the NS3 ATPase activity for the naphthoquinones 9c and 9b is in agreement with the difference in the IC50 values obtained for the DENV-2 replication in cells. Again, the 1421373-65-0 supplier naphthoquinone 9c was more effective in inhibiting both the NS3 ATPase activity and the replication of DENV-2 in Vero cells than the naphthoquinone 9b. Efforts to elucidate the mechanism of action of compounds 9b and 9c on the double-stranded nucleic acid unwind activity of the helicase of NS3 were carried out. Helicase assays using nucleic acid labeled with ATP at its 5��end together with the T4 polynucleotide kinase, and 5��Cy3/3��BHQ2 labeled by fluorescence resonance energy transfer were performed as described by Benarroch and co-workers and Boguszewska-Chachulska and co-workers, respectively. However, due to the low solubility of compounds in water and the increasing of ATPase activity in the presence of nucleic acids, it was not possible to assess the inhibitory potential of these compounds in the NS3 helicase activity. We also evaluated whether the compounds were able to suppress the proteolytic activity of DENV-2 NS3. Several Table 1. Inhibitory effect of pyran naphthoquinones on DENV-2 replication. Pyran naphthoquinone % of Inhibition of DENV-2 Replication of Inhibition of DENV-2 Replication Screening assays were performed with qPCR for quantification of the produced viral progeny in DENV-2 infected Vero cells at the highest non-toxic concentration of the pyran naphthoquinone compounds, followed by confirmation with Tissue Culture Infectious Dose of 50 assays. For those compounds that did not show inhibition in the qPCR assay the TCID50 assay was not performed. NT-not tested for viral replication due to its high cytotoxicity both in Vero and HepG2 cells. studies have shown that the NS2B cofactor is required by the protease domain to form a proteolytic active domain. Therefore, the NS3 protease domain linked to the NS2B cofactor was purified and the inhibition assay was performed as described by Leung and co-workers. We did not observe any effect of compounds 9b and 9c against the NS3 protease activity even in the presence of the NS2b cofactor. Therefore, the activity of the naphtoquinones 9b and 9c against the NS3 from DENV is specific and does not involve the proteolytic domain. This study is one of the first to demons
