ABSTRACT
Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/ß-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/ß-catenin signaling within CSCs. Disruption of CBP-ß-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA-Binding Proteins/metabolism , Neoplastic Stem Cells/metabolism , Peptide Fragments/metabolism , RNA-Binding Proteins/metabolism , Sialoglycoproteins/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adult , Aged , Animals , Apoptosis/drug effects , Azabicyclo Compounds/pharmacology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Differentiation/drug effects , Cells, Cultured , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Mice , Mice, Inbred NOD , Middle Aged , Neoplastic Stem Cells/cytology , Neoplastic Stem Cells/transplantation , Organophosphates/pharmacology , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/genetics , Proto-Oncogene Proteins c-myc/metabolism , Pyrimidinones/pharmacology , RNA Interference , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/genetics , Sialoglycoproteins/antagonists & inhibitors , Sialoglycoproteins/genetics , Sumoylation/drug effects , Transcriptome/drug effects , Wnt Signaling Pathway/drug effects , beta Catenin/metabolismABSTRACT
Inherited and somatic mutations in the APC gene, a human tumor-suppressor, occur in a large percentage of colon cancers, leading to elevated levels of nuclear beta-Catenin, and to activation of TCF/beta-Catenin-responsive genes including cyclin D1 and c-myc. To identify small molecule antagonists of this pathway, we screened transformed colorectal cells with a secondary structure-templated chemical library, in search of compounds that attenuated a TCF/beta-Catenin-responsive reporter gene. From this library we selected ICG-001 (IC50=3 microM) as a lead compound. Design and synthesis of the chemical library and some preliminary biological evaluation is described.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Pyrimidinones/pharmacology , TCF Transcription Factors/antagonists & inhibitors , TCF Transcription Factors/metabolism , beta Catenin/antagonists & inhibitors , beta Catenin/metabolism , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Cell Line , Cells, Cultured , Drug Design , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Structure-Activity Relationship , TCF Transcription Factors/genetics , Transcription, Genetic/drug effects , beta Catenin/geneticsABSTRACT
Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.
Subject(s)
Antineoplastic Agents/metabolism , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cytoskeletal Proteins/metabolism , Gene Expression Regulation , Pyrimidinones/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Adenomatous Polyposis Coli/genetics , Adenomatous Polyposis Coli/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Apoptosis/physiology , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Cell Line , Colon/anatomy & histology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclic AMP Response Element-Binding Protein/genetics , Cyclin D1/genetics , Cyclin D1/metabolism , Cytoskeletal Proteins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Inhibitor of Apoptosis Proteins , Lymphoid Enhancer-Binding Factor 1 , Male , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Molecular Structure , Neoplasm Proteins , Pyrimidinones/chemistry , Pyrimidinones/therapeutic use , Signal Transduction/physiology , Survivin , Trans-Activators/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , beta CateninABSTRACT
Opioid analgesics provide outstanding benefits for relief of severe pain. The mechanisms of the analgesia accompanied with some side effects have been investigated by many scientists to shed light on the complex biological processes at the molecular level. New opioid drugs and therapies with more desirable properties can be developed on the bases of accurate insight of the opioid ligand-receptor interaction and clear knowledge of the pharmacological behavior of opioid receptors and the associated proteins. Toward this goal, recent advances in selective opioid receptor agonists and antagonists including opioid ligand-receptor interactions are summarized in this review article.
Subject(s)
Ligands , Morphine/chemistry , Narcotic Antagonists , Opioid Peptides/chemistry , Analgesics, Opioid/therapeutic use , Animals , Binding Sites , Forecasting , Humans , Morphine/pharmacology , Opioid Peptides/pharmacology , Receptors, Opioid/chemistry , Research , Sensitivity and Specificity , Structure-Activity RelationshipABSTRACT
There is increasing evidence that redox regulation of transcription, particularly activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), is important in inflammatory diseases. Human thioredoxin (TRX), a member of the oxidoreductase superfamily, was initially identified, as a factor which augments the production of interleukin-2 receptor alpha (IL-2R alpha) in human T-cell lymphotropic virus type 1 (HTLV-1) infected patient T-cells. Substrates for the redox activity of TRX bind the active site cleft in extended strand structure. The rapid generation of high numbers of peptide secondary structure mimetics through solid-phase synthesis is a key technology for the identification of pharmaceutical leads based on such protein-peptide interactions. In this manuscript, we describe a chemogenomic approach utilizing an extended strand templated library to develop small molecule inhibitors to validate oxidoreductase molecular targets in a murine asthma model.
Subject(s)
Molecular Mimicry , Peptides/chemistry , Peptides/pharmacology , Animals , Asthma/drug therapy , Combinatorial Chemistry Techniques , Enzyme Inhibitors , Humans , Mice , Peptide Library , Peptides/therapeutic use , Protein Structure, Secondary , Receptors, G-Protein-Coupled/agonists , Transcription, Genetic/drug effectsABSTRACT
Asthma is characterized by an oxidantantioxidant imbalance in the lungs leading to activation of redox-sensitive transcription factors, nuclear factor kappaB (NF-kappaB), and activator protein-1 (AP-1). To develop therapeutic strategies for asthma, we used a chemogenomics approach to screen for small molecule inhibitor(s) of AP-1 transcription. We developed a beta-strand mimetic template that acts as a reversible inhibitor (pseudosubstrate) of redox proteins. This template incorporates an enedione moiety to trap reactive cysteine nucleophiles in the active sites of redox proteins. Specificity for individual redox factors was achieved through variations in X and Y functionality by using a combinatorial library approach. A limited array (2 x 6) was constructed where X was either NHCH(3) or NHCH(2) Ph and Y was methyl, phenyl, m-cyanophenyl, m-nitrophenyl, m-acetylaniline, or m-methylbenzoate. These analogs were evaluated for their ability to inhibit transcription in transiently transfected human lung epithelial A549 cells from either an AP-1 or NF-kappaB reporter. A small-molecule inhibitor, PNRI-299, was identified that selectively inhibited AP-1 transcription (IC(50) of 20 microM) without affecting NF-kappaB transcription (up to 200 microM) or thioredoxin (up to 200 microM). The molecular target of PNRI-299 was determined to be the oxidoreductase, redox effector factor-1 by an affinity chromatography approach. The selective redox effector factor-1 inhibitor, PNRI-299, significantly reduced airway eosinophil infiltration, mucus hypersecretion, edema, and IL-4 levels in a mouse asthma model. These data validate AP-1 as an important therapeutic target in allergic airway inflammation.
Subject(s)
Asthma/drug therapy , Carbon-Oxygen Lyases/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase , Transcription Factor AP-1/metabolism , Animals , Binding Sites , Bronchoalveolar Lavage Fluid , Cell Nucleus/metabolism , Chromatography , Cysteine/chemistry , Cytosol/metabolism , Female , Genes, Reporter , Humans , Inhibitory Concentration 50 , Interleukin-4/metabolism , Lung/drug effects , Mice , Mice, Inbred BALB C , Models, Chemical , Models, Molecular , NF-kappa B/antagonists & inhibitors , Oxidation-Reduction , Peptide Library , Thioredoxins/antagonists & inhibitors , Tumor Cells, CulturedABSTRACT
The secondary structure peptidomimetic approach is a rational way to develop novel nonpeptide pharmaceutical agents based upon biologically significant proteinaceous leads. A part of this approach elaborated in this laboratory over the past ten years is reviewed along with the recent developments in this field.
Subject(s)
Drug Design , Molecular Mimicry , Peptides/chemical synthesis , Crystallography, X-Ray , Models, Molecular , Peptides/chemistry , Peptides/pharmacology , Protein Structure, SecondaryABSTRACT
We have identified a mu-selective opioid receptor agonist without a cationic amino group in the molecule from libraries of bicyclic beta-turn peptidomimetics. The biologically active conformation of the lead is proposed to mimic an endomorphin type III 4 --> 1 beta-turn conformation.
