Limits...
Biochemical Effect of Resistance Mutations against Synergistic Inhibitors of RSV RNA Polymerase.

Deval J, Fung A, Stevens SK, Jordan PC, Gromova T, Taylor JS, Hong J, Meng J, Wang G, Dyatkina N, Prhavc M, Symons JA, Beigelman L - PLoS ONE (2016)

Bottom Line: AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation.The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP.Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication.

View Article: PubMed Central - PubMed

Affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.

ABSTRACT
ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.

No MeSH data available.


Related in: MedlinePlus

Synergistic antiviral effect of ALS-8112 and AZ-27 on RSV replication.(A) Isobologram analysis of ALS-8112 and AZ-27 interaction in HEp-2 cells against RSV replication. AZ-27 and ALS-8112 concentrations were on the x- and y- axis respectively. The three lines intersecting both axes represent additivity for EC50 (red), EC75 (green) and EC90 (blue). The calculated CI for EC50, EC75, and EC90 all fall below their respective additivity lines, indicating a synergy (0.3–0.7). (B) Prichard’s model for 3-D drug interaction dose-responses. The synergy volume was above the 0% plane and falls within 50–100 range, indicating a significant synergy (n = 5).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4862670&req=5

pone.0154097.g007: Synergistic antiviral effect of ALS-8112 and AZ-27 on RSV replication.(A) Isobologram analysis of ALS-8112 and AZ-27 interaction in HEp-2 cells against RSV replication. AZ-27 and ALS-8112 concentrations were on the x- and y- axis respectively. The three lines intersecting both axes represent additivity for EC50 (red), EC75 (green) and EC90 (blue). The calculated CI for EC50, EC75, and EC90 all fall below their respective additivity lines, indicating a synergy (0.3–0.7). (B) Prichard’s model for 3-D drug interaction dose-responses. The synergy volume was above the 0% plane and falls within 50–100 range, indicating a significant synergy (n = 5).

Mentions: Antiviral combination studies were performed in vitro to measure the drug-drug interaction between ALS-8112 and AZ-27. HEp-2 cells were infected with recombinant RSV containing renilla luciferase as a reporter, in the presence of various concentrations of ALS-8112 and AZ-27 added individually or in combinations. Cell viability during the course of the experiment was >80% in all combinations of drug concentrations, indicating no increase in cytotoxicity when ALS-8112 and AZ-27 were combined together in HEp-2 cells (S3 Fig). The drug combination effect was first determined by the isobologram and combination index (CI) methods of Chou and Talalay [13,14], which provide quantitative estimation of the extent of synergy or antagonism. A CI of 1 indicates an additive effect between two compounds, whereas a CI< 1 or CI >1 indicates synergism or antagonism, respectively. With this analysis, the combination of ALS-8112 and AZ-27 resulted in significant synergy in antiviral effect, as determined by a CI = 0.38 for EC50, CI = 0.44 for EC75, and CI = 0.52 for EC90 (Fig 7A). Data were also analyzed using the Bliss-Independence model using Prichard’s MacSynergy II software [15]. This method uses a three-dimensional graphical layout representing the calculated additive surface predicting additive interaction as a horizontal plane at 0%. Peaks above this plane are indicative of synergy, and depressions below the horizontal plane indicative of antagonism. In the case of ALS-8112 combined with AZ-27, two peaks above the horizontal plane were observed with a total peak volume of 55.2 μM2% (Fig 7B). This volume is reflective of significant synergy of inhibition between the two drugs.


Biochemical Effect of Resistance Mutations against Synergistic Inhibitors of RSV RNA Polymerase.

Deval J, Fung A, Stevens SK, Jordan PC, Gromova T, Taylor JS, Hong J, Meng J, Wang G, Dyatkina N, Prhavc M, Symons JA, Beigelman L - PLoS ONE (2016)

Synergistic antiviral effect of ALS-8112 and AZ-27 on RSV replication.(A) Isobologram analysis of ALS-8112 and AZ-27 interaction in HEp-2 cells against RSV replication. AZ-27 and ALS-8112 concentrations were on the x- and y- axis respectively. The three lines intersecting both axes represent additivity for EC50 (red), EC75 (green) and EC90 (blue). The calculated CI for EC50, EC75, and EC90 all fall below their respective additivity lines, indicating a synergy (0.3–0.7). (B) Prichard’s model for 3-D drug interaction dose-responses. The synergy volume was above the 0% plane and falls within 50–100 range, indicating a significant synergy (n = 5).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862670&req=5

pone.0154097.g007: Synergistic antiviral effect of ALS-8112 and AZ-27 on RSV replication.(A) Isobologram analysis of ALS-8112 and AZ-27 interaction in HEp-2 cells against RSV replication. AZ-27 and ALS-8112 concentrations were on the x- and y- axis respectively. The three lines intersecting both axes represent additivity for EC50 (red), EC75 (green) and EC90 (blue). The calculated CI for EC50, EC75, and EC90 all fall below their respective additivity lines, indicating a synergy (0.3–0.7). (B) Prichard’s model for 3-D drug interaction dose-responses. The synergy volume was above the 0% plane and falls within 50–100 range, indicating a significant synergy (n = 5).
Mentions: Antiviral combination studies were performed in vitro to measure the drug-drug interaction between ALS-8112 and AZ-27. HEp-2 cells were infected with recombinant RSV containing renilla luciferase as a reporter, in the presence of various concentrations of ALS-8112 and AZ-27 added individually or in combinations. Cell viability during the course of the experiment was >80% in all combinations of drug concentrations, indicating no increase in cytotoxicity when ALS-8112 and AZ-27 were combined together in HEp-2 cells (S3 Fig). The drug combination effect was first determined by the isobologram and combination index (CI) methods of Chou and Talalay [13,14], which provide quantitative estimation of the extent of synergy or antagonism. A CI of 1 indicates an additive effect between two compounds, whereas a CI< 1 or CI >1 indicates synergism or antagonism, respectively. With this analysis, the combination of ALS-8112 and AZ-27 resulted in significant synergy in antiviral effect, as determined by a CI = 0.38 for EC50, CI = 0.44 for EC75, and CI = 0.52 for EC90 (Fig 7A). Data were also analyzed using the Bliss-Independence model using Prichard’s MacSynergy II software [15]. This method uses a three-dimensional graphical layout representing the calculated additive surface predicting additive interaction as a horizontal plane at 0%. Peaks above this plane are indicative of synergy, and depressions below the horizontal plane indicative of antagonism. In the case of ALS-8112 combined with AZ-27, two peaks above the horizontal plane were observed with a total peak volume of 55.2 μM2% (Fig 7B). This volume is reflective of significant synergy of inhibition between the two drugs.

Bottom Line: AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation.The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP.Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication.

View Article: PubMed Central - PubMed

Affiliation: Alios BioPharma, Inc., part of the Janssen Pharmaceutical Companies, South San Francisco, California, United States of America.

ABSTRACT
ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.

No MeSH data available.


Related in: MedlinePlus