Chemical structure of CRDS and antiviral activity based on DENV-2 MTT assay using LLC-MK2 cells.

<p>Structure of CRDS (A) and dose-dependent inhibition of DENV-2 infection by CRDS by MTT assay (B).</p

Efficient inhibitory effect of CRDS on DENV replication in monocytic HL-60 cells.

<p>Anti-DENV-2 was monitored at 100 µg/mL of CRDS 4 days after infection in HL-60 cells.</p

Effect of CRDS on low pH-induced fusion of DENV-2 infected C6/36 cells.

<p>The cells were incubated with DENV at 4°C for 1.5 h during viral adsorption in the presence of 100 µg/mL of either CRDS or heparin, followed by a temperature shift to 28°C, and cultured for 4 days to monitor virus-induced cell fusion (A). In the experiment shown in B, the compound was added at the time of temperature shift to 28°C, after viral attachment.</p

LigPlot⁺ representation of the hydrophobic interactions between CRDS and the residues of the binding pocket.

<p>The hydrophobic contacts are represented by dotted lines. The LigPlot⁺<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002188#pntd.0002188-Laskowski1" target="_blank">[49]</a> was used to map the interactions.</p

Proposed binding model of CRDS to DENV envelope protein obtained by Induced fit docking.

<p>The coordinates of the DENV E protein were obtained from PDB from the crystal structure 1OKE <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002188#pntd.0002188-Modis1" target="_blank">[13]</a>. Detailed information is described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002188#s2" target="_blank">Materials and Methods</a>.</p

Antiviral activity of CRDS on the four dengue virus serotypes.

<p>Antiviral activity of CRDS on the four dengue virus serotypes.</p

Hydrogen bonding interactions of the inhibitor with the residues in the binding pocket.

<p>Strong H-bonds (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002188#pntd-0002188-t003" target="_blank">Table 3</a>) are formed with conserved residues His 244, Lys 310, Asn 153 and Lys 247 (A). Residues lining the CRDS-binding pocket of the E protein (B).</p

Effect of CRDS on DENV-2 infection in DC-SIGN-expressing cells.

<p>The effect of CRDS was evaluated on DENV infection in DC-SIGN expressing cells. Raji-DC-SIGN cells were infected with large amount of DENV (1000 pfu) in the presence of CRDS. The cultures were analyzed for DENV infection by FACS assays 4 days later.</p

CRDS inhibits both attachment as well as an early post-attachment step of entry.

<p>Time of addition experiments were performed to identify the step at which CRDS exerts its effect (A) and efficacy of inhibitors during attachment and post-attachment (B). Studies were performed using the LLC-MK2 cells as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002188#s2" target="_blank">Materials and Methods</a> in detail.</p

Structure-activity–relationship of the CRDS.

a<p>: Measured in H₂0.</p>b<p>: Molecular weight.</p>c<p>: Degree of sulfation.</p>d<p>: 50% effective concentration.</p>e<p>: 50% cytotoxic concentration.</p>f<p>: 50% cytotoxic concentration/50% effective concentration.</p