Redox absorbance assay with different conditions.

<p>To determine the source of discrepancies between the redox absorbance assay results and known mechanisms, the absorbance assay was carried out in different conditions. The patterns of redox absorbance kinetics in substrate-depleted conditions were tested with (A) zileuton and (B) DMSO at 80 minutes after the start of the reaction. The absorbance of zileuton increased slowly over time. (C) For CDC, which showed the strongest redox activity in the fluorescence assay, the absorbance assay was carried out in the buffer used in the fluorescence assay. However, redox activity was not detected.</p

Kinetic curves of the redox absorbance assay.

<p>The kinetic curves of 13(S)-HpODE are depicted for 10 µM of (A) zileuton, (B) PF4191834, (C) YS121, (D) caffeic acid, (E) CDC, (F) CAY10606, (G) CAY10649, (H) NDGA, and (I) DMSO control. The amount of substrate was measured by absorbance at 234 nm, and measurements were recorded at every second for 3 minutes. The values were normalized by subtracting the absorbance at the start of the reactions. The average absorbance change of three independent tests were summarized in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087708#pone-0087708-t001" target="_blank">Table 1</a>. Redox inhibitors and non-redox inhibitors showed decreasing and increasing patterns, respectively.</p

Comparison of the assay results with the known redox mechanisms of 5-LO inhibitors.

<p>The results of two redox assays and the known redox mechanisms of tested compounds were compared. The lower EC₅₀ value from fluorescence assay signifies the stronger redox activity. When the calculated EC₅₀ was over the experimental range, it was indicated as having >100 µM of EC₅₀. The change in absorbance (Δ abs) of each inhibitor was calculated by subtracting the absorbance at 180 seconds from the starting absorbance value. The tests were performed in triplicate, and their averages and standard deviations are presented. The known mechanisms are explained in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087708#s2" target="_blank">materials and methods</a> section.</p><p>The redox fluorescence assay correctly distinguished the three non-redox inhibitors by having high EC₅₀ values. Meanwhile, the redox absorbance assay predicted contradictory results to known mechanism for several redox compounds, such as NDGA and CDC.</p

Dose-response curves of the redox fluorescence assay.

<p>The amount of remaining fluorescence represented the redox activity of the inhibitor. The concentration of inhibitors varied from 1.69 µM, and dose-response curves were generated for (A) zileuton, (B) PF4191834, (C) YS121, (D) caffeic acid, (E) CDC, (F) CAY10606, (G) CAY10649, and (H) NDGA. The tests were carried out in duplicate and each curve was fitted by three-parameter logistic regression, using the Prism software. Calculated EC₅₀ values from three independent tests are averaged and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087708#pone-0087708-t001" target="_blank">Table 1</a>.</p

Discovery of Phenylaminopyridine Derivatives as Novel HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors

We identified a novel class of aryl-substituted triazine compounds as potent non-nucleoside reverse transcriptase inhibitors (NNRTIs) during a high-throughput screening campaign that evaluated more than 200000 compounds for antihuman immunodeficiency virus (HIV) activity using a cell-based full replication assay. Herein, we disclose the optimization of the antiviral activity in a cell-based assay system leading to the discovery of compound <b>27</b>, which possessed excellent potency against wild-type HIV-1 (EC₅₀ = 0.2 nM) as well as viruses bearing Y181C and K103N resistance mutations in the reverse transcriptase gene. The X-ray crystal structure of compound <b>27</b> complexed with wild-type reverse transcriptase confirmed the mode of action of this novel class of NNRTIs. Introduction of a chloro functional group in the pyrazole moiety dramatically improved hERG and CYP inhibition profiles, yielding highly promising leads for further development