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.
A series of phosphonamide-based hydroxamate derivatives were synthesized, and the inhibitory activities were evaluated against various metalloproteinases in order to clarify its selectivity profile. Among the four diastereomeric isomers resulting from the chirality at the C-3 and P atoms, the compound with a (R,R)-configuration both at the C-3 position and the phosphorus atom was found to be potently active, while the other diastereomeric isomers were almost inactive. A number of (R,R)-compounds synthesized here exhibited broad spectrum activities with nanomolar K(i) values against MMP-1, -3, -9, and TACE and also showed nanomolar IC(50) values against HB-EGF shedding in a cell-based inhibition assay. The modeling study using X-ray structure of MMP-3 suggested the possible binding mode of the phosphonamide-based inhibitors.
Angiogenic homeostasis is maintained by a balance between vascular endothelial growth factor (VEGF) and Notch signaling in endothelial cells (ECs). We screened for molecules that might mediate the coupling of VEGF signal transduction with down-regulation of Notch signaling, and identified B-cell chronic lymphocytic leukemia/lymphoma6-associated zinc finger protein (BAZF). BAZF was induced by VEGF-A in ECs to bind to the Notch signaling factor C-promoter binding factor 1 (CBF1), and to promote the degradation of CBF1 through polyubiquitination in a CBF1-cullin3 (CUL3) E3 ligase complex. BAZF disruption in vivo decreased endothelial tip cell number and filopodia protrusion, and markedly abrogated vascular plexus formation in the mouse retina, overlapping the retinal phenotype seen in response to Notch activation. Further, impaired angiogenesis and capillary remodeling were observed in skin-wounded BAZF−/− mice. We therefore propose that BAZF supports angiogenic sprouting via BAZF-CUL3-based polyubiquitination-dependent degradation of CBF1 to down-regulate Notch signaling.
A disintegrin and metalloprotease 12 (ADAM12/meltrin ␣) is a key enzyme implicated in the ectodomain shedding of membrane-anchored heparin-binding epidermal growth factor (EGF)-like growth factor (proHB-EGF)-dependent epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 are obscure. To determine how ADAM12 is activated, we screened proteins that bind to the cytoplasmic domain of ADAM12 using a yeast twohybrid system and identified a protein called PACSIN3 that contains a Src homology 3 domain. An analysis of interactions between ADAM12 and PACSIN3 using glutathione S-transferase fusion protein revealed that a proline-rich region (amino acid residues 829 -840) of ADAM12 was required to bind PACSIN3. Furthermore, co-immunoprecipitation and co-localization analyses of ADAM12 and PACSIN3 proteins also revealed their interaction in mammalian cells expressing both of them. The overexpression of PACSIN3 in HT1080 cells enhanced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced proHB-EGF shedding. Furthermore, knockdown of endogenous PACSIN3 by small interfering RNA in HT1080 cells significantly attenuated the shedding of proHB-EGF induced by TPA and angiotensin II. Our data indicate that PACSIN3 has a novel function as an up-regulator in the signaling of proHB-EGF shedding induced by TPA and angiotensin II. The transactivation of epidermal growth factor receptor (EGFR)1 by G-protein-coupled receptor (GPCR) agonists is a critical element in various responses of diverse cell types including fibroblasts, keratinocytes, astrocytes, and smooth muscle cells (1, 2). A novel mechanistic concept of the EGFR transactivation-signaling pathway involves the proteolytic release of heparin-binding EGF-like growth factor (HB-EGF) at the surface of cells stimulated with GPCR agonists (3). HB-EGF is a member of the EGF family that directly binds EGFR and thereby enhances its phosphorylation, resulting in cell growth and differentiation (4). Like other members of the EGF family, HB-EGF is synthesized as a membrane-anchored form (proHB-EGF) and then proteolytically processed to become a bioactive soluble form, a process that is called ectodomain shedding. The ectodomain shedding of proHB-EGF is an important posttranslational modification that converts a tethered insoluble juxtacrine growth factor into a soluble ligand leading to the autocrine or paracrine activation of EGFR.Studies of GPCR mitogenic signaling have proven that EGFR transactivation is dependent on HB-EGF in smooth muscle cells (2), cardiac endothelial cells (5), and cardiomyocytes (6), as well as in various pathological processes such as cardiac hypertrophy (6), chronic active gastritis associated with Helicobacter pylori (7), and cystic fibrosis (8).Growing evidence points to a disintegrin and metalloproteases (ADAMs) as key enzymes of proHB-EGF shedding in EGFR transactivation signaling. All ADAMs have an extracellular portion with a metalloprotease domain, a transmembrane region and a cytoplasmic tail, and several ADAMs h...
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