The specificities of nine approved tyrosine kinase inhibitors (imatinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, sorafenib, sunitinib, and pazopanib) were determined by activity-based kinase profiling using a large panel of human recombinant active kinases. This panel consisted of 79 tyrosine kinases, 199 serine/threonine kinases, three lipid kinases, and 29 disease-relevant mutant kinases. Many potential targets of each inhibitor were identified by kinase profiling at the K m for ATP. In addition, profiling at a physiological ATP concentration (1 mM) was carried out, and the IC 50 values of the inhibitors against each kinase were compared with the estimated plasma-free concentration (calculated from published pharmacokinetic parameters of plasma C trough and C max values). This analysis revealed that the approved kinase inhibitors were well optimized for their target kinases. This profiling also implicates activity at particular off-target kinases in drug side effects. Thus, large-scale kinase profiling at both K m and physiological ATP concentrations could be useful in characterizing the targets and off-targets of kinase inhibitors.

The small genome of fission yeast Schizosaccharomyces pombe contains 4824 predicted genes and gene disruption suggests that ∼ ∼ ∼ ∼ 850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/ cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype.

In evaluating kinase inhibitors, kinetic parameters such as association/dissociation rate constants are valuable information, as are equilibrium parameters K D and IC 50 values. Surface plasmon resonance (SPR) is a powerful technique to investigate these parameters. However, results are often complicated because of impaired conformations by inappropriate conditions required for protein immobilization and/or heterogeneity of the orientation of immobilization. In addition, conventional SPR experiments are generally time-consuming. Here we introduce the use of single-site specifically biotinylated kinases combined with a multichannel SPR device to improve such problems. Kinetic parameters of four compounds-staurosporine, dasatinib, sunitinib, and lapatinib-against six kinases were determined by the ProteOn XPR36 system. The very slow off-rate of lapatinib from the epidermal growth factor receptor and dasatinib from Bruton's tyrosine kinase and colony stimulating factor 1 receptor (CSF1R) were confirmed. Furthermore, IC 50 values were determined by an activity-based assay. Evaluating both physicochemical and biochemical properties would help to understand the detailed character of the compound.

Background: Neural crest cells (NC cells) are highly migratory multipotent cells. Their multipotency is transient at the early stage of their generation; soon after emerging from the neural tube, these cells turn into lineage-restricted precursors. However, recent studies have disputed this conventionally believed paradigm. In this study, we analyzed the differentiation potency of NC-derived cells after their arrival at target tissues. Results: Using Sox10-IRES-Venus mice, we found that the NC-derived cells in the skin, DRG, and inner ear could be divided into two populations: Sox10-positive/Kit-negative cells (Sox101/Kitcells) and Sox10-and Kit-positive cells (Sox101/Kit1 cells). Only the Sox101/Kit-cells were detected in the intestines. Unexpectedly, the Sox101/Kit1 cells differentiated into neurons, glial cells, and melanocytes, showing that they had maintained their multipotency even after having entered the target tissues. The Sox101/Kit1 cells in the DRG maintained their multipotency for a restricted period during the earlier embryonic stages, whereas those in the skin and inner ear were multipotent yet even in later embryonic stages. Conclusions: We showed that NC-derived Sox101/Kit1 cells maintained their multipotency even after entry into the target tissues. This unexpected differentiation potency of these cells in tissues seems to have been strictly restricted by the tissue microenvironment. Developmental Dynamics 243:368-380, 2014. V C 2013 Wiley Periodicals, Inc.Key words: neural crest; kit; Sox10; mouse Key findings:NC-derived cells in skin, DRG, and inner ear are divided into 2 populations: Sox101/Kit-cells and Sox101/ Kit1 cells. Sox101/Kit-cells in the skin and Sox101/Kit1 cells in DRGs and inner ear are previously unidentified NC-derived cells. The NC-derived Sox101/Kit1 cells maintain their multipotency even after having entered the target tissues. The differentiation potency of NC-derived cells in tissues is restricted depending on the tissue environment.

It is known that some kinase inhibitors are sensitive to the phosphorylation state of the kinase, and therefore those compounds can discriminate between a phosphorylated and unphosphorylated protein. In this study, we prepared two colony stimulating factor-1 receptor (CSF-1R) tyrosine kinase proteins: one highly phosphorylated by autophosphorylation and the other dephosphorylated by phosphatase treatment. These kinases were subjected to an activity-based assay to investigate the effect of their phosphorylation state on the potency of several kinase inhibitors. Dasatinib, sorafenib, PD173074 and staurosporine showed similar inhibition against different phosphorylation states of CSF-1R, but pazopanib, sunitinib, GW2580 and imatinib showed more potent inhibition against dephosphorylated CSF-1R. Binding analysis of the inhibitors to the two different phosphorylation forms of CSF-1R, using surface plasmon resonance spectrometry, revealed that staurosporine bound to both forms with similar affinity, but sunitinib bound to the dephosphorylated form with higher affinity. Thus, these observations suggest that sunitinib binds preferentially to the inactive form, preventing the activation of CSF-1R. Screening against different activation states of kinases should be an important approach for prioritizing compounds and should facilitate inhibitor design.