the 2'Me-moiety led to a lack of recognition by RSV polymerase (Figs 5 and 6A).


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 dual RSV/HCV polymerase inhibitors.(A) Incorporation by recombinant RSV polymerase of CTP analogs with a ribose containing either an OH, F, diF, or F-Me at the 2'-position, and either an H, N3, or ClCH2 at the 4'-position. Immediate chain terminators form a product at the +4 position, while natural substrate (CTP) fully extends the primer to the +7 position. (B) Comparative analysis of IC50 values of CTP analogs tested against RSV RNP complex versus HCV polymerase. Inhibitors of RSV polymerase are circled in green, and inhibitors of HCV polymerase are circled in red.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004995.g005: Identification of dual RSV/HCV polymerase inhibitors.(A) Incorporation by recombinant RSV polymerase of CTP analogs with a ribose containing either an OH, F, diF, or F-Me at the 2'-position, and either an H, N3, or ClCH2 at the 4'-position. Immediate chain terminators form a product at the +4 position, while natural substrate (CTP) fully extends the primer to the +7 position. (B) Comparative analysis of IC50 values of CTP analogs tested against RSV RNP complex versus HCV polymerase. Inhibitors of RSV polymerase are circled in green, and inhibitors of HCV polymerase are circled in red.
Mentions: To understand at the molecular level why ALS-8112-TP, unlike mericitabine-TP, is a substrate for RSV polymerase, we analyzed the incorporation profile of a series of structural intermediates between ALS-8112-TP and mericitabine-TP. Nine CTP analogs were modified to contain either an OH, F, diF, or F-Me at the 2'-position, and either an H, N3, or ClCH2 at the 4'-position (Fig 5A). Some of these molecules included the triphosphate form of other clinically relevant molecules such as gemcitabine (2'diF-CTP) and balapiravir (4'N3-CTP). We found that 2'F- and 2'diF-CTP were substrates for RSV polymerase, but did not cause immediate chain termination (Fig 5A). In contrast, 2'F-4'ClCH2- (ALS-8112-TP), 4'N3-, 2'F-4'N3- and 2'diF-4'N3-CTP were both substrates and chain terminators of recombinant RSV polymerase. Finally, 4'ClCH2-CTP seemed less efficiently recognized by the enzyme, while 2'diF-4'ClCH2- and 2'F-2'Me-CTP (mericitabine-TP) were almost completely inactive. Balapiravir-TP (4'N3-CTP) is a known inhibitor of HCV polymerase [26], which suggested that other CTP analogs could have dual RSV/HCV inhibition properties. This was further investigated by measuring the IC50 values of all compounds against HCV polymerase and RSV RNP complex. As expected, 2'F- and 2'diF-CTP did not efficiently inhibit either of the two polymerases, most likely because they did not cause immediate chain termination (Fig 5B) [27]. In contrast, 4'N3-, 2'F-4'N3-, 2'diF-4'N3-, and 4'ClCH2-CTP were all dual RSV/HCV polymerase inhibitors. These results were in agreement with the dual RSV/HCV antiviral effect of balapiravir in subgenomic replicons (Table 3). From this series of molecules, 2'F-4'ClCH2- (ALS-8112-TP) was the only CTP analog to efficiently inhibit RSV but not HCV polymerase. We conclude that, at the structural level, the selectivity of ALS-8112 towards RSV is provided by a combination of the 2'F- and the 4'ClCH2 moieties on the ribose group (Fig 6A and Fig H in S1 Text). On the other hand, the presence of the 2'Me-moiety led to a lack of recognition by RSV polymerase (Figs 5 and 6A).

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