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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

Inhibition of RSV replication by ALS-8112 and ALS-8176.(A) Chemical structure of 2'-fluoro-4'-chloromethyl (2'F-4'ClCH2) cytidine, or ALS-8112. (B) In vitro inhibition potency of ALS-8112 against the RSV A2 and B1 strains grown in HEp-2 cells. The viral RNA level was measured by qRT-PCR, and reported as percentage of the uninhibited condition (n = 3). The effect of ALS-8112 on the viability of human epithelial HEp-2 cells was also evaluated. The highest concentration of ALS-8112 used to measure the concentration resulting in 50% cytotoxicity (CC50) was 100 μM (n = 3). (C) In vitro efficacy of ALS-8112 in a three-dimensional lung model. Primary human tracheal/bronchial epithelial cells from three individual human donors were infected on the apical side with the RSV A2 strain, while increasing concentrations of ALS-8112 were added to the basal medium. (D) Fluorescence microphotographs of HEp-2 cells infected with recombinant RSV-mKate2, in the presence of DMSO, 10 μM ALS-8112, or 10 μM ALS-8176. (E) African Green monkey efficacy model. ALS-8176 was administered BID for a total of 6 days. At the end of treatment (Day 5 post-infection), RSV RNA titers were measured in bronchoalveolar lavage (BAL) and nasopharyngeal (NP) swab samples for each group (vehicle and drug) containing four animals. Limit of detection (LOD) for qRT-PCR analysis was 50 copies/mL (dashed line).
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ppat.1004995.g001: Inhibition of RSV replication by ALS-8112 and ALS-8176.(A) Chemical structure of 2'-fluoro-4'-chloromethyl (2'F-4'ClCH2) cytidine, or ALS-8112. (B) In vitro inhibition potency of ALS-8112 against the RSV A2 and B1 strains grown in HEp-2 cells. The viral RNA level was measured by qRT-PCR, and reported as percentage of the uninhibited condition (n = 3). The effect of ALS-8112 on the viability of human epithelial HEp-2 cells was also evaluated. The highest concentration of ALS-8112 used to measure the concentration resulting in 50% cytotoxicity (CC50) was 100 μM (n = 3). (C) In vitro efficacy of ALS-8112 in a three-dimensional lung model. Primary human tracheal/bronchial epithelial cells from three individual human donors were infected on the apical side with the RSV A2 strain, while increasing concentrations of ALS-8112 were added to the basal medium. (D) Fluorescence microphotographs of HEp-2 cells infected with recombinant RSV-mKate2, in the presence of DMSO, 10 μM ALS-8112, or 10 μM ALS-8176. (E) African Green monkey efficacy model. ALS-8176 was administered BID for a total of 6 days. At the end of treatment (Day 5 post-infection), RSV RNA titers were measured in bronchoalveolar lavage (BAL) and nasopharyngeal (NP) swab samples for each group (vehicle and drug) containing four animals. Limit of detection (LOD) for qRT-PCR analysis was 50 copies/mL (dashed line).

Mentions: A series of ribonucleoside analogs was recently identified as inhibiting the replication of RSV, and the optimization of the precursor molecules led to the chemical synthesis of 2'-fluoro-4'-chloromethyl (2'F-4'ClCH2) cytidine, referred to as ALS-8112 (Fig 1A) [18]. The nucleoside analog ALS-8112 did not significantly decrease the viability of human epithelial (HEp-2) cells after 5 days (with the concentration resulting in 50% cytotoxicity [CC50] > 100 μM) (Fig 1B). Using the same cell type and assay duration, ALS-8112 inhibited the RNA replication of RSV A2 and B1 strains with concentration resulting in 50% inhibition (EC50) values of 0.153 ± 0.076 μM, and 0.132 ± 0.055 μM, respectively (Fig 1B). In addition, ALS-8112 demonstrated potent inhibition of a range of diverse RSV clinical isolates with comparable EC50 values (Table A in S1 Text). To understand the role of 5'-triphosphate formation in antiviral effect, we synthesized ALS-8112-I, an analog of ALS-8112 in which the 5'-hydroxyl group was replaced by iodine (Fig AA in S1 Text). Because of this modification, ALS-8112-I cannot form any triphosphate in vitro. As expected, ALS-8112-I did not significantly inhibit the luciferase activity in the RSV replicon (Fig AB in S1 Text). The in vitro antiviral activity of ALS-8112 was also characterized in a three-dimensional tissue culture system. This system consists of normal, human-derived tracheal/bronchial epithelial cells cultured to form a pseudo-stratified cell arrangement closely resembling the epithelial tissue of the respiratory tract [20,21]. The apical surface of the cultures contains numerous microvilli and cilia and the presence of tight junctions resembles the normal epithelial tissue of the lung. In this in vitro three-dimensional lung model, ALS-8112 was added to the basal media and incubated overnight before RSV strain A2 was added to the apical side of the system. The antiviral activity of ALS-8112 in human donor cells (n = 3) is described in Fig 1C and Fig B in S1 Text. In the donor cells, ALS-8112 inhibited RSV RNA replication with an EC50 ranging between 0.09 and 0.73 μM, and 90% inhibition (EC90) between 1.3 and 2.7 μM. We conclude that ALS-8112 is a pan-strain inhibitor of RSV replication in vitro, and that the antiviral activity of ALS-8112 is dependent upon the formation of its 5'-triphosphate metabolite.


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)

Inhibition of RSV replication by ALS-8112 and ALS-8176.(A) Chemical structure of 2'-fluoro-4'-chloromethyl (2'F-4'ClCH2) cytidine, or ALS-8112. (B) In vitro inhibition potency of ALS-8112 against the RSV A2 and B1 strains grown in HEp-2 cells. The viral RNA level was measured by qRT-PCR, and reported as percentage of the uninhibited condition (n = 3). The effect of ALS-8112 on the viability of human epithelial HEp-2 cells was also evaluated. The highest concentration of ALS-8112 used to measure the concentration resulting in 50% cytotoxicity (CC50) was 100 μM (n = 3). (C) In vitro efficacy of ALS-8112 in a three-dimensional lung model. Primary human tracheal/bronchial epithelial cells from three individual human donors were infected on the apical side with the RSV A2 strain, while increasing concentrations of ALS-8112 were added to the basal medium. (D) Fluorescence microphotographs of HEp-2 cells infected with recombinant RSV-mKate2, in the presence of DMSO, 10 μM ALS-8112, or 10 μM ALS-8176. (E) African Green monkey efficacy model. ALS-8176 was administered BID for a total of 6 days. At the end of treatment (Day 5 post-infection), RSV RNA titers were measured in bronchoalveolar lavage (BAL) and nasopharyngeal (NP) swab samples for each group (vehicle and drug) containing four animals. Limit of detection (LOD) for qRT-PCR analysis was 50 copies/mL (dashed line).
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ppat.1004995.g001: Inhibition of RSV replication by ALS-8112 and ALS-8176.(A) Chemical structure of 2'-fluoro-4'-chloromethyl (2'F-4'ClCH2) cytidine, or ALS-8112. (B) In vitro inhibition potency of ALS-8112 against the RSV A2 and B1 strains grown in HEp-2 cells. The viral RNA level was measured by qRT-PCR, and reported as percentage of the uninhibited condition (n = 3). The effect of ALS-8112 on the viability of human epithelial HEp-2 cells was also evaluated. The highest concentration of ALS-8112 used to measure the concentration resulting in 50% cytotoxicity (CC50) was 100 μM (n = 3). (C) In vitro efficacy of ALS-8112 in a three-dimensional lung model. Primary human tracheal/bronchial epithelial cells from three individual human donors were infected on the apical side with the RSV A2 strain, while increasing concentrations of ALS-8112 were added to the basal medium. (D) Fluorescence microphotographs of HEp-2 cells infected with recombinant RSV-mKate2, in the presence of DMSO, 10 μM ALS-8112, or 10 μM ALS-8176. (E) African Green monkey efficacy model. ALS-8176 was administered BID for a total of 6 days. At the end of treatment (Day 5 post-infection), RSV RNA titers were measured in bronchoalveolar lavage (BAL) and nasopharyngeal (NP) swab samples for each group (vehicle and drug) containing four animals. Limit of detection (LOD) for qRT-PCR analysis was 50 copies/mL (dashed line).
Mentions: A series of ribonucleoside analogs was recently identified as inhibiting the replication of RSV, and the optimization of the precursor molecules led to the chemical synthesis of 2'-fluoro-4'-chloromethyl (2'F-4'ClCH2) cytidine, referred to as ALS-8112 (Fig 1A) [18]. The nucleoside analog ALS-8112 did not significantly decrease the viability of human epithelial (HEp-2) cells after 5 days (with the concentration resulting in 50% cytotoxicity [CC50] > 100 μM) (Fig 1B). Using the same cell type and assay duration, ALS-8112 inhibited the RNA replication of RSV A2 and B1 strains with concentration resulting in 50% inhibition (EC50) values of 0.153 ± 0.076 μM, and 0.132 ± 0.055 μM, respectively (Fig 1B). In addition, ALS-8112 demonstrated potent inhibition of a range of diverse RSV clinical isolates with comparable EC50 values (Table A in S1 Text). To understand the role of 5'-triphosphate formation in antiviral effect, we synthesized ALS-8112-I, an analog of ALS-8112 in which the 5'-hydroxyl group was replaced by iodine (Fig AA in S1 Text). Because of this modification, ALS-8112-I cannot form any triphosphate in vitro. As expected, ALS-8112-I did not significantly inhibit the luciferase activity in the RSV replicon (Fig AB in S1 Text). The in vitro antiviral activity of ALS-8112 was also characterized in a three-dimensional tissue culture system. This system consists of normal, human-derived tracheal/bronchial epithelial cells cultured to form a pseudo-stratified cell arrangement closely resembling the epithelial tissue of the respiratory tract [20,21]. The apical surface of the cultures contains numerous microvilli and cilia and the presence of tight junctions resembles the normal epithelial tissue of the lung. In this in vitro three-dimensional lung model, ALS-8112 was added to the basal media and incubated overnight before RSV strain A2 was added to the apical side of the system. The antiviral activity of ALS-8112 in human donor cells (n = 3) is described in Fig 1C and Fig B in S1 Text. In the donor cells, ALS-8112 inhibited RSV RNA replication with an EC50 ranging between 0.09 and 0.73 μM, and 90% inhibition (EC90) between 1.3 and 2.7 μM. We conclude that ALS-8112 is a pan-strain inhibitor of RSV replication in vitro, and that the antiviral activity of ALS-8112 is dependent upon the formation of its 5'-triphosphate metabolite.

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