17 research outputs found
Surfing the Protein-Protein Interaction Surface Using Docking Methods: Application to the Design of PPI Inhibitors
PI3K/AKT signaling modulates transcriptional expression of EWS/FLI1 through specificity protein 1
Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition
Thienoquinolines as Novel Disruptors of the PKC epsilon/RACK2 Protein-Protein Interaction
Ten protein kinase C (PKC) isozymes play divergent roles in signal transduction. Because of sequence similarities, it is particularly difficult to generate isozyme-selective small molecule inhibitors. In order to identify such a selective binder, we derived a pharmacophore model from the peptide EAVSLKPT, a fragment of PKCĪµ that inhibits the interaction of PKCĪµ and receptor for activated C-kinase 2 (RACK2). A database of 330ā000 molecules was screened in silico, leading to the discovery of a series of thienoquinolines that disrupt the interaction of PKCĪµ with RACK2 in vitro. The most active molecule, N-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxino[2,3-g]thieno[2,3-b]quinoline-8-carboxamide (8), inhibited this interaction with a measured IC50 of 5.9 Ī¼M and the phosphorylation of downstream target Elk-1 in HeLa cells with an IC50 of 11.2 Ī¼M. Compound 8 interfered with MARCKS phosphorylation and TPA-induced translocation of PKCĪµ (but not that of PKCĪ“) from the cytosol to the membrane. The compound reduced the migration of HeLa cells into a gap, reduced invasion through a reconstituted basement membrane matrix, and inhibited angiogenesis in a chicken egg assay
3D Pharmacophore Modeling Techniques in Computer-Aided Molecular Design Using LigandScout
This chapter reviews theory related to pharmacophore models and guide the user through six essential workflows using LigandScout: structureābased pharmacophore modeling, ligandābased pharmacophore modeling, creating sharedāfeature pharmacophore models, accurate virtual screening and pharmacophore editing in the active site, Hit analysis and parallel virtual screening. It includes some tutorials involving the creation of 3Dāchemical feature pharmacophore models and the use of these models to find biologically active molecules using virtual screening methods. Pharmacophore modeling together with virtual screening have become increasingly popular in the last decades and matured to a valuable and efficient basis for a wide variety of computerāaided drug design projects. The most common representation of pharmacophores is a spatial arrangement of soācalled chemical (or pharmacophoric) features that describe essential structural elements and/or observed ligandāreceptor interactions by means of geometric entities. The chapter provides an overview of the methodological details and applicability of the most commonly used approaches for creating pharmacophore models
Thienoquinolines as Novel Disruptors of the PKCĪµ/RACK2 ProteināProtein Interaction
Ten
protein kinase C (PKC) isozymes play divergent roles in signal transduction.
Because of sequence similarities, it is particularly difficult to
generate isozyme-selective small molecule inhibitors. In order to
identify such a selective binder, we derived a pharmacophore model
from the peptide EAVSLKPT, a fragment of PKCĪµ that inhibits
the interaction of PKCĪµ and receptor for activated C-kinase
2 (RACK2). A database of 330ā000 molecules was screened in
silico, leading to the discovery of a series of thienoquinolines that
disrupt the interaction of PKCĪµ with RACK2 in vitro. The most
active molecule, <i>N</i>-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxinoĀ[2,3-<i>g</i>]ĀthienoĀ[2,3-<i>b</i>]Āquinoline-8-carboxamide
(<b>8</b>), inhibited this interaction with a measured IC<sub>50</sub> of 5.9 Ī¼M and the phosphorylation of downstream target
Elk-1 in HeLa cells with an IC<sub>50</sub> of 11.2 Ī¼M. Compound <b>8</b> interfered with MARCKS phosphorylation and TPA-induced translocation
of PKCĪµ (but not that of PKCĪ“) from the cytosol to the
membrane. The compound reduced the migration of HeLa cells into a
gap, reduced invasion through a reconstituted basement membrane matrix,
and inhibited angiogenesis in a chicken egg assay
Thienoquinolines as Novel Disruptors of the PKCĪµ/RACK2 ProteināProtein Interaction
International audienceTen protein kinase C (PKC) isozymes play divergent roles in signal transduction. Because of sequence similarities, it is particularly difficult to generate isozyme-selective small molecule inhibitors. In order to identify such a selective binder, we derived a pharmacophore model from the peptide EAVSLKPT, a fragment of PKCĪµ that inhibits the interaction of PKCĪµ and receptor for activated C-kinase 2 (RACK2). A database of 330ā000 molecules was screened in silico, leading to the discovery of a series of thienoquinolines that disrupt the interaction of PKCĪµ with RACK2 in vitro. The most active molecule, N-(3-acetylphenyl)-9-amino-2,3-dihydro-1,4-dioxino[2,3-g]thieno[2,3-b]quinoline-8-carboxamide (8), inhibited this interaction with a measured IC50 of 5.9 Ī¼M and the phosphorylation of downstream target Elk-1 in HeLa cells with an IC50 of 11.2 Ī¼M. Compound 8 interfered with MARCKS phosphorylation and TPA-induced translocation of PKCĪµ (but not that of PKCĪ“) from the cytosol to the membrane. The compound reduced the migration of HeLa cells into a gap, reduced invasion through a reconstituted basement membrane matrix, and inhibited angiogenesis in a chicken egg assay