Design, Synthesis, and Characterization of a Highly Effective Hog1 Inhibitor: A Powerful Tool for Analyzing MAP Kinase Signaling in Yeast Peter Dine´ r1., Jenny Veide Vilg2., Jimmy Kjelle´ n2., Iwona Migdal3, Terese Andersson 1 , Marinella Gebbia4, Guri Giaever4, Corey Nislow5, Stefan Hohmann 2 , Robert Wysocki3, Markus J. Tama´ s2, Morten Grøtli1* 1 Medicinal Chemistry, Department of Chemistry, University of Gothenburg, Go¨ teborg, Sweden, 2 Microbiology, Department of Cell and Molecular Biology, University of Gothenburg, Go¨ teborg, Sweden, 3 Institute of Plant Biology, Department of Genetics and Cell Physiology, University of Wroclaw, Wroclaw, Poland, 4 Department of Pharmaceutical Sciences, University of Toronto, Toronto, Canada, 5 Department of Molecular Genetics, University of Toronto, Toronto, Canada Abstract The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G1 checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action. Citation: Dine´r P, Veide Vilg J, Kjelle´n J, Migdal I, Andersson T, et al. (2011) Design, Synthesis, and Characterization of a Highly Effective Hog1 Inhibitor: A Powerful Tool for Analyzing MAP Kinase Signaling in Yeast. PLoS ONE 6(5): e20012. doi:10.1371/journal.pone.0020012 Editor: Daniel Lew, Duke University Medical Center, United States of America Received January 21, 2011; Accepted April 8, 2011; Published May 31, 2011 Copyright: 2011 Dine´r et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was financed by the European Commission (the CELLCOMPUT project, Contract No. 043310 to MG and SH), the Chemical Biology Platform at the University of Gothenburg (MG, SH, MJT), the Olle Engkvist Byggma¨stare Foundation (MJT), the Signhild Engkvist Foundation (JVV), the Polish Ministry of Science and Higher Education (Grant No. N303027137 to IM and RW), and the CIHR (to GG (81340) and CN (84305)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: grotli@chem.gu.se . These authors contributed equally to this work. Introduction Protein kinases have crucial roles in virtually all signaling pathways and they regulate diverse cellular functions, such as cell cycle pro- gression, apoptosis, metabolism, differentiation, cell morphology and migration, and secretion of cellular proteins [1]. Many kinases are highly conserved throughout the eukaryotic kingdoms and constitute an important field of research because of their involvement in disease processes. For instance, abnormal signaling is the cause of many human diseases, while activation of signal transduction pathways is a major survival response during drug therapies. The present under- standing of cellular signal transduction is in most cases restricted to the wiring schemes of signaling pathways, while little is known about their dynamic operation and time-dependent parameters for signaling output. The latter has been difficult to explore since traditional analy- sis has relied upon gene deletion/knock-out mutants, and in such mutants, cells can compensate for the lack of the kinase by rewiring signaling pathways or by adapting by other means [2]. An alternative approach is to use highly selective, cell-permeable, and fast-acting inhibitors of individual kinases to systematically investigate the cellular function of a kinase in real time. Protein kinases share common sequences and structural ho- mology in their ATP-binding sites. Many ATP competitive kinase inhibitors lack selectivity because the catalytic cleft is highly conserved in sequence and conformation [3]. Nevertheless, despite this high degree of conservation in the ATP-binding site, highly selective small molecules with favorable pharmaceutical properties have been developed [4]. One approach that has been used successfully with high inhibition selectivity is the so-called ASKA technology [5]. This approach involves modifying a kinase inhibitor to eliminate its binding affinity for its native target and subsequent mutation of a protein kinase to allow it to recognize the orthogonal inhibitor. The basic idea relies on the assumption that the so-called ‘‘gate-keeper’’ residue blocks access to an additional hydrophobic pocket in the ATP cleft and that the mutation (from a larger to a smaller residue, typically glycine) contributes to a stronger binding of the orthogonal inhibitor. Shokat and colleagues have used this approach extensively to study protein kinases [6]. Recently, ASKA technology has been used to identify novel targets and to provide novel insights into the mechanisms that control signaling through the Saccharomyces cerevisiae (budding yeast) HOG MAPK pathway [7,8,9]. PLoS ONE | www.plosone.org 1 May 2011 | Volume 6 | Issue 5 | e20012