Hit to lead account of the discovery of a new class of inhibitors of Pim kinases and crystallographic studies revealing an unusual kinase binding mode.

A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR.

Assay optimization: a statistical design of experiments approach.

With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses.

Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase.

Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity, we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds, seven hits were identified.

Three mechanistically distinct kinase assays compared: Measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors.

Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity).

Discovery of potent and selective PKC-theta inhibitors.

An uHTS campaign was performed to identify selective inhibitors of PKC-theta. Initial triaging of the hit set based on selectivity and historical analysis led to the identification of 2,4-diamino-5-nitropyrimidines as potent and selective PKC-theta inhibitors. A homology model and initial SAR is presented demonstrating that a 2-arylalkylamino substituent in conjunction with suitable 4-diamino substituent are essential for achieving selectivity over many kinases.