Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1T100G). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

This work was financed by the European Commission (QUASI, contract LSHG-CT2003-503230 and CellComput, contract 043310). CN is supported by a grant from the Canadian Institutes for Health Research (#84305) and from the Canadian Cancer Society (grant#020380) to GG. FP’s laboratory is also supported by the FP7 UNICELLSYS grant (#201142) and the Fundación Marcelino Botín (FMB). FP is the recipient of an ICREA Acadèmia (Generalitat de Catalunya). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript