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An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Actin has an ill-defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1-overexpressing mice, insulin-stimulated glucose uptake was increased; while Tpm3.1-null mice they were more sensitive to the impact of high-fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3-L1 adipocytes abrogates insulin-stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.
© 2015 The Authors. Traffic published by John Wiley &amp; Sons Ltd.

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Significant Differences in the Development of Acquired Resistance to the MDM2 Inhibitor SAR405838 between In Vitro and In Vivo Drug Treatment.

SAR405838 is a potent and specific MDM2 inhibitor currently being evaluated in Phase I clinical trials for the treatment of human cancer. Using the SJSA-1 osteosarcoma cell line which harbors an amplified MDM2 gene and wild-type p53, we have investigated the acquired resistance mechanisms both in vitro and in vivo to SAR405838. Treatment of SJSA-1 cells with SAR405838 in vitro leads to dose-dependent cell growth inhibition, cell cycle arrest and robust apoptosis. However, prolonged treatment of SJSA-1 cells in vitro with SAR405838 results in profound acquired resistance to the drug. Analysis of in vitro-derived resistant cell lines showed that p53 is mutated in the DNA binding domain and can no longer be activated by SAR405838. Treatment of the parental SJSA-1 xenograft tumors with SAR405838 in mice yields rapid tumor regression but the tumors eventually regrow. Culturing the regrown tumors established a number of sublines, which showed only modest (3-5 times) loss of sensitivity to SAR405838 in vitro. Sequencing of the p53 showed that it retains its wild-type status in these in vivo sublines, with the exception of one subline, which harbors a single heterozygous C176F p53 mutation. Using xenograft models of two in vivo derived sublines, which has either wild-type p53 or p53 containing a single heterozygous C176F mutation, we showed that while SAR405838 effectively achieves partial tumor regression in these models, it no longer induces complete tumor regression and tumors resume growth once the treatment is stopped. Harvesting and culturing tumors obtained from a prolonged treatment with SAR405838 in mice established additional in vivo sublines, which all contain a single heterozygous C176F mutation with no additional p53 mutation detected. Interestingly, SAR405838 can still effectively activate p53 in all sublines containing a single heterozygous C176F mutation, with a moderately reduced potency as compared to that in the parental cell line. Consistently, SAR405838 is 3-5 times less effective in all the in vivo derived sublines containing a single heterozygous C176F p53 mutation than in the SJSA-1 parental cell line in assays of cell growth and apoptosis. Computational modeling suggested that a p53 tetramer containing two wild-type p53 molecules and two C176F mutated molecules can maintain the structural stability and interactions with DNA by formation of additional hydrophobic and cation-π interactions which compensate for the loss of sulphur-zinc coordination. Our data thus show that SJSA-1 tumor cells acquire very different levels of resistance in vitro and in vivo to the MDM2 inhibitor SAR405838. Our present study may have a significant implication for the investigation of resistant mechanisms for other classes of anticancer drugs.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor which has been implicated in numerous solid and hematologic cancers. ALK mutations are reported in about 5-7% of neuroblastoma cases but the ALK-positive percentage increases significantly in the relapsed patient population. Crizotinib, the first clinically approved ALK inhibitor for the treatment of ALK-positive lung cancer has had less dramatic responses in neuroblastoma. Here we investigate the efficacy of a second-generation ALK inhibitor, brigatinib, in a neuroblastoma setting. Employing neuroblastoma cell lines, mouse xenograft and Drosophila melanogaster model systems expressing different constitutively active ALK variants, we show clear and efficient inhibition of ALK activity by brigatinib. Similar abrogation of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. These results suggest that brigatinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma that should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop.

Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, ∼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.
Copyright © 2016 Elsevier Ltd. All rights reserved.

Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis.

Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been approved for cancer treatment. However, VEGF inhibitors alone were shown to promote tumor invasion and metastasis by increasing intratumoral hypoxia in some preclinical and clinical studies. Emerging reports suggest that Delta-like ligand 4 (Dll4) is a promising target of angiogenesis inhibition to augment the effects of VEGF inhibitors. To evaluate the effects of simultaneous blockade against VEGF and Dll4, we developed a bispecific antibody, HD105, targeting VEGF and Dll4. The HD105 bispecific antibody, which is composed of an anti-VEGF antibody (bevacizumab-similar) backbone C-terminally linked with a Dll4-targeting single-chain variable fragment, showed potent binding affinities against VEGF (KD: 1.3 nM) and Dll4 (KD: 30 nM). In addition, the HD105 bispecific antibody competitively inhibited the binding of ligands to their receptors, i.e., VEGF to VEGFR2 (EC50: 2.84 ± 0.41 nM) and Dll4 to Notch1 (EC50: 1.14 ± 0.06 nM). Using in vitro cell-based assays, we found that HD105 effectively blocked both the VEGF/VEGFR2 and Dll4/Notch1 signaling pathways in endothelial cells, resulting in a conspicuous inhibition of endothelial cell proliferation and sprouting. HD105 also suppressed Dll4-induced Notch1-dependent activation of the luciferase gene. In vivo xenograft studies demonstrated that HD105 more efficiently inhibited the tumor progression of human A549 lung and SCH gastric cancers than an anti-VEGF antibody or anti-Dll4 antibody alone. In conclusion, HD105 may be a novel therapeutic bispecific antibody for cancer treatment.

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Category: Uncategorized

An actin filament population defined by the tropomyosin Tpm3.1 regulates glucose uptake.

Significant Differences in the Development of Acquired Resistance to the MDM2 Inhibitor SAR405838 between In Vitro and In Vivo Drug Treatment.

Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice.

Outcome of the First wwPDB/CCDC/D3R Ligand Validation Workshop.

Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis.