S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

<div><p>The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.</p> </div

Atomic decomposition of the vacuum interaction energy between the AdoHcy and SIN ligands and the WNV MTase.

<p>Color coded projection onto the individual ligand atoms of the interaction energy between AdoHcy (panel <b>A</b>), SIN (panel <b>B</b>), and the difference (SIN-AdoHcy projected on SIN, panel <b>C</b>), and the WNV MTase. Panel <b>D</b> shows the numerical value of the interaction energy difference between the two ligands with the chemically different atoms bounded by a green box. The chemical difference is localized to atoms 13-17: atom 13 is a sulfur in AdoHcy, while atoms 13-17 are CHNH2 in SIN. The atoms numbers are indicated by green labels in panel C. All values are in kcal/mol.</p

Inhibition of the N-7 activities of the WNV and DENV2 MTases by synthesized derivatives at 20 µM or 75 µM concentrations.

<p>The methylation activity without compounds was set at 100%. Details were described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076900#pone-0076900-g003" target="_blank">Figure <b>3</b></a>.</p

Chemical structures of AdoHcy and its four derivatives synthesized.

<p>Groups of AdoHcy that were modified were in red color.</p

Inhibition of the N-7 and 2’-O methylation activities of the flavivirus MTases by AdoHcy.

<p>(<b>A</b>) TLC analysis of inhibition of the N-7 methylation activity of the WNV MTase by AdoHcy was analyzed on TLC plates. The spots representing different cap structures on TLC plates were quantified by a PhosphorImager. The N-7 methylation was measured by conversion of G*pppA-RNA→m⁷G*pppA-RNA (e.g., the specific activity (%) = Intensity (m⁷G*pppA)/(Intensity (G*pppA) +Intensity (m⁷G*pppA)) *100) (Here and after, the asterisk indicates that the following phosphate is ³²P labeled; the RNA represents the first 90 nucleotides of the WNV genome). The relative methylation activity without AdoHcy was set at 100%, and the relative methylation activity with a particular compound was defined as specific activity (compound)/specific activity (no compound) * 100. (<b>B</b>) TLC analysis of inhibition of the 2’-O methylation activity of the WNV MTase by AdoHcy. The 2’-O methylation was measured by conversion of m⁷G*pppA-RNA→m⁷G*pppAm-RNA (e.g., the specific activity (%) = Intensity (m⁷G*pppAm)/(Intensity (m⁷G*pppA) +Intensity (m⁷G*pppAm)) *100). The methylation activity without AdoHcy was set at 100%, and the relative 2’-O methylation activity with compounds was defined the same way as in panel A. The migration positions of the G*pppA and m⁷G*pppA molecules are labeled on the side of the TLC images. (<b>C</b>-<b>D</b>) TLC analysis of inhibition of the N-7 (<b>C</b>) and 2’-O (<b>D</b>) methylation activities of flavivirus MTases in the presence or absence of 150 µM SIN or 150 µM AdoHcy. The methylation activity for each MTase without compound was set at 100%. The relative methylation activity for each MTase with compound (SIN or AdoHcy) was calculated as percentage to the activity without any compound. The migration positions of the G*pppA, m⁷G*pppA, and m⁷G*pppAm molecules are labeled on the side of the TLC images. VP39 was included in Panel <b>D</b> as a positional control for the 2’-O methylation reaction.</p

Cytotoxicity and antiviral analyses for AdoHcy.

<p>(<b>A</b>) Cytotoxicity of AdoHcy. A549 cells were incubated with various concentrations of AdoHcy and then assayed for viability at 42 h postincubation. (<b>B</b>) Inhibition of the WNV replication by AdoHcy. (<b>C</b>) Inhibition of the DENV2 replication by AdoHcy. A549 cells were infected with WNV or DENV2 at an multiplicity of infection of 0.1, in the presence or absence of AdoHcy. At 42 h post-infection, viral titers in culture fluids were quantified by plaque assays on Vero cells.</p