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Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A.

Kolocouris A, Tzitzoglaki C, Johnson FB, Zell R, Wright AK, Cross TA, Tietjen I, Fedida D, Busath DD - J. Med. Chem. (2014)

Bottom Line: The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1).Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding.The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens , Athens 15771, Greece.

ABSTRACT
A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

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CPT structure of the HA trimer, producedby Gamblin et al. (1RVX,A/Puerto Rico/8/1934)21 with a bound NAG-GAL-SIAligand as ball-and-stick (red) and with the two nearby 6-resistance sites highlighted in blue, residues 159 (above ligand)and 186 (left of ligand). The third 6-resistance site,324, also in blue, is near the bottom of the structure. Because ofa common insertion after residue 133 found in A/Calif/07/2009 (H1N1),these correspond to N160, S187, and I325, respectively, in the A/Calif/07/2009 6-resistant mutants.
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fig2: CPT structure of the HA trimer, producedby Gamblin et al. (1RVX,A/Puerto Rico/8/1934)21 with a bound NAG-GAL-SIAligand as ball-and-stick (red) and with the two nearby 6-resistance sites highlighted in blue, residues 159 (above ligand)and 186 (left of ligand). The third 6-resistance site,324, also in blue, is near the bottom of the structure. Because ofa common insertion after residue 133 found in A/Calif/07/2009 (H1N1),these correspond to N160, S187, and I325, respectively, in the A/Calif/07/2009 6-resistant mutants.

Mentions: In the amantadine–H3N2 system,drug resistance appearedafter one passage in the presence of drug, with no detectable activityof amantadine 1 against the progeny from passage 1 orpassage 2 at 50 μM but normal amantadine 1 activityagainst the original virus post hoc (EC50 = 3.0 ±0.5 μM; N = 9). In contrast, in the 6–H1N1 system, virus progeny produced in the presence of drugat passages 1–5 maintained full drug sensitivity (EC50 = 2.1–5.4 μM). Resistance to 6 developedsteadily between passage 6 and passage 12, becoming significant afterpassage 10. Without any drug in the medium, the development of viralresistance to compound 6 was negligible; i.e., the EC50 retested at passage 0 was 4.7 ± 0.7 μM, at passage10 was 3.0 ± 0.3, and at passage 30 was 7.7 ± 0.6 μM.Resistance to amantadine develops rapidly in vitro,17 in mice,18 and in the clinicalsetting19 through a small set of mutations,primarily L26F, V27A, V27T, A30T, S31N, and G34E.20 These are residues whose side chains are near the 4-foldsymmetric amantadine binding site.5 Nochanges from the parent A/California/07/2009 were observed for theamino acid translation of the M-segment of the passage-12 6-resistant strain for residues sequenced, 10–73. Hence, resistancedid not develop by selection of additional amantadine-resistance mutationsin M2. Sequencing of segment 4 (HA gene), however, revealed threeamino acid substitutions (Figure 2, Table S1) compared to the parental A/California/07/2009sequence, i.e., N160D, S187P, and I325S (numbering started after the13-residue HA signal sequence).


Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A.

Kolocouris A, Tzitzoglaki C, Johnson FB, Zell R, Wright AK, Cross TA, Tietjen I, Fedida D, Busath DD - J. Med. Chem. (2014)

CPT structure of the HA trimer, producedby Gamblin et al. (1RVX,A/Puerto Rico/8/1934)21 with a bound NAG-GAL-SIAligand as ball-and-stick (red) and with the two nearby 6-resistance sites highlighted in blue, residues 159 (above ligand)and 186 (left of ligand). The third 6-resistance site,324, also in blue, is near the bottom of the structure. Because ofa common insertion after residue 133 found in A/Calif/07/2009 (H1N1),these correspond to N160, S187, and I325, respectively, in the A/Calif/07/2009 6-resistant mutants.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127532&req=5

fig2: CPT structure of the HA trimer, producedby Gamblin et al. (1RVX,A/Puerto Rico/8/1934)21 with a bound NAG-GAL-SIAligand as ball-and-stick (red) and with the two nearby 6-resistance sites highlighted in blue, residues 159 (above ligand)and 186 (left of ligand). The third 6-resistance site,324, also in blue, is near the bottom of the structure. Because ofa common insertion after residue 133 found in A/Calif/07/2009 (H1N1),these correspond to N160, S187, and I325, respectively, in the A/Calif/07/2009 6-resistant mutants.
Mentions: In the amantadine–H3N2 system,drug resistance appearedafter one passage in the presence of drug, with no detectable activityof amantadine 1 against the progeny from passage 1 orpassage 2 at 50 μM but normal amantadine 1 activityagainst the original virus post hoc (EC50 = 3.0 ±0.5 μM; N = 9). In contrast, in the 6–H1N1 system, virus progeny produced in the presence of drugat passages 1–5 maintained full drug sensitivity (EC50 = 2.1–5.4 μM). Resistance to 6 developedsteadily between passage 6 and passage 12, becoming significant afterpassage 10. Without any drug in the medium, the development of viralresistance to compound 6 was negligible; i.e., the EC50 retested at passage 0 was 4.7 ± 0.7 μM, at passage10 was 3.0 ± 0.3, and at passage 30 was 7.7 ± 0.6 μM.Resistance to amantadine develops rapidly in vitro,17 in mice,18 and in the clinicalsetting19 through a small set of mutations,primarily L26F, V27A, V27T, A30T, S31N, and G34E.20 These are residues whose side chains are near the 4-foldsymmetric amantadine binding site.5 Nochanges from the parent A/California/07/2009 were observed for theamino acid translation of the M-segment of the passage-12 6-resistant strain for residues sequenced, 10–73. Hence, resistancedid not develop by selection of additional amantadine-resistance mutationsin M2. Sequencing of segment 4 (HA gene), however, revealed threeamino acid substitutions (Figure 2, Table S1) compared to the parental A/California/07/2009sequence, i.e., N160D, S187P, and I325S (numbering started after the13-residue HA signal sequence).

Bottom Line: The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1).Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding.The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens , Athens 15771, Greece.

ABSTRACT
A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH2 group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

Show MeSH
Related in: MedlinePlus