Inhibition of the ATPase activity of full-length NS3 by the compounds 9b and 9c.

<p>Increasing concentrations of naphthoquinones 9b and 9c were tested for their activity against ATPase site. The reaction was performed in buffer containing 40 mM Tris-HCl (pH 7.5), 5 mM DTT, 100 mM KCl, and 5 mM MgCl₂. The compounds were pre-incubated with 600nM NS3FL for 10 min at 30 °C followed by the addition of 1 mM ATP. Each point represents the average of three independent replicates in different concentrations of the compounds.</p

Inhibition of the ATPase activity of purified NS3 by naphthoquinones.

<p>(A) NaphSDS-Page analysis of the purification full-length NS3 (NS3FL) from <i>E coli</i>. Lane 1, molecular weight marker; lane 2, 10xHis-NS3FL (10xHis-Ubiquitin site) after refolding by dialysis; lane 3 and 4, NS3FL after 10xHis-Ubiquitin site tag excision by YUH in ratio 1:5 and 1:1, respectively; and lane 5, purified NS3FL protein following His-TRAP column chromatography. The gel was stained with Comassie brilhant blue G. (B) A range of pyran naphthoquinones was tested for their activity against ATPase site. The screening was performed in buffer containing 40 mM Tris-HCl (pH 7.5), 5 mM DTT, 100 mM KCl, and 5 mM MgCl₂. The compounds were pre-incubated with 600nM NS3FL for 10 min at 30 °C followed by the addition of 1 mM ATP. The screening was carried out in duplicate and the unpaired t-test was used for the statistical analysis. Those compounds showing statistical difference from the control (p < 0.05) were labeled with an asterisk.</p

General scheme used for preparation of pyran naphthoquinones.

<p>The o-QM intermediate is generated in situ by Knoevenagel condensation of 2-hydroxy-1,4-naphthoquinone (7) with formaldehyde and arylaldehydes. The subsequent hetero Diels-Alder reaction with substituted dienophiles in aqueous ethanol media yields high amounts of α- and β-lapachone derivatives.</p

Dose-response curves and citotoxicity of 1,4-pyran naphthoquinones 9b and 9c in DENV-2-infected Vero cells.

<p>Vero cells were infected with DENV-2 at an m.o.i of 0.1 in the presence of increasing concentrations of the 1,4-pyran naphthoquinones 9b and 9c. 72 hours after infection the cell-free supernatants were harvested and processed for the quantification of produced viral progeny by qPCR. (A) The percentage of inhibition was calculated using the ΔΔCT values. (B) The concentration of each compound that inhibit 50% of the viral replication (IC₅₀) was determined by a Hill’s regression curve after logarithmic interpolation of the data using the GraphPad 6 software. (C) The potential cytotoxic effect of each compound was evaluated in uninfected cells by the vital neutral red staying and represented as percentage of the untreated control. Data represent mean values ± standard deviations (SD) for three independent experiments.</p