ATP K_m determination for TBK1 and IKKε.

<p>Enzymatic reactions of A) TBK1 or B) IKKε were incubated at room temperature with 10 ATP concentrations varying from 333 µM to 0.017 µM in three fold dilutions. Reactions were sampled on the Caliper EZReader system at 9.35 minute intervals over a 3 hour period. Percent conversions were calculated from relative heights of product and substrate peaks and used to calculate velocity and ATP K_m in Graph Pad Prism.</p

Activity comparison for TBK1 and IKKε.

<p>Number of active compounds (N) detected in each screen for total number detected (unfiltered), number after drug like filtering (filtered.drug-like), hits from the LOPAC and Kinase libraries, and the number of chemical clusters and singleton hits as described in the text.</p

Distribution of compound activity.

<p>A–B) The kinase library was screened at 10 µM in a single concentration format against enzymatic reactions of A) TBK1 and B) IKKε. The distribution of activity is shown as a frequency histogram based on the number of compounds active at each level (% Inhibition). The data follow a normal distribution.</p

Identification of the optimal phosphorylation motif for TBK1.

<p>A-B) The positional scanning peptide library technology was used to determine the optimal phosphorylation motif for recombinant A) GST-TBK1 WT or B) kinase-dead GST-TBK1 K38A as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Hutti3" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041494#pone.0041494-Turk1" target="_blank">[28]</a>. Briefly, 198 peptide libraries were phosphorylated in individual kinase assays. The sequence for these libraries is Y-A-X-X-X-Z-X-S/T-X-X-X-X-A-G-K-K-biotin (Z =  fixed amino acid, X =  equimolar mixture of amino acids excluding Ser, Thr, and Cys). After binding to a streptavidin-coated membrane, phosphorylation was visualized by the incorporation of ³²P. C) Primary and secondary selections for TBK1, as determined in A). D) 50 µM of the indicated peptide was phosphorylated in an <i>in vitro</i> kinase assay with recombinant GST-TBK1 for 30 min. Phosphorylation of each peptide is shown as a percentage of the rate of phosphorylation of TBK1-Tide, the optimal peptide substrate for TBK1. Error bars are standard deviation.</p

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone <b>1</b> makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design

Novel Modes of Inhibition of Wild-Type Isocitrate Dehydrogenase 1 (IDH1): Direct Covalent Modification of His315

IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,β-unsaturated enone <b>1</b> makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design