Although ASKA technology has turned out to be very useful for studying protein kinases in general, it would be more convenient to use wt kinase inhibitors and thereby circumvent the need to generate cells that express the as version of the protein kinase of interest. Furthermore, it cannot be excluded that the as-mutation alters kinase activity and/or stability to some extent. Hence, by interfering less with the natural biological system, the experimental data will probably be more relevant. Therefore, we were interested in the development of a specific wtHog1 inhibitor that would allow us to study the dynamic behavior of this kinase. Exposure of yeast to high osmolarity triggers rapid phosphor- ylation, activation, and nuclear translocation of Hog1, the MAPK of the HOG pathway [10,11,12]. In the nucleus, Hog1 associates with stress-responsive promoters via specific transcription factors and stimulates gene expression by recruiting general transcription factors, chromatin-modifying enzymes, and RNA polymerase II [10,11]. Activation of gene expression is not the sole mechanism by which Hog1 controls osmoadaptation; a plasma membrane anchored version of Hog1 is biologically active and such cells can withstand osmotic stress [13]. Hog1 has been shown to regulate a number of cytosolic proteins [14,15,16] and to delay cell cycle progression by negatively regulating the activity of cyclin-depen- dent kinase complexes through a number of different mechanisms [17,18,19]. Hog1 is also activated in the presence of the metalloid arsenite [As(III)], and its kinase activity is required for cell survival during As(III) exposure. Interestingly, the dynamics of Hog1 activation by osmotic stress and As(III) are different, and As(III)- activation does not involve a Hog1-dependent induction of gene expression. Instead, Hog1 contributes to As(III) tolerance by restricting As(III) uptake into cells and by controlling cell cycle progression [14,20]. The MAPK p38 is the mammalian ortholog of yeast Hog1 and is extensively studied due to its involvement in chronic inflammatory diseases [21]. p38 is also activated by As(III) [22] and triggers cell cycle arrest, differentiation, or mitochondrial apoptotic cell death [23,24]. One class of selective p38 inhibitors is the pyridinylimidazole-based compounds (SB) [25,26]. Several of these compounds are highly potent and inhibit p38 at nanomolar concentrations (Figure 1). However, these inhibitors cannot be used for in vivo inhibition of Hog1 since they do not accumulate in yeast cells (see Uptake of inhibitors by yeast cells). Recently, we took advantage of the structural similarities between 4- and 5- substituted 1,2,3-triazoles and pyridinylimidazole-based inhibitors in the design of new inhibitors of p38, which prompted us to explore the use of triazoles as potential Hog1 inhibitors [27]. Herein, we report the design, synthesis, and biological evaluation of potent and selective 4- and 5-substituted 1,2,3-triazoles as wtHog1 inhibitors. Using two of these novel inhibitors, we de- monstrate that Hog1 controls the exit from As(III)-induced cell cycle arrest. Results and Discussion Design So far, there is no structural information (X-ray or NMR) available for Hog1. Nonetheless, Hog1 is highly similar to mam- malian p38a, with 51% identity at the amino acid level, and we built homology models of Hog1 based on structural information from crystallographic data for p38a (1a9u). The homology model showed high conservation of the amino acid residues in the ATP- binding cleft between Hog1 and p38a, suggesting that the binding motif of inhibitors in p38a could potentially be used to guide the development of Hog1 inhibitors. A Hog1 Inhibitor for Analyzing Kinase Signaling Figure 1. p38 kinase inhibitors. SB 203580 is a pyridinyl imidazole inhibitor of p38 MAPK that specifically blocks its kinase activity and is widely used as a research tool. Compounds 1a–1e were recently described as p38a inhibitors. doi:10.1371/journal.pone.0020012.g001 A new series of 4- and 5-substituted 1,2,3-triazoles (compounds 4a–e) were designed to have amine functionality in the 2-position of the pyridine ring that could potentially form an extra hydrogen bond with the hinge region (Figure 2A). The new triazole com- pounds 4a–e were docked into the homology model of Hog1 [28,29]. The binding mode of the amine-containing triazoles (yellow) is similar to the binding mode of the SB203580 inhibitor (a known inhibitor of p38a, p38b, and AKT/PKB; in blue); i.e., the 4-flourophenyl group interacts with hydrophobic region I and the nitrogen in the pyridine group hydrogen bonds to the amide of Gln103 in the hinge region (Figure 2B). In addition, the docking studies showed that the amine fun- ctionality hydrogen bonds to the carbonyl group of Gln103 in the hinge region, which could potentially increase the binding affinity [30]. Synthesis Previously, we had recognized the Cu(I)-catalyzed azide-alkyne 1,3-dipolar [2+3]-cycloaddition reaction as the key step in forming five-membered 4- and 5-substituted 1,2,3-triazoles, which can easily be coupled via a Suzuki coupling to yield compounds that have been evaluated as p38a inhibitors (compounds 1a–1e; see Figure 1) [27]. By the use of the bifunctional 2-chloropyridine boronic acid in the Suzuki coupling, the reaction sequence can be extended via a Hartwig-Buchwald C-N bond coupling, yielding the target compounds containing the amine substituent in the 2- position of the pyridine ring (Figure 3) [31]. The synthesis of the target compounds is shown in Figure 4. Compound 2 was prepared as previously described [27]. The synthesis of the 4- and 5-substituted 1,2,3-triazole intermediate 3 was completed in high yield (89%) via a palladium-catalyzed Suzuki coupling reaction between the halogenated 4-aryl substi- tuted 5-iodo-1,2,3-triazole (1) and 2-chloropyridin-4-ylboronic acid in the presence of Pd(PPh3)4 (2 mol%) and K2CO3 at 150uC in the microwave. The subsequent Hartwig-Buchwald C-N bond coupling be- tween 3 and various amines using 5 as a catalyst furnished the target compounds 4a–e in good yields (64–74%, Figure 4). PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20012