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Characterization of potent fusion inhibitors of influenza virus.

Rowse M, Qiu S, Tsao J, Xian T, Khawaja S, Yamauchi Y, Yang Z, Wang G, Luo M - PLoS ONE (2015)

Bottom Line: Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells.Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change.This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, 1025 18th Street South, Birmingham, Alabama, 35294, United States of America.

ABSTRACT
New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes.

No MeSH data available.


Related in: MedlinePlus

Structure and EC50 values of inhibitors.The structures of compounds 136 (A) and 211 (D). X-31 virus plaque reduction assays were performed using monolayers of MDCK-2 cells. For X-31 virus, the EC50 of 136 and 211 was calculated to be 48 pM and 140 nM, respectively (B and E). VSV plaque reduction assays were performed using monolayers of HeLa cells. For VSV, the EC50 of 136 and 211 was calculated to be 130 pM and 1.2 μM, respectively (C and F). Representative data are shown from 3 independent experiments. Data points are the average of 3 replicates ± SD.
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pone.0122536.g001: Structure and EC50 values of inhibitors.The structures of compounds 136 (A) and 211 (D). X-31 virus plaque reduction assays were performed using monolayers of MDCK-2 cells. For X-31 virus, the EC50 of 136 and 211 was calculated to be 48 pM and 140 nM, respectively (B and E). VSV plaque reduction assays were performed using monolayers of HeLa cells. For VSV, the EC50 of 136 and 211 was calculated to be 130 pM and 1.2 μM, respectively (C and F). Representative data are shown from 3 independent experiments. Data points are the average of 3 replicates ± SD.

Mentions: Compound P25H2 was previously found to inhibit cell infection of multiple influenza virus strains with high potency [15]. Derivatives of P25H2 were synthesized and tested for anti-influenza activities in plaque reduction assays [13]. Fig. 1A shows the structure of a particularly potent derivative, 136. A less potent derivative (compound 211) with a similar structure is used for a control because it inhibits virus only at a much higher concentration than 136 (Fig. 1D). Using X-31 virus the EC50 values of 136 and 211 were calculated by plaque reduction assays and the results are shown in Fig. 1B and E, respectively. 136 has an EC50 value of 48 picomolar whereas 211 has an EC50 value of 140 nanomolar, a difference of approximately 2900 fold. Plaque reduction assays with vesicular stomatitis virus (VSV) were also performed with 136 and 211 (Fig. 1C and F). 136 and 211 inhibited VSV with an EC50 of 130 pM and 1.2 μM, respectively. An approximately three fold greater concentration of 136 is required for inhibition of VSV as compared to X-31 virus. Table 1 summarizes the EC50 of 136 against many other influenza virus strains and includes the 95% confidence interval for all virus strains tested. S1 Fig. shows the plaque reduction assay results for the additional virus strains tested. Table 2 summarizes the EC50 values of 211 and includes the 95% confidence interval for the virus strains tested. Additionally, the pH of virus preparations were unaltered by 136 (S1 Table) and the cellular toxicity (CC50) of 136 was determined to be 50 μM by a MTT assay (Fig. 2A). The selectivity index of 136 is calculated to be 1x106. Because of the high potency and low cellular toxicity of 136, it was selected for further characterization.


Characterization of potent fusion inhibitors of influenza virus.

Rowse M, Qiu S, Tsao J, Xian T, Khawaja S, Yamauchi Y, Yang Z, Wang G, Luo M - PLoS ONE (2015)

Structure and EC50 values of inhibitors.The structures of compounds 136 (A) and 211 (D). X-31 virus plaque reduction assays were performed using monolayers of MDCK-2 cells. For X-31 virus, the EC50 of 136 and 211 was calculated to be 48 pM and 140 nM, respectively (B and E). VSV plaque reduction assays were performed using monolayers of HeLa cells. For VSV, the EC50 of 136 and 211 was calculated to be 130 pM and 1.2 μM, respectively (C and F). Representative data are shown from 3 independent experiments. Data points are the average of 3 replicates ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4372562&req=5

pone.0122536.g001: Structure and EC50 values of inhibitors.The structures of compounds 136 (A) and 211 (D). X-31 virus plaque reduction assays were performed using monolayers of MDCK-2 cells. For X-31 virus, the EC50 of 136 and 211 was calculated to be 48 pM and 140 nM, respectively (B and E). VSV plaque reduction assays were performed using monolayers of HeLa cells. For VSV, the EC50 of 136 and 211 was calculated to be 130 pM and 1.2 μM, respectively (C and F). Representative data are shown from 3 independent experiments. Data points are the average of 3 replicates ± SD.
Mentions: Compound P25H2 was previously found to inhibit cell infection of multiple influenza virus strains with high potency [15]. Derivatives of P25H2 were synthesized and tested for anti-influenza activities in plaque reduction assays [13]. Fig. 1A shows the structure of a particularly potent derivative, 136. A less potent derivative (compound 211) with a similar structure is used for a control because it inhibits virus only at a much higher concentration than 136 (Fig. 1D). Using X-31 virus the EC50 values of 136 and 211 were calculated by plaque reduction assays and the results are shown in Fig. 1B and E, respectively. 136 has an EC50 value of 48 picomolar whereas 211 has an EC50 value of 140 nanomolar, a difference of approximately 2900 fold. Plaque reduction assays with vesicular stomatitis virus (VSV) were also performed with 136 and 211 (Fig. 1C and F). 136 and 211 inhibited VSV with an EC50 of 130 pM and 1.2 μM, respectively. An approximately three fold greater concentration of 136 is required for inhibition of VSV as compared to X-31 virus. Table 1 summarizes the EC50 of 136 against many other influenza virus strains and includes the 95% confidence interval for all virus strains tested. S1 Fig. shows the plaque reduction assay results for the additional virus strains tested. Table 2 summarizes the EC50 values of 211 and includes the 95% confidence interval for the virus strains tested. Additionally, the pH of virus preparations were unaltered by 136 (S1 Table) and the cellular toxicity (CC50) of 136 was determined to be 50 μM by a MTT assay (Fig. 2A). The selectivity index of 136 is calculated to be 1x106. Because of the high potency and low cellular toxicity of 136, it was selected for further characterization.

Bottom Line: Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells.Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change.This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, 1025 18th Street South, Birmingham, Alabama, 35294, United States of America.

ABSTRACT
New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes.

No MeSH data available.


Related in: MedlinePlus