Integrated single-dose kinome profiling data is predictive of cancer cell line sensitivity to kinase inhibitors.

Protein kinase activity forms the backbone of cellular information transfer, acting both individually and as part of a broader network, the kinome. Their central role in signaling leads to kinome dysfunction being a common driver of disease, and in particular cancer, where numerous kinases have been identified as having a causal or modulating role in tumor development and progression. As a result, the development of therapies targeting kinases has rapidly grown, with over 70 kinase inhibitors approved for use in the clinic and over double this number currently in clinical trials. Understanding the relationship between kinase inhibitor treatment and their effects on downstream cellular phenotype is thus of clear importance for understanding treatment mechanisms and streamlining compound screening in therapy development. In this work, we combine two large-scale kinome profiling data sets and use them to link inhibitor-kinome interactions with cell line treatment responses (AUC/IC50). We then built computational models on this data set that achieve a high degree of prediction accuracy (R2 of 0.7 and RMSE of 0.9) and were able to identify a set of well-characterized and understudied kinases that significantly affect cell responses. We further validated these models experimentally by testing predicted effects in breast cancer cell lines and extended the model scope by performing additional validation in patient-derived pancreatic cancer cell lines. Overall, these results demonstrate that broad quantification of kinome inhibition state is highly predictive of downstream cellular phenotypes.

The phosphotransferase system gene ptsH affects persister formation in Klebsiella pneumoniae by regulating cyclic adenosine monophosphate levels.

Klebsiella pneumoniae is one of the most common opportunistic pathogens causing hospital- and community-acquired infections. Antibiotic resistance in K. pneumoniae has emerged as a major clinical and public health threat. Persisters are specific antibiotic-tolerant bacterial cells. Studies on the mechanism underlying their formation mechanism and growth status are scarce. Therefore, it is urgent to explore the key genes and signalling pathways involved in the formation and recovery process of K. pneumoniae persisters to enhance the understanding and develop relevant treatment strategies. In this study, we treated K. pneumoniae with a lethal concentration of levofloxacin. It resulted in a distinct plateau of surviving levofloxacin-tolerant persisters. Subsequently, we obtained bacterial samples at five different time points during the formation and recovery of K. pneumoniae persisters to perform transcriptome analysis. ptsH gene was observed to be upregulated during the formation of persisters, and down-regulated during the recovery of the persisters. Further, we used CRISPR-Cas9 to construct ΔptsH, the ptsH-knockout K. pneumoniae strain, and to investigate the effect of ptsH on the persister formation. We observed that ptsH can promote the formation of persisters, reduce accumulation of reactive oxygen species, and enhance antioxidant capacity by reducing cyclic adenosine monophosphate (cAMP) levels. To the best of our knowledge, this is the first study to report that ptsH plays a vital role in forming K. pneumoniae persisters. This study provided important insights to further explore the mechanism underlying the formation of K. pneumoniae persisters and provided a potential target for treating infection with K. pneumoniae persisters.

Design, Synthesis and Biological Evaluation of 1-Phenyl-2-(phenylamino) Ethanone Derivatives as Novel MCR-1 Inhibitors.

Polymyxins are considered to be the last-line antibiotics that are used to treat infections caused by multidrug-resistant (MDR) gram-negative bacteria; however, the plasmid-mediated transferable colistin resistance gene (mcr-1) has rendered polymyxins ineffective. Therefore, the protein encoded by mcr-1, MCR-1, could be a target for structure-based design of inhibitors to tackle polymyxins resistance. Here, we identified racemic compound 3 as a potential MCR-1 inhibitor by virtual screening, and 26 compound 3 derivatives were synthesized and evaluated in vitro. In the cell-based assay, compound 6g, 6h, 6i, 6n, 6p, 6q, and 6r displayed more potent activity than compound 3. Notably, 25 µΜ of compound 6p or 6q combined with 2 µg·mL-1 colistin could completely inhibit the growth of BL21(DE3) expressing mcr-1, which exhibited the most potent activity. In the enzymatic assay, we elucidate that 6p and 6q could target the MCR-1 to inhibit the activity of the protein. Additionally, a molecular docking study showed that 6p and 6q could interact with Glu246 and Thr285 via hydrogen bonds and occupy well the cavity of the MCR-1 protein. These results may provide a potential avenue to overcome colistin resistance, and provide some valuable information for further investigation on MCR-1 inhibitors.

Light-activated kinases enable temporal dissection of signaling networks in living cells.

We report a general strategy for creating protein kinases in mammalian cells that are poised for very rapid activation by light. By photoactivating a caged version of MEK1, we demonstrate the specific, rapid, and receptor independent activation of an artificial subnetwork within the Raf/MEK/ERK pathway. Time-lapse microscopy allowed us to precisely characterize the kinetics of elementary steps in the signaling cascade and provided insight into adaptive feedback and rate-determining processes in the pathway.

Targeted covalent drugs of the kinase family.

In the past decade tremendous progress has been made toward a new class of therapeutics termed 'targeted covalent drugs', in which structure-based approaches are employed to create small molecules that inactivate their protein target through targeted covalent attachment to a specific cysteine. In the kinase field, this approach is demonstrating promise in overcoming the potency, selectivity, and efficacy challenges currently faced by reversible kinase inhibitors, with several advancing into late stage clinical testing. This design paradigm has been successfully applied to making drug candidates for epidermal growth factor receptor (EGFR), Her2, and Bruton's tyrosine kinase (Btk). Here we review recent pre-clinical and clinical advances with targeted covalent kinase inhibitors, and the potential for broader application of the approach.

Antidiabetic drug voglibose is protective against ischemia-reperfusion injury through glucagon-like peptide 1 receptors and the phosphoinositide 3-kinase-Akt-endothelial nitric oxide synthase pathway in rabbits.

Glucagon-like peptide 1 (GLP-1) reportedly exerts a protective effect against cardiac ischemia. We hypothesized that the alpha-glucosidase inhibitor voglibose, an unabsorbable antidiabetic drug with cardioprotective effects, may act through stimulation of GLP-1 receptors. The results of the present study suggest oral administration of voglibose reduces myocardial infarct size and mitigates cardiac dysfunction in rabbits after 30 minutes of coronary occlusion and 48 hours of reperfusion. Voglibose increased basal and postprandial plasma GLP-1 levels and reduced postprandial plasma glucose levels. The infarct size-reducing effect of voglibose was abolished by treatment with exendin(9-39), wortmannin, Nomega-nitro-L-arginine methylester, or 5-hydroxydecanoate), which inhibit GLP-1 receptors, phosphoinositide 3-kinase, nitric oxide synthase, and K(ATP) channels, respectively. Western blot analysis showed that treatment with voglibose upregulated myocardial levels of phospho-Akt, phosphoendothelial nitric oxide synthase after myocardial infarction. The upregulation of phospho-Akt was inhibited by exendin(9-39) and wortmannin. These findings suggest that voglibose reduces myocardial infarct size through stimulation of GLP-1 receptors, activation of the phosphoinositide 3-kinase-Akt-endothelial nitric oxide synthase pathways, and the opening of mitochondrial K(ATP) channels. These findings may provide new insight into therapeutic strategies for the treatment of patients with coronary artery disease.

Overexpression of human beta-defensin-3 in oral dysplasia: potential role in macrophage trafficking.

Human beta-defensins (hBDs) are small, cationic antimicrobial peptides produced by oral and other mucosal epithelia. More recently, hBDs have been shown to regulate adaptive immunity. In this study, we provide new information about the potential role of hBD-3 in the progression of oral cancer. In normal human oral epithelia, hBD-3 is produced by mitotically active cells in the basal layers of oral epithelium, whereas hBD-1 and -2 are coexpressed in the differentiated spinosum and granulosum layers. Interestingly, premalignant cells in carcinoma in situ lesions overexpress hBD-3, but not hBD-1 and hBD-2, correlating with specific recruitment and infiltration of macrophages. Our in vitro studies demonstrate that hBD-3 chemoattracts THP-1 monocytic cells and that epidermal growth factor (EGF) significantly induces hBD-3 expression in oral epithelial cells via mitogen-activated protein kinase (MAPK) kinase MEK1/2, p38 MAPK, protein kinase C (PKC), and phosphoinositide 3 kinase (PI3K), but not via Janus kinase (JAK) and signal transducer and activator of transcription (STATs). These results suggest that hBD-3 serves as a mitogen responsive gene in the initiation of oral cancer and may act as a motility signal to recruit tumor-associated macrophages.

Probing the human kinome for kinases involved in pancreatic cancer cell survival and gemcitabine resistance.

Except for gemcitabine, chemotherapeutic agents are ineffective with pancreatic adenocarcinoma because this cancer is resistant to apoptosis induction. Involvement of specific kinases in such resistance is likely. We developed a systematic strategy to screen the human kinome and select kinases whose inhibition in pancreatic cancer cells can increase 1) spontaneous apoptosis or 2) gemcitabine-induced apoptosis. The pancreatic adenocarcinoma cell line MiaPaCa-2 was transfected with 645 pairs of siRNAs directed to all human kinases. The same experiment was conducted in cells treated with 150 microM gemcitabine. Apoptosis was measured after 2 days and the results were normalized for cell viability. A panel of 56 kinases whose inhibition increased spontaneous apoptosis by at least 50% was established. Ten of them gave similar results on Panc1 and BxPC3 pancreatic adenocarcinoma cell lines. A panel of 83 kinases whose inhibition increased gemcitabine-induced apoptosis by 50% or more was also established. Twelve kinases appeared in both panels. A cumulative increase in apoptosis was observed when inhibiting simultaneously several kinases. Such a systematic approach allowed characterization of all kinases involved in pancreatic cancer cell survival and resistance to gemcitabine. Inhibitors of these kinases, used alone or in combination, might improve the treatment of pancreatic adenocarcinoma.

Apoptosis pathway-targeted drugs--from the bench to the clinic.

It is an exciting time for cancer researchers in the field of apoptotic cell death. The avalanche of discoveries over the past decade or so regarding how apoptosis is regulated begins to be exploited for therapeutic benefit as the first apoptosis-targeted drugs enter early clinical trials. This chapter provides a selective review on the development of such drugs. We also outline issues regarding the regulation and design of early clinical trials of this type of molecularly targeted agent. Finally, we discuss the biomarkers and surrogate pharmacodynamic endpoint assays currently available to chart the efficacy of apoptosis-inducing anticancer therapy.

Biology of gastrointestinal stromal tumors.

Once a poorly defined pathologic oddity, in recent years, gastrointestinal stromal tumor (GIST) has emerged as a distinct oncogenetic entity that is now center stage in clinical trials of kinase-targeted therapies. This review charts the rapid progress that has established GIST as a model for understanding the role of oncogenic kinase mutations in human tumorigenesis. Approximately 80% to 85% of GISTs harbor activating mutations of the KIT tyrosine kinase. In a series of 322 GISTs (including 140 previously published cases) studied by the authors in detail, mutations in the KIT gene occurred with decreasing frequency in exons 11 (66.1%), 9 (13%), 13 (1.2%), and 17 (0.6%). In the same series, a subset of tumors had mutations in the KIT-related kinase gene PDGF receptor alpha (PDGFRA), which occurred in either exon 18 (5.6%) or 12 (1.5%). The remainder of GISTs (12%) were wild type for both KIT and PDGFRA. Comparative studies of KIT-mutant, PDGFRA-mutant, and wild-type GISTs indicate that there are many similarities between these groups of tumors but also important differences. In particular, the responsiveness of GISTs to treatment with the kinase inhibitor imatinib varies substantially depending on the exonic location of the KIT or PDGFRA mutation. Given these differences, which have implications both for the diagnosis and treatment of GISTs, we propose a molecular-based classification of GIST. Recent studies of familial GIST, pediatric GIST, and variant forms of GIST related to Carney's triad and neurofibromatosis type 1 are discussed in relationship to this molecular classification. In addition, the role of mutation screening in KIT and PDGFRA as a diagnostic and prognostic aid is emphasized in this review.

Switching on kinases: oncogenic activation of BRAF and the PDGFR family.

The cytoplasmic serine/threonine kinase BRAF and receptor tyrosine kinases of the platelet-derived growth factor receptor (PDGFR) family are frequently activated in cancer by mutations of an equivalent amino acid. Structural studies have provided important insights into why these very different kinases share similar oncogenic hot spots and why the PDGFR juxtamembrane region is also a frequent oncogenic target. This research has implications for other kinases that are mutated in human tumours and for the treatment of cancer using kinase inhibitors.

[Major histocompatibility complex (MHC) class II: are lipid rafts the missing link?]. / Complexe majeur d'histocompatibilité de classe II : diversité fonctionnelle.

Aside from their crucial roles in the presentation of nominal antigen to CD4+ T cells and susceptibility to autoimmune diseases, substantial evidences suggest that MHC class II molecules act as signal transducer receptors as well. The signals transmitted affect diverse biological functions. Paradoxically, the cytoplasmic and transmembrane domains of these molecules are devoid of classic signaling motifs. The recent discovery of the presence of membrane microdomains, also called lipid rafts, that are enriched in kinases and adaptor molecules, may contribute to the elucidation of the mechanisms by which MHC class II molecules transmit their signals.

Activation of the cAMP-dependent protein kinase signaling pathway by luteinizing hormone in trout theca layers.

In the fish ovary, LH is the main factor regulating the production of steroids during the periovulatory period and its effects are believed to be mediated, at least partially, through the cAMP-dependent protein kinase (PKA) signaling pathway. However, there is no direct evidence for the presence of PKA in the fish ovary nor on the regulation of its activity by fish LH. Here, we show the identification of regulatory (R) and catalytic (C) subunits of PKA in trout theca cells by immunoblotting. DEAE-cellulose chromatography of theca cell extracts indicated the presence of PKA type I and II and showed that trout theca cells display PKA-specific phosphotransferase and cAMP-binding activities. Salmon LH (sLH) stimulated PKA activity and increased the levels of immunoreactive RIIalpha, RIIbeta and C subunits in trout theca layers. These observations, coupled with the sLH-dependent decrease in the half-life of the C subunit, as shown by pulse-chase experiments, strongly suggest that sLH activates PKA in trout theca cells. Furthermore, our results suggest that ovarian PKA activity and its regulation by LH has been well conserved from fish to humans.

Kinases, intracellular calcium, and apolipophorin-III influence the adhesion of larval hemocytes of the lepidopterous insect, Galleria mellonella.

Based on the results from the use of selective inhibitors and activators, active protein kinase A, protein tyrosine kinase, and protein kinase C (PKC) isoforms decreased the adhesion of larval Galleria mellonella hemocytes to glass slides. The protein kinase A inhibitor at all concentrations increased granular cell adhesion only whereas protein tyrosine kinase elevated both granular and plasmatocyte attachment at the lowest concentration. Active, Ca(2+)- and lipid-dependent PKC isoforms limited plasmatocyte and granular cell adhesion whereas PKC that was inhibited by selected compounds (with differed modes of PKC inhibition) enhanced hemocyte attachment. The granular cells were more sensitive to the PKC inhibitors than were plasmatocytes. Phospholipase C and its diacylglyceride product were necessary to reduce hemocyte adhesion and maintain PKC activity. Extracellular Ca(2+), possibly transported through L-channels, was required for plasmatocyte attachment. In contrast, lowering the levels of cytosolic Ca(2+) was associated with decreased PKC activity and was required for hemocyte adhesion.

Fanconi anemia complementation group A cells are hypersensitive to chromium(VI)-induced toxicity.

Fanconi anemia (FA) is an autosomal recessive disorder characterized by diverse developmental abnormalities, progressive bone marrow failure, and a markedly increased incidence of malignancy. FA cells are hypersensitive to DNA cross-linking agents, suggesting a general defect in the repair of DNA cross-links. Some forms of hexavalent chromium [Cr(VI)] are implicated as respiratory carcinogens and induce several types of DNA lesions, including ternary DNA-Cr-DNA interstrand cross-links (Cr-DDC). We hypothesized that human FA complementation group A (FA-A) cells would be hypersensitive to Cr(VI) and Cr(VI)-induced apoptosis. Using phosphatidylserine translocation and caspase-3 activation, human FA-A fibroblasts were found to be markedly hypersensitive to chromium-induced apoptosis compared with CRL-1634 cells, which are normal human foreskin fibroblasts (CRL). The clonogenicity of FA-A cells was also significantly decreased compared with CRL cells after Cr(VI) treatment. There was no significant difference in either Cr(VI) uptake or Cr-DNA adduct formation between FA-A and CRL cells. These results show that FA-A cells are hypersensitive to Cr(VI) and Cr-induced apoptosis and that this hypersensitivity is not due to increased Cr(VI) uptake or increased Cr-DNA adduct formation. The results also suggest that Cr-DDC may be proapoptotic lesions. These results are the first to show that FA cells are hypersensitive to an environmentally relevant DNA cross-linking agent.

Signaling from toxic metals to NF-kappaB and beyond: not just a matter of reactive oxygen species.

The nuclear factor kappa B (NF-kappaB) family of transcription factors controls expression of a number of early response genes associated with inflammatory responses, cell growth, cell cycle progression, and neoplastic transformation. These genes include a multitude of cytokines, chemokines, adhesion molecules, immune receptors, stress proteins, apoptotic or anti-apoptotic regulators, and several oncogenes. Accumulating evidence indicates that a variety of toxic metals are able to affect the activation or activity of NF-kappaB, but the molecular mechanisms involved in this process remain largely unknown. The signaling pathways mediating cytokine- or microorganism-induced NF-kappaB activation have been well established recently. Whether the same signaling systems are involved in metal-induced NF-kappaB activation, however, is unclear. In the present review, we have attempted to evaluate and update the possible mechanisms of metal signals on the activation and function of NF-kappaB.

Do phosphatidylinositides modulate vertebrate phototransduction?

Mammalian rod cyclic nucleotide gated (CNG) channels (i.e., alpha plus beta subunits) are strongly inhibited by phosphatidylinositol 4, 5-bisphosphate (PIP(2)) when they are expressed in Xenopus oocytes and studied in giant membrane patches. Cytoplasmic Mg-ATP inhibits CNG currents similarly, and monoclonal antibodies to PIP(2) reverse the effect and hyperactivate currents. When alpha subunits are expressed alone, PIP(2) inhibition is less strong; olfactory CNG channels are not inhibited. In giant patches from rod outer segments, inhibition by PIP(2) is intermediate. Other anionic lipids (e.g., phosphatidyl serine and phosphatidic acid), a phosphatidylinositol-specific phospholipase C, and full-length diacylglycerol have stimulatory effects. Although ATP also potently inhibits cGMP-activated currents in rod patches, the following findings indicate that ATP is used to transphosphorylate GMP, generated from cGMP, to GTP. First, a phosphodiesterase (PDE) inhibitor, Zaprinast, blocks inhibition by ATP. Second, inhibition can be rapidly reversed by exogenous regulator of G-protein signaling 9, suggesting G-protein activation by ATP. Third, the reversal of ATP effects is greatly slowed when cyclic inosine 5'-monophosphate is used to activate currents, as expected for slow inosine 5' triphosphate hydrolysis by G-proteins. Still, other results remain suggestive of regulatory roles for PIP(2). First, the cGMP concentration producing half-maximal CNG channel activity (K(1/2)) is decreased by PIP(2) antibody in the presence of PDE inhibitors. Second, the activation of PDE activity by several nucleotides, monitored electrophysiologically and biochemically, is reversed by PIP(2) antibody. Third, exogenous PIP(2) can enhance PDE activation by nucleotides.

The plasma membrane calcium pump, its role and regulation: new complexities and possibilities.

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2(+)-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2(+)-ATPase controls resting levels of [Ca2+]CYT has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2(+)-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors.

The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate: glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0.3 mM glucose. Likewise, addition of > 0.3 mM glucose, but not < 0.3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C] glucose assimilation in bacteria passing through the 0.5 to 0.3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)Glc PTS is 96-97% saturated at 0.3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0.3 mM, a second shift occurred below 1 muM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.