A Hog1 Inhibitor for Analyzing Kinase Signaling Figure 3. Retro-synthetic analysis of the target compounds. doi:10.1371/journal.pone.0020012.g003 Figure 2. The ATP binding site of Hog1. A) Schematic picture of the ATP binding site of Hog1 from homology modeling using p38a (1a9u) as the template. B) Docking of triazole-based inhibitors 4a–e (yellow) together with SB203580 (blue) into the ATP-binding site of Hog1. doi:10.1371/journal.pone.0020012.g002 Efficacy The effect of the compounds on Hog1 activity was evaluated using in vitro kinase assays. For this, we incubated purified in vitro- activated Hog1 together with a biotinylated peptide as a substrate. The initial assays were performed in the presence of 0.1 mM of the compounds 1a–1e, 4a–4e, and SB203580, and the efficacy of these compounds to reduce phosphorylation of the Hog1 substrate was measured (Figure 5A). We found that compounds 1a–1e were less efficient in inhibiting substrate phosphorylation (50–70% remaining activity) compared to the reference compound SB203580 (40% remaining activity). Of the new compounds 4a–4e, compound 4c had a weak effect on Hog1 activity (about 75% remaining activity) while 4d and 4e were similar to SB203580 (35–40% remaining activity). Importantly, compounds 4a and 4b showed a significant decrease in substrate phosphorylation at a concentration of Figure 4. Scheme for the synthesis of target compounds 4a–e. doi:10.1371/journal.pone.0020012.g004 PLoS ONE | www.plosone.org 3 May 2011 | Volume 6 | Issue 5 | e20012