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Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase.

Brecher M, Chen H, Liu B, Banavali NK, Jones SA, Zhang J, Li Z, Kramer LD, Li H - PLoS ONE (2015)

Bottom Line: The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2'-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor.In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity.The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY, 12208 United States of America.

ABSTRACT
The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2'-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition.

No MeSH data available.


Related in: MedlinePlus

Inhibition of the N7 and 2’-O methylation activities of the WNV MTase by 42 top ranking compounds at 150 μM concentration.Inhibitions of the N7 and 2’-O methylation activities of the WNV MTase were analyzed on TLC plates. The N7 methylation was measured by conversion of G*pppA-RNA→m7G*pppA-RNA; the 2’-O methylation was measured by conversion of m7G*pppA-RNA→m7G*pppAm-RNA (the asterisk indicates that the following phosphate is 32P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The relative methylation activity without compounds was set at 100%, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) * 100.
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pone.0130062.g002: Inhibition of the N7 and 2’-O methylation activities of the WNV MTase by 42 top ranking compounds at 150 μM concentration.Inhibitions of the N7 and 2’-O methylation activities of the WNV MTase were analyzed on TLC plates. The N7 methylation was measured by conversion of G*pppA-RNA→m7G*pppA-RNA; the 2’-O methylation was measured by conversion of m7G*pppA-RNA→m7G*pppAm-RNA (the asterisk indicates that the following phosphate is 32P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The relative methylation activity without compounds was set at 100%, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) * 100.

Mentions: A suitable ligand binding pocket for virtual screening (VS) is provided by the crystal structures for SAH and 36A ligands bound to the DENV3 MTase (PDB ID: 3P8Z) [39]. The DENV3 MTase-inhibitor co-structure was chosen because the SAH-derivative inhibitor occupied a flavivirus-conserved pocket [34] and clearly defined the co-factor binding pocket [39]. We first optimized the docking parameters for AutoDock Vina by re-docking SAH and 36A into the SAM-binding site of the MTase. The root-mean-square deviation (RMSD) between the re-docked and crystallography-determined conformations of SAH and 36A was 1.2 Å and 1.7 Å, respectively (fig 1). These numbers are comparable to the ones published previously, by using different structures as models [25–27]. We then applied these optimized parameters to dock the NCI diversity set II library into the binding sites of both monomers in the DENV3 MTase structure, using AutoDock Vina. We selected 42 top-ranked compounds with better scores than the SAH control for further investigation (fig 2).


Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase.

Brecher M, Chen H, Liu B, Banavali NK, Jones SA, Zhang J, Li Z, Kramer LD, Li H - PLoS ONE (2015)

Inhibition of the N7 and 2’-O methylation activities of the WNV MTase by 42 top ranking compounds at 150 μM concentration.Inhibitions of the N7 and 2’-O methylation activities of the WNV MTase were analyzed on TLC plates. The N7 methylation was measured by conversion of G*pppA-RNA→m7G*pppA-RNA; the 2’-O methylation was measured by conversion of m7G*pppA-RNA→m7G*pppAm-RNA (the asterisk indicates that the following phosphate is 32P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The relative methylation activity without compounds was set at 100%, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) * 100.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476580&req=5

pone.0130062.g002: Inhibition of the N7 and 2’-O methylation activities of the WNV MTase by 42 top ranking compounds at 150 μM concentration.Inhibitions of the N7 and 2’-O methylation activities of the WNV MTase were analyzed on TLC plates. The N7 methylation was measured by conversion of G*pppA-RNA→m7G*pppA-RNA; the 2’-O methylation was measured by conversion of m7G*pppA-RNA→m7G*pppAm-RNA (the asterisk indicates that the following phosphate is 32P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The relative methylation activity without compounds was set at 100%, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) * 100.
Mentions: A suitable ligand binding pocket for virtual screening (VS) is provided by the crystal structures for SAH and 36A ligands bound to the DENV3 MTase (PDB ID: 3P8Z) [39]. The DENV3 MTase-inhibitor co-structure was chosen because the SAH-derivative inhibitor occupied a flavivirus-conserved pocket [34] and clearly defined the co-factor binding pocket [39]. We first optimized the docking parameters for AutoDock Vina by re-docking SAH and 36A into the SAM-binding site of the MTase. The root-mean-square deviation (RMSD) between the re-docked and crystallography-determined conformations of SAH and 36A was 1.2 Å and 1.7 Å, respectively (fig 1). These numbers are comparable to the ones published previously, by using different structures as models [25–27]. We then applied these optimized parameters to dock the NCI diversity set II library into the binding sites of both monomers in the DENV3 MTase structure, using AutoDock Vina. We selected 42 top-ranked compounds with better scores than the SAH control for further investigation (fig 2).

Bottom Line: The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2'-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor.In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity.The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY, 12208 United States of America.

ABSTRACT
The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2'-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition.

No MeSH data available.


Related in: MedlinePlus