Figure 5. In vitro Hog1 kinase activity assays. (A) Efficacy of compounds 1a–e, 4a–e, and SB203580. (B) IC50 curves for com- pounds 4a, 4b, and SB203580. Kinase assays were performed in a kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT) containing 0.4 mg GST-Hog1, 0.2 mM ATP, 0.1 mCi/nmol [32cP]ATP, and 100 mM peptide substrate, and Hog1 activity was determined as described in the Experimental section. Kinase reactions were performed A Hog1 Inhibitor for Analyzing Kinase Signaling in the presence of 0.1 mM inhibitor (A) or with a range of inhibitor concentrations (B). The concentration of the DMSO vehicle was identical in all reactions (1% final). The results are the average of three independent experiments and the error bars represent the standard deviation (s.d.). doi:10.1371/journal.pone.0020012.g005 0.1 mM (25–30% remaining activity), suggesting stronger inhibi- tion compared to SB203580. IC50 determination In order to compare the potency of compounds 4a, 4b, and SB203580, their IC50-values were determined. For this, we added these inhibitors to kinase reactions at concentrations ranging from 0.10 nM to 10 mM, measured substrate phosphorylation, plotted the remaining activity against inhibitor concentration, and cal- culated the IC50-values (Figure 5B). The IC50-values for com- pounds 4a and 4b were determined as 7.460.41 nM and 6.262.2 nM respectively, giving approximately 7-fold stronger inhibition than the reference compound SB203580 (49.561.6 nM). Hence, 4a and 4b are more potent inhibitors of Hog1 activity in vitro compared to the reference inhibitor SB203580. Uptake of inhibitors by yeast cells To be useful in vivo, the inhibitors need to enter yeast cells. To test this, a lawn of yeast cells was spread on solid medium and filter discs containing various concentrations of compounds 4a, 4b, and SB203580 were placed on top of the lawn (Figure 6A). Survival of yeast cells in the presence of osmotic stress requires Hog1 activity; hence, inhibition of Hog1 can be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress. Thus, the size of the halo is a measure of Hog1 inhibition in this assay. When wild-type yeast was exposed to high osmolarity, a clear halo was formed around compounds 4a and 4b at concentrations down to 0.1 mM (Figure 6A). No growth inhibition was observed around the filter discs in the absence of osmotic stress (Figure 6A), indicating that these compounds are not toxic to cells at these concentrations. In a reciprocal experiment, we tested whether these compounds could alleviate growth inhibition caused by overexpression of the kinases Ssk1 and Pbs2; these kinases act upstream of Hog1 and their overexpression results in Hog1 hyperactivation. In turn, hyperactivated Hog1 inhibits growth while inactivation of Hog1 (by deletion of the HOG1 gene) partially suppresses the phenotypes caused by Ssk1 or Pbs2 overexpression [32,33]. Importantly, the presence of 4a or 4b alleviated the growth inhibition caused by Ssk1 and Pbs2 overexpression (Figures 6B, S6 and S7), indicating that 4a and 4b are taken up by cells and inhibit Hog1 activity. In the case of SB203580, no halo was formed around osmo- stressed cells (Figure 5A), indicating that this compound does not enter cells or that it is efficiently exported. To distinguish between these possibilities, we repeated the halo assay using the pdr5D mutant that lacks a major multidrug export protein Pdr5 [34]. 4a and 4b also inhibited Hog1 activity in pdr5D cells, since clear halos were formed even at 0.02 mM (Figure S1). In contrast, no halo was formed in the presence of SB203580, suggesting that it does not accumulate in yeast cells. In order to study if the pyridinylimidazole-based kinase inhibitors in general have a poor uptake into yeast cells, we carried out another halo assay using wild-type yeast cells and four different commercially available pyridinylimidazole-based kinase inhibitors (Figure S2). We also included a commercially available inhibitor with a completely different structure that inhibits p38 MAP kinase by utilizing an PLoS ONE | www.plosone.org 4 May 2011 | Volume 6 | Issue 5 | e20012