Activity-based kinase profiling of approved tyrosine kinase inhibitors.

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.

Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state.

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the γ-phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.

Characterization of kinase inhibitors using different phosphorylation states of colony stimulating factor-1 receptor tyrosine kinase.

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.

Diacylglycerol kinase ß knockout mice exhibit lithium-sensitive behavioral abnormalities.

BACKGROUND: Diacylglycerol kinase (DGK) is an enzyme that phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). DGKß is widely distributed in the central nervous system, such as the olfactory bulb, cerebral cortex, striatum, and hippocampus. Recent studies reported that the splice variant at the COOH-terminal of DGKß was related to bipolar disorder, but its detailed mechanism is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we performed behavioral tests using DGKß knockout (KO) mice to investigate the effects of DGKß deficits on psychomotor behavior. DGKß KO mice exhibited some behavioral abnormalities, such as hyperactivity, reduced anxiety, and reduced depression. Additionally, hyperactivity and reduced anxiety were attenuated by the administration of the mood stabilizer, lithium, but not haloperidol, diazepam, or imipramine. Moreover, DGKß KO mice showed impairment in Akt-glycogen synthesis kinase (GSK) 3ß signaling and cortical spine formation. CONCLUSIONS/SIGNIFICANCE: These findings suggest that DGKß KO mice exhibit lithium-sensitive behavioral abnormalities that are, at least in part, due to the impairment of Akt-GSK3ß signaling and cortical spine formation.

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state.

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.

Selection of inhibitory peptides for Aurora-A kinase from a phage-displayed library of helix-loop-helix peptides.

Conformationally constrained peptide libraries have been made by grafting randomized amino acid sequences onto a rigid scaffold derived from natural proteins. Here, as a library scaffold, we propose a de novo designed helix-loop-helix motif. We constructed a peptide library of the loop region and screened against Aurora-A, which is a member of the Aurora family of serine/threonine protein kinases, to successfully isolate the inhibitory peptides. A semi-rational strategy, which combines phage-displayed libraries and de novo designed peptides, would provide a new way to generate selective peptide inhibitors for the protein kinase family.

A silent mutation made possible efficient production of active human Frk tyrosine kinase in Escherichia coli.

Fyn-related kinase (Frk) was first identified using human breast cancer cells. It shares 51% identity with c-Src. Like all members of the Src family, Frk is thought to cause several cancers via dysregulations in signal transduction from cell-surface receptors. The excess activity of Frk on beta-cells has a crucial role in type-I diabetes. A silent mutation at Ile229 conferred a bacterial expression system on the kinase domains of Frk, which allowed for the quick expression and purification of one unphosphorylated and two mono-phosphorylated kinase domains. The C-terminal catalytic segment of the human Frk kinase conjugating hexahistidine purification tag (His-tag) was expressed in Escherichia coli. After first-step purification utilizing the His-tag, an anion-exchange chromatogram yielded three major peaks that had distinguishable phosphorylation characteristics as judged by Western blot analysis and measurement of kinase activity. This result of active protein production should promote drug discovery studies, including highthrough-put screening and structure-based drug design.

Structural basis for the inhibitor recognition of human Lyn kinase domain.

Human Lyn tyrosine kinase is expressed in hematopoietic tissues and plays crucial roles in the signal transduction of hematopoietic immune system. Its excess activity is involved in several tumors. The crystal structure has revealed that the potent inhibitor staurosporine binds to human Lyn kinase domain at the ATP-binding site. The remarkable structural features of the staurosporine-binding region will offer valuable structural insights for the structure-based design of novel Lyn-selective inhibitors.

CB-12181, a new azasugar-based matrix metalloproteinase/tumor necrosis factor-alpha converting enzyme inhibitor, inhibits vascular endothelial growth factor-induced angiogenesis in vitro and retinal neovascularization in vivo.

To evaluate the anti-angiogenic efficacy of CB-12181 [an azasugar derivative that has inhibitory actions against matrix metalloproteinases (MMPs) and tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE)], we investigated the suppressing ability on in vitro (tube formation by endothelial cells) and in vivo (retinal neovascularization on murine ischemia-induced proliferative retinopathy) models of angiogenesis. For in vitro analysis, a capillary-like tube formation model using human umbilical vein endothelial cells (HUVECs) and fibroblasts co-culture assay was employed. Tube formation of HUVECs was stimulated by vascular endothelial growth factor (VEGF) and incubated with different concentrations of CB-12181 (0.1-100 microM) for 11 days. For in vivo analysis, mice were exposed to 75% oxygen between postnatal days 7 and 12 (P7 to P12). Then, the mice were removed from the oxygen treatment and treated with CB-12181 (1, 15, or 50 mg/kg) by daily subcutaneous injection from the time of reintroduction to room air at P12 until P16. At P17, pathological and physiological angiogenesis was quantified using retinal flat-mounts visualized by fluorescent angiography. In the in vitro angiogenesis model, CB-12181 significantly suppressed VEGF-induced HUVEC tube formation. Furthermore, in the in vivo angiogenesis model, administration of CB-12181 significantly suppressed retinal neovascularization without any apparent side effects on physiological revascularization to the oxygen-induced obliteration area. These results suggest that CB-12181 might be useful in the treatment of various diseases that depend on pathologic angiogenesis, and especially valuable for the treatment of diabetic retinopathy and retinopathy of prematurity.

Crystal structure of human mono-phosphorylated ERK1 at Tyr204.

Extracellular signal-regulated kinase (ERK) is a member of the MAP kinase family, and can regulate several cellular responses. The isoforms ERK1 and ERK2 have markedly similar amino acid sequences, but exhibit distinctive physiological functions. As well as ERK2, ERK1 was auto- and mono-phosphorylated at Tyr204 in the activation loop during Escherichia coli production, resulting in basal level activity, approximately 500-fold less compared with fully-active ERK1 dual-phosphorylated at Thr202 and Tyr204. Crystal structure demonstrated that the mono-phosphorylated ERK1 kinase possessed a novel conformation distinguishable from the un-phosphorylated (inactive) and the dual-phosphorylated (full-active) forms. The characteristic structural features in both the C-helix and the activation loop likely contribute to the basal activity of the mono-phosphorylated ERK1. The structural dissection of ERK1 compared to ERK2 suggests that the structural differences in the D-motif binding site and in the backside binding site are putative targets for development of selective ERK1/ERK2 inhibitors.

Protein purification and preliminary crystallographic analysis of human Lyn tyrosine kinase.

Lyn is a member of the Src family of non-receptor protein kinase. As well as all members of the Src family, Lyn is thought to participate in signal transduction from cell surface receptors. The crystal structure of Lyn would have a better understanding of Lyn function in various cells. For the purpose of crystallization, C-terminal catalytic segment of human Lyn kinase conjugating hexahistidine purification tag (His-tag) was expressed in Sf21 insect cells. After first step purification utilizing His-tag, an anion-exchange chromatogram yielded four major peaks which had distinguishable phosphorylation manner as judged by Western blot analysis, Native-PAGE analysis and kinase activity measurements. The fractioned samples were separately examined for crystallization screening using a commercial available screening kit. The mono-phosphorylated protein was crystallized with a small rod-shaped and needle clusters. The higher phosphorylated samples corresponding to the other three fractions on the anion-exchange chromatogram were aggregated or precipitated under the above conditions. A crystal of the mono-phosphorylated sample was diffracted to 3.2A with synchrotron source at Photon Factory and a complete X-ray diffraction data set was collected. The coarse structure was solved by a molecular replacement method and further structural refinement is currently underway.

A unique monoclonal antibody 29A stains the cytoplasm of amniotic epithelia and cutaneous basement membrane.

BACKGROUND: The basic function of epithelia is to provide a boundary between tissue and its external environment, and is achieved by a wide variety of components including extracellular molecules. Multiple monoclonal antibodies raised against epithelial antigens have helped identify a range of distinct, novel protein epitopes. OBJECT: In this study, we raised a monoclonal antibody to detect a novel epithelial molecular component. METHODS: We have produced a mouse monoclonal antibody using normal human amniotic tissue as an immunogen. The monoclonal antibody was subsequently immunohistochemically screened, and the target antigen was cloned using an immunoscreening method. RESULT: In the course of the screening, we identified unique antibody staining patterns within the cytoplasm of a subset of amniotic cells at intervals within the normal placental epithelia. By immunoscreening, we identified this candidate gene as laminin receptor (LR). By dot blot analysis, this antibody reacted with recombinant LR. The same localization of the antigen and LR was proved by a double staining immunofluorescence test in the placenta. This monoclonal antibody unexpectedly demonstrated linear staining within the dermal-epidermal junction of normal human skin but failed to react within the keratinocyte cytoplasm. CONCLUSION: We have produced and characterized a novel monoclonal antibody 29A that recognizes an LR-related molecule, which demonstrated a unique staining pattern. This monoclonal antibody might be a useful tool for further investigations into the epithelial tissues and the cutaneous basement membrane (BM).

Heparin-binding EGF-like growth factor accelerates keratinocyte migration and skin wound healing.

Members of the epidermal growth factor (EGF) family are the most important growth factors involved in epithelialization during cutaneous wound healing. Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is thought to play an important role in skin wound healing. To investigate the in vivo function of HB-EGF in skin wound healing, we generated keratinocyte-specific HB-EGF-deficient mice using Cre/loxP technology in combination with the keratin 5 promoter. Studies of wound healing revealed that wound closure was markedly impaired in keratinocyte-specific HB-EGF-deficient mice. HB-EGF mRNA was upregulated at the migrating epidermal edge, although cell growth was not altered. Of the members of the EGF family, HB-EGF mRNA expression was induced the most rapidly and dramatically as a result of scraping in vitro. Combined, these findings clearly demonstrate, for the first time, that HB-EGF is the predominant growth factor involved in epithelialization in skin wound healing in vivo and that it functions by accelerating keratinocyte migration, rather than proliferation.

Tyrosinase gene analysis in Japanese patients with oculocutaneous albinism.

BACKGROUND: Oculocutaneous albinism (OCA) is a heterogeneous congenital disorder. Tyrosinase is a key enzyme in melanin biosynthesis, and tyrosinase gene mutations cause the OCA1 subtype. OBJECTIVE: This study was intended evaluate the frequency and details of tyrosinase gene mutations in Japanese OCA patients. PATIENTS AND METHODS: We examined nine non-consanguineous OCA families, sequenced the tyrosinase gene of the patients and also confirmed a splicing site mutation using exon trapping system. RESULTS: Tyrosinase gene mutations were identified in five out of nine OCA families (55%). IVS2-10deltt-7t-a was present in 3 out of 18 alleles in three families (16%), P310insC was present in three alleles in three families (16%) and R278X was found in three alleles (16%), including those in one heterozygous and one compound homozygous patient. G97V (290 G-T) was found in 1 out of 18 alleles, and we could not find G97V in the mutation database. We have added this mutation as 9th mutation of Japanese OCA1 patients. In 8 of 18 alleles, four families, no tyrosinase mutations were identified. They were presumed not to be OCA1, but other subtypes of OCA. Exon trapping system demonstrated IVS2-10deltt-7t-a mutation generated the abnormal splicing site, and inserted the codon 4 bases in mRNA level resulting in premature termination codon downstream. CONCLUSION: This study provided new information about OCA1 mutations, and highlights the requirement of broader detailed search to make precise diagnosis of OCA.

Limited role for interleukin-18 in the host protection response to pulmonary infection with Pseudomonas aeruginosa in mice.

We report that clearance of Pseudomonas aeruginosa, accumulation of neutrophils, and synthesis of tumor necrosis factor alpha and macrophage inflammatory protein 2 in the infected lung were not largely different in interleukin-18 (IL-18) knockout or transgenic mice compared with control mice. Our results suggest a limited role for IL-18 in the host defense against P. aeruginosa.

Exacerbated and prolonged allergic and non-allergic inflammatory cutaneous reaction in mice with targeted interleukin-18 expression in the skin.

Interleukin 18 induces both T helper 1 and T helper 2 cytokines, proinflammatory cytokines, chemokines, and IgE and IgG1 production. A role of interleukin 18 in inflammatory cutaneous reactions is still unclear, however. Here we generated keratin 5/interleukin 18 transgenic mice overexpressing mature murine interleukin 18 in the skin using a human keratin 5 promoter. In the contact hypersensitivity model, trinitrochlorobenzene elicited a stronger ear swelling in keratin 5/interleukin 18 transgenic mice compared with control littermate wild-type or immunoglobulin/interleukin 18 transgenic mice in which mature interleukin 18 was expressed by B and T cells under the control of the immunoglobulin promoter. Application of an irritant, croton oil, induced stronger and more sustained ear swelling in keratin 5/interleukin 18 transgenic mice than in immunoglobulin/interleukin 18 transgenic or wild-type mice. Repetitive topical application (weekly for six consecutive weeks) of trinitrochlorobenzene to their ears also elicited a stronger cutaneous inflammation in keratin 5/interleukin 18 transgenic mice than seen in immunoglobulin/interleukin 18 transgenic or wild-type mice. After these six trinitrochlorobenzene applications, the expression of interferon-gamma, interleukin-4, and CCL20 mRNA in the ear tissue was increased and dermal changes, such as acanthosis and eosinophilic, neutrophilic, and mast cell infiltration, were greater in keratin 5/interleukin 18 transgenic mice than in wild-type mice. Furthermore, the repetitive application elicited a significant increase in serum IgE levels and the number of B cells in the draining lymph node in keratin 5/interleukin 18 transgenic mice. These results suggest that overexpression of interleukin 18 in the skin aggravates allergic and nonallergic cutaneous inflammation, which is accompanied by high expression of T helper 1 and T helper 2 cytokines and chemokines in the skin.