WO2007149550A2 - Modulation de la différenciation et des fonctions d'une cellule par fox01 et signalisation notch - Google Patents

Modulation de la différenciation et des fonctions d'une cellule par fox01 et signalisation notch Download PDF

Info

Publication number
WO2007149550A2
WO2007149550A2 PCT/US2007/014537 US2007014537W WO2007149550A2 WO 2007149550 A2 WO2007149550 A2 WO 2007149550A2 US 2007014537 W US2007014537 W US 2007014537W WO 2007149550 A2 WO2007149550 A2 WO 2007149550A2
Authority
WO
WIPO (PCT)
Prior art keywords
foxol
csi
compound
cell
polypeptide
Prior art date
Application number
PCT/US2007/014537
Other languages
English (en)
Other versions
WO2007149550A3 (fr
Inventor
Domenico Accili
Tadahiro Kitamura
Yashuhiro Funahashi
Jan K. Kitajewski
Original Assignee
The Trustees Of Columbia University In The City Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Trustees Of Columbia University In The City Of New York filed Critical The Trustees Of Columbia University In The City Of New York
Publication of WO2007149550A2 publication Critical patent/WO2007149550A2/fr
Publication of WO2007149550A3 publication Critical patent/WO2007149550A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This application relates to the field of cell signaling.
  • ForkheadO 1 A (FOXO 1 A, forkhead in rhabdomyosarcoma, forkhead box 01 A, FKHR, FKHl) is a member of the O sub-family of forkhead (Fox) proteins that regulate hormone-induced differentiation. FoxO proteins are subject to post-translational modifications including phosphorylation and acetylation. FoxOl phosphorylation regulates myoblast and pre-adipocyte differentiation, and gain-of-function mutations in FoxOl prevent both processes.
  • the Notch pathway plays a role in differentiation, e.g., neural, vascular, muscular, and endocrine differentiation, during embryogenesis.
  • the intracellular domain of the Notch receptor (Notch-IC) translocates to the nucleus, where it interacts with the DNA binding protein CsI (which stands for CBFl, Suppressor of Hairless, and Lag-1 ; also termed Rbp-J ⁇ ) changing the transcriptional properties of Notch from a suppressor to an activator of transcription.
  • CsI targets include the Hairy and Enhancer of Split (Hes) genes. Hesl controls gut endoderm, preadipocyte, and myoblast differentiation. Myogenic differentiation is regulated via Hesl inhibition of the myogenic effector MyoD.
  • FoxOl A has been reported to be of importance in some alveolar rhabdosarcomas, type 11 diabetes, cancer, muscular dystrophy, autoimmunity, and premature ovarian failure.
  • the invention relates to the finding that FoxO land Notch cooperate in the regulation of differentiation. FoxOl regulates the Notch signaling pathway by direct interaction with the Notch target CsI to activate Hesl transcription. Accordingly, the invention relates to a method for identifying a compound capable of modulating Notch activity.
  • the method includes contacting a FoxOl polypeptide or fragment of a FoxOl polypeptide with a CsI polypeptide or fragment of a CsI polypeptide under protein binding conditions, and optionally, admixing a test compound with the FoxOl polypeptide and the CsI polypeptide, and determining whether the test compound inhibits the binding of FoxOl polypeptide with CsI polypeptide when compared to the binding in the absence of the compound, such that a test compound that can inhibit the binding is a compound capable of modulating Notch activity.
  • the test compound is admixed with one or more of the polypeptides before contacting, during contacting, after contacting, or any combination thereof.
  • a fragment of a FoxOl polypeptide can, in some embodiments, bind a CsI polypeptide (e.g., a CsI polypeptide that is between about 10 amino acids to about 500 amino acids, or a CsI polypeptide that is at least about 500 amino acids in length, for example, a CsI polypeptide or fragment that includes amino acids 179-272 of a full-length CsI protein.
  • the FoxOl polypeptide includes from about 1 to about 300 contiguous amino acids from the amino terminus of a FoxOl sequence.
  • the FoxOl polypeptide can include, in some cases, from about 20 to about 300 contiguous amino acids from the amino terminus of a FoxOl sequence.
  • the method can also include determining one or more of the following; whether the test compound modulates Hesl expression in a cell, or whether the compound modulates expression of at least one of: an angiogenesis gene, a myogenesis gene, a neurogenesis gene, a gut development gene, an adipogenic gene, or a pancreatic ⁇ cell differentiation gene.
  • the contacting occurs in a cell, e.g., a myoblast, a pre-adipocyte, a neuronal precursor cell, a PC-12 cell, a gut endoderm cell, an embryonic stem cell, or a pancreatic duct cell, or a derivative of any of the foregoing cell types.
  • a cell e.g., a myoblast, a pre-adipocyte, a neuronal precursor cell, a PC-12 cell, a gut endoderm cell, an embryonic stem cell, or a pancreatic duct cell, or a derivative of any of the foregoing cell types.
  • Another aspect of the invention includes a method for identifying a compound capable of inhibiting binding of a FoxOl protein and a CsI protein.
  • the method includes admixing a compound with a FoxOl polypeptide or a fragment thereof and a CsI polypeptide under protein-binding conditions, and measuring the amount of FoxOl polypeptide bound to CsI, such that a decrease in the amount of FoxOl bound to CsI in the presence of the compound compared to the amount of FoxOl bound in the absence of the compound indicates that the compound is capable of inhibiting the interaction between CsI and FoxOl .
  • the FoxOl is a fragment of FoxOl that can bind to CsI.
  • the method includes determining one or more of: whether the test compound modulates Notch activity; whether the test compound modulates Hesl expression in a cell; or whether the compound modulates expression of at least one of: an angiogenesis gene, a myogenesis gene, a neurogenesis gene, a gut development gene, an adipogenic gene, a pancreatic ⁇ cell differentiation gene, or any combination thereof.
  • Yet other embodiments include determining whether the compound inhibits binding of the CsI protein to the N-terminal region of the FoxOl protein, whether the compound binds to the N-terminal region of the FoxOl protein whether the test compound binds to the CsI protein, determining whether the test compound binds to all or a portion of amino acids 1-300 of a CsI protein, determining whether the test compound binds to all or a portion of amino acids 179-272 of a human CsI or a homolog thereof.
  • the cell is a myoblast, a pre-adipocyte, a neuronal precursor cell, a PC-12 cell, a pancreatic ⁇ cell precursor, a gut endoderm cell, an embryonic stem cell, or a pancreatic duct cell, or a derivative of any of the foregoing cell types.
  • Another aspect of the invention is a compound capable of inhibiting an interaction between FoxOl and CsI comprising a peptide from about amino acid position 1 to about amino acid position 300 of a FoxOl protein, an antibody, an antibody fragment, a peptide, a peptoid, a non-nucleic acid small organic molecule, or other small molecule.
  • the peptide is derived from a protein sequence represented by Genbank accession no. NM_019739.
  • Yet another aspect of the invention is a method for inhibiting muscle differentiation.
  • the method includes contacting a myoblast with a compound that can enhance the interaction between FoxOl and CsI.
  • the myoblast is in a mammal, e.g., a human.
  • the compound includes one or more of a FoxOl polypeptide, a Csl-binding fragment of FoxOl polypeptide, a nucleic acid encoding a FoxOl polypeptide or a fragment thereof, a CsI polypeptide, a FoxOl -binding fragment of a CsI polypeptide, a nucleic acid encoding a CsI polypeptide or fragment thereof, or any combination thereof.
  • the invention also relates to a method for increasing muscle differentiation.
  • the method includes contacting a myoblast with a compound that can inhibit the interaction between FoxOl and CsI.
  • the myoblast is in a mammal, e.g., a human.
  • the invention also relates to a method of modulating differentiation.
  • the method includes contacting an undifferentiated cell with a compound that modulates the interaction between CsI and FoxOl .
  • the compound inhibits the interaction between FoxO 1 and CsI and differentiation is enhanced.
  • the compound enhances myoblast differentiation, adipogenesis, angiogenesis, pancreatic ⁇ cell differentiation, or neurite sprouting.
  • the compound enhances the interaction between FoxO 1 and CsI and differentiation is decreased.
  • the compound inhibits myoblast differentiation, angiogenesis, pancreatic ⁇ cell differentiation, or neurite sprouting.
  • Another aspect of the invention is a method of inhibiting expression of Hesl.
  • the method includes contacting a cell that can express Hesl with a compound that inhibits FoxOl protein expression or activity.
  • the compound includes an siRNA or interfering RNA (RNAi) that specifically inhibits expression of a FoxOl gene.
  • FIG. IA is a reproduction of a set of micrographs of C 2 C12 cells transduced with adenoviruses as indicated and immunostained with anti-myosin and DAPI (4', 6-diamidino- 20-phenylinole).
  • FlG. IB is a reproduction of a micrograph of C 2 C 12 cells transduced with HA- FoxOl-ADA or HA-Notchl-lC adenovirus and immunostained with anti-HA antibody and DAPI.
  • FIG. ID is a reproduction of a Western blot Of C 2 Ci 2 cells co-transfected with FoxOl siRNA and a sequence encoding FLAG-FoxOl or Green Fluorescent Protein (GFP). The ability of Foxo 1 siRN A to inhibit expression of endogenous (left panel) and transfected (right panel) Foxol following adenoviral transduction was tested.
  • FIG. IE is a reproduction of a semiquantitiave RT-PCR analysis ofMy/5, MyoD, and myosin expression during embryogenesis.
  • FIG. 1 G is a reproduction of a photomicrograph of C2C12 cells transduced with HA-FoxOl-ADA or HA-Notchl-IC adenovirus and stained with anti-HA antibody and DAPI.
  • FIG. IH is a reproduction of a Western blot of FoxOl, FoxO3, and FoxO4 expression in C 2 Ci 2 cells transfected with FoxOl siRNA.
  • FIG. II is a reproduction of a photomicrograph Of C 2 Ci 2 cells transduced with lacZ, FoxOl-ADA, orNotchl-IC adenovirus, stained with anti-Ki67 antibody and DAPI.
  • the numbers indicate the Ki67 labeling index as a percentage of Ki67-positive cells (at least 1,000 cells counted.)
  • FIG. IJ is a reproduction of a Western blot of FoxOl-ADA and siRNA-resistant FoxOl-ADA in cells transfected with FoxOl siRNA.
  • FIG. IK represents a bar graph of a morphometric analysis of Myosin-positive cells. Results from differentiation experiments were analyzed by scoring the number of Myosin-immunostained cells as percentage of all DAPI-positive cells.
  • FIG. IL depicts DBD-FoxolADA reporter gene assays that were carried out using the canonical Foxol -responsive Igfbpl promoter (left panel) and the Hesl promoter (right panel) in cells co-transfected with Foxol -ADA or DBD-FoxolADA.
  • Western blot (inset) demonstrates that expression levels of the two proteins are similar.
  • An asterisk indicates PO.01 by ANOVA.
  • FIG. 2 A is a reproduction of the result of a co-immunoprecipitation experiment in which endogenous FoxOl and CsI expressing C 2 Ci 2 cells were co-cultured with LacZ- (-) or Jagged 1 -expressing HEK293 cells (+).
  • IP immunoprecipitation
  • FIG. 2B is a reproduction of the result of a co-immunoprecipitation experiment in C 2 Ci 2 cells.
  • IP is immunoprecipitation
  • IB is immunoblotting
  • TCL total cellular lysate.
  • FIG. 2C is a reproduction of the result of a co-immunoprecipitation experiment using C 2 Ci 2 cells.
  • FTG. 2D is a reproduction of the result of an experiment in which cells were co- transfected with FLAG-CsI, HA-FoxOl , or HA-Notchl-IC.
  • FIG. 2E is a reproduction of the result of an experiment in which cells were co- transfected with FLAG-CsI, HA-FoxOl, or HA-Notchl-IC.
  • FIG. 2F is a reproduction of the result of a co-immunoprecipitation experiment in which C 2 Ci 2 cells were co-transfected with FLAG-CsI and the truncated mutant Myc- or HA-tagged ⁇ 256 FoxOl.
  • FIG. 2G is a reproduction of the result of a co-immunoprecipitation experiment in which C 2 C 12 cells were co-transfected with FLAG-CsI and the truncated mutant Myc- or HA-tagged ⁇ 256 FoxOl .
  • FIG. 2 J is a reproduction of the immunoblot result of an experiment in which full-length and truncated fragments of GST-FoxOl and GST-FLAG/Csl were purified from bacteria, co-incubated, and CsI isolated using an anti-FLAG antibody. Immunoprecipitates were detected with anti-FoxOl or anti-FLAG antibodies
  • FIG. 2K is a reproduction of an experiment in which a Hesl promoter ChIP spanning the CsI binding site in C 2 Ci 2 cells was used to detect endogenous FoxOl, CsI, and Notch 1 (Endog.); or following transduction with FoxOl-ADA (FoxOlOADA during myoblast differentiation.
  • Input represents DNA extracted from chromatin prior to immunoprecipitation.
  • Hesl expression (semiquantitative RT-PCR) and myosin expression (Western blot) corresponding to each time point are shown.
  • Day 0 is the time when cells are serum-deprived to induce myoblast fusion.
  • FIG. 2L is a reproduction of the results of experiments in which cells were co- transfected with FoxO3, FoxO4 expression vectors and a CsI expression vector, immunoprecipitated with antibody specific to a FoxO, and the immunoprecipitates assayed for co-immunoprecipitation of the specific FoxOl and CsI.
  • FIG. 2M is a bar graph depicting the results of assays of reporter gene activity using promoter assays of murine CsI promoter (-1536 to 22) following co-transfection of C 2 Ci 2 cells with wild-type FoxOl or FoxOl-ADA.
  • FIG. 3 A is a bar graph depicting the results of Hesl reporter gene assays in HEK293 cells transduced with FoxOl-ADA, Notch 1-IC, FoxOl, siRNA GFP siRNA, or control plasmid. Luciferase activity was assayed and normalized to ⁇ -galactosidase activity. The data are represented by arbitrary units relative to control empty vector.
  • FIG. 3B is a reproduction of the result of an experiment in which Hesl, Hes5, and Heyl expression was measured by semiquantitative RT-PCR in C 2 C 1 2 cells transduced with FoxOl-ADA or Notchl-IC following transfection of GFP, FoxOl , or CsI siRNA as indicated.
  • FIG. 3C is a bar graph depicting the results of assays of reporter gene activity using promoter assays of a synthetic Hesl reporter gene containing four tandem repeats of a CsI binding site and co-transfection with FoxOl and Notchl-IC in C 2 Ci 2 cells.
  • FIG. 3D is a pair of bar graphs depicting the results of assays of reporter gene activity using the FoxOl -responsive Igfbpl promoter (left graph) and the Hesl (right graph) in cells co-transfected with FoxOI-ADA or DBD-FoxOl ADA.
  • the inset is a reproduction of a Western blot detecting expression levels of FoxOI-ADA and DBD-FoxOlADA.
  • An asterisk indicates that P ⁇ 0.01 by ANOVA.
  • FIG. 4 A is a reproduction of the results of a ChIP assay of endogenous FoxOl and Notchl in C 2 C 12 cells co-cultured with LacZ- (-) or Jagged 1 -expressing HEK293 cells (+) in the absence (lanes 1-2) and presence (lanes 3-4) of CsI siRNA.
  • FIG. 4B is a reproduction of the results of ChIP assays of endogenous Notchl in a co-culture system in the absence (lanes 1-2) and presence (lanes 3-4) of FoxOl siRNA.
  • FIG. 4C is a bar graph depicting the results of Hesl promoter assays following co-culture in the absence or presence of FoxOl GFP siRNA.
  • FlG. 4D is a reproduction of the results of ChIP assays of NcoR, SMRT, and MAMLl binding to Hesl in the co-culture system in the absence (lanes 1-2) and presence (lanes 3-4) of FoxOl siRNA.
  • FIG. 4F is a drawing depicting a model of FoxOl and Notch regulation of the Hesl promoter.
  • FIG. 4G is a reproduction of a Western blot depicting the CsI levels in C 2 Ci 2 cells following transfection with CsI siRNA at various concentrations.
  • FIG. 5 A is a reproduction of a set of micrographs of PC 12 cells transduced with FoxOl -ADA, Notchl -IC, or Notch decoy using adenoviruses in the presence or absence of FoxOl siRNA as indicated.
  • FIG. 5B is a reproduction of a set of micrographs of 3T3-F442A preadipocytes transduced with FoxOl -ADA, Notch 1 -IC, or Notch decoy using adenoviruses and in the presence or absence of FoxOl siRNA as indicated. Triglyceride accumulation in 3T3- F442A was assayed using oil red-O staining.
  • FIG. 5C is a reproduction of a set of micrographs of HUVECs transduced with FoxOl -ADA, Notchl-IC, or Notch decoy using retroviruses in the presence or absence of FoxOl siRNA as indicated.
  • PSI is the presenilin ( ⁇ -secretase) inhibitor compound E (5 ⁇ M).
  • FIG. 5D is a bar graph depicting the percentage of PCl 2 cells transduced with FoxOl and Notch expression constructs, and induced with nerve growth factor (NGF) that exhibited neurite outgrowth.
  • NGF nerve growth factor
  • FIG. 5F is a bar graph depicting the percentage of HUVECs transduced with FoxOl and Notch expression constructs that exhibited tube formation. An asterisk indicates PO.01 by ANOVA.
  • FIG. 6 A is a reproduction of results from a Western blot where Foxol and Foxo4 expression levels were analyzed.
  • FIG. 6B is a reproduction of metachromatic and immunohistochemical micrographs depicting soleus and plantaris muscle from Myog-Foxol mice and control (lox/lox) littermates.
  • FoxO transcription factors convey anti-differentiation signals that can be removed via Akt-dependent phosphorylation.
  • Notch signaling plays a role in cell fate determination in multiple lineages, including myoblasts, endothelial cells, and pre- adipocytes.
  • FoxOl regulates the Notch pathway by engaging the Notch target CsI to activate hesl transcription.
  • Constitutively active FoxOl can inhibit differentiation and such inhibition can be at least partially reversed with a Notch antagonist.
  • suppression of FoxOl expression prevents Notch inhibition by decreasing Notch binding to CsI and thus decreasing expression of Hesl and its target genes. It has also been found that the effect of FoxOl on transcription is mediated via a protein-protein interaction, not a protein-DNA interaction.
  • the invention provides methods (also referred to herein as "screening assays") for identifying modulators, i.e., candidate compounds (e.g., proteins, peptides, peptidomimetics, peptoids, small non-nucleic acid small organic molecules, small inorganic molecules, heteroorganic molecules, organometallic molecules, nucleic acids (e.g., oligonucleotides, siRNA, and nucleic acids containing non-naturally occurring nucleic acids) or other drugs) that can modulate the binding of a FoxOl protein and a CsI protein.
  • Compounds thus identified can be used to modulate the activity of FoxOl and CsI, for example resulting in modulation of Notch activity.
  • a compound that interferes with the interaction between a FoxOl protein and a CsI protein can inhibit one or more activities associated with Notch, e.g., the differentiation process is inhibited by Notch-IC.
  • the invention provides assays for screening test compounds to identify compounds that bind to a FoxOl polypeptide or a CsI polypeptide. Such compounds can then be tested for their ability to increase or decrease the interaction between a FoxOl polypeptide and a CsI polypeptide.
  • an assay is conducted to identify compounds that promote or inhibit the interaction between a FoxOl and a CsI polypeptide.
  • an assay is a cell-based assay in which a cell that expresses a FoxOl protein or biologically active portion thereof and a CsI protein or biologically active portion thereof is contacted with a test compound, and the ability of the test compound to modulate Notch activity is determined. Determining the ability of the test compound to modulate Notch activity can be accomplished by monitoring, for example, the activity of Hesl or by monitoring the ability of the cell to differentiate in the presence and absence of the test compound. Alternatively, the efficacy of a test compound can be determined by measuring the binding of proteins known as transcriptional coactivators and corepressors to the Notch target, Hesl .
  • induction or expression or activity of one or more markers associated with differentiation of the specific cell type can be assayed, cell morphology can be assayed (e.g., sprouting by a PC- 12 cell), or a function of the cell can be monitored.
  • the cell for example, can be from an invertebrate, a non-human mammal such as a mouse, rat, rabbit, sheep, cow, horse, pig, or non-human primate, or a human.
  • Markers of differentiation that are useful in the assays described herein include, but are not limited to, myogenin, myosin, glucose transporter 4, leptin, peroxisome proliferation-activating receptor gamma (PP AR ⁇ ), and myelin basic protein.
  • the ability of the test compound to modulate binding of a FoxOl polypeptide and a CsI polypeptide can also be evaluated. Pairs of FoxOl and CsI polypeptides that can bind to each other are referred to as binding partners.
  • An assay can be performed, for example, by coupling one of the polypeptides with a label (e.g., a radioisotope or enzymatic label) such that binding of the labeled polypeptide with the partner can be determined by detecting the labeled compound in a complex.
  • a label e.g., a radioisotope or enzymatic label
  • the binding partners are incubated in the presence and absence (control) of a test compound, the bound material separated from the unbound material, and the amount of labeled binding partner associated with the unlabeled binding partner is assayed. In some cases, the amount of labeled binding partner that is unbound is assayed. In either case, the amount of binding in the presence of the test compound is compared to the amount of binding in the absence of the test compound. A difference in the amount of binding between the binding partners in the presence of the test compound compared to the amount of binding in the absence of the test compound indicates that the test compound can modulate the interaction between the binding partners. For example, a test compound that can decrease the binding between a FoxOl and a CsI polypeptide is a candidate compound for promoting differentiation, e.g., in a cell for which differentiation is inhibited by Notch.
  • a polypeptide can be labeled with a radiolabel such as 125 ⁇ 35$ ⁇ 14 ⁇ or ⁇ H, either directly or indirectly, and the radioisotope detected by direct counting or detection of radioemission or by scintillation counting.
  • a polypeptide can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a polypeptide is synthesized as a heterologous polypeptide that includes a detectable label such as a FLAG peptide or a green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • the ability of a test compound to modulate the interaction between the binding partners can be performed without the labeling of any of the interactants.
  • the ability of a test compound to modulate the interaction of a FoxOl polypeptide and a CsI polypeptide can be detected, e.g., using a microphysiometer, without the labeling of either the compound or the FoxOl polypeptide (McConnell et al., 1992, Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potent ⁇ ometric sensor
  • cell-free assays involve preparing and incubating a reaction mixture of a FoxOl polypeptide and a CsI polypeptide under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
  • a test compound is incubated with one of the binding partners prior to, during, or after the binding partners interact, and the amount of interaction in the presence and absence of the test compound is assayed.
  • a test compound that can alter the interaction e.g., the amount of binding, Kd, or Ka
  • FoxOl and CsI are used in approximately equimolar concentrations.
  • a cell-free assay in which a FoxOl protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the FoxOl protein or biologically active portion thereof is evaluated.
  • Biologically active portions of a FoxOl protein to be used in an assay of the present invention include fragments that participate in interactions with a CsI molecule. Once a compound that can bind to the FoxOl is identified, it is generally tested for its ability to affect at least one of; the activity of a FoxOl or the ability to modulate the interaction between a FoxOl and a CsI.
  • the interaction between two molecules can also be detected, e.g., using fluorescence resonance energy transfer (FRET)(for example, Lakowicz et al., U.S. Patent No. 5,631,169; Stavrianopoulos et al., U.S. Patent No. 4,868,103; and fretimaging.org/mcnamaraintro.html).
  • FRET fluorescence resonance energy transfer
  • a fluorophore label on the first, 'donor' molecule is selected such that the emitted fluorescent energy of the donor is absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn fluoresces due to the absorbed energy.
  • the 'donor' protein molecule can utilize the natural fluorescent energy of tryptophan residues.
  • Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label can be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal.
  • An FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter).
  • determining the ability of binding partners to interact in the presence and absence of a test compound, or the ability of a test compound to bind to one of the binding partners can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (e.g., Sjolander and Urbaniczky, 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, Curr. Opin. Struct. Biol. 5:699-705).
  • BiA Biomolecular Interaction Analysis
  • one of the binding partners is anchored onto a solid phase.
  • the binding partner/test compound complexes anchored on the solid phase can be detected after a binding reaction.
  • the binding protein can be anchored onto a solid surface, and the test compound (which is not anchored) can be labeled, either directly or indirectly, with detectable labels discussed herein.
  • the binding partner to be immobilized can be anchored directly to a solid phase (e.g., a particle, bead, plate, slide, or other suitable surface), or can be indirectly immobilized, for example, via an antibody that recognizes the binding partner and is attached to the solid phase.
  • Formats in which one of the binding partners is immobilized are useful to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • an antibody used in such an assay specifically recognizes one of the binding partners, but does not interfere with binding of the binding partners to each other.
  • Such antibodies can be derivatized to the wells of the plate, and the binding partner trapped in the wells by antibody conjugation.
  • Binding of a test compound to a binding partner, or the interaction of binding partners in the presence and absence of a test compound can be accomplished in any vessel suitable for containing the reactants.
  • vessels include microtiter plates, test tubes, slides, chips, and micro-centrifuge tubes. Suitable modifications can be made to one or both of the binding partners to facilitate an assay using a particular vessel or surface.
  • a fusion protein can be provided that adds a domain to a binding partner that allows binding to a matrix.
  • a glutathione-S-transferase/FoxOl fusion protein or a glutathione-S-transferase/Csl fusion protein can be adsorbed onto glutathione Sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non- adsorbed binding partner, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, an the amount of complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of interaction between the binding partners determined using techniques known in the art.
  • binding partners on a matrix include using conjugation of biotin and streptavidin.
  • One of the binding partners is biotinylated, for example, using biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemicals).
  • the non-immobilized component is added to the surface containing the anchored component and the two components are incubated for sufficient time and under conditions the permit binding. After the incubation is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished using methods known in the art. When the initially non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody).
  • the amount of complex formed in the presence and absence of a test compound can be determined to identify test compounds that increase binding between the binding partners, or to identify compounds that decrease binding between the binding partners.
  • Cell free assays can be conducted in a liquid phase.
  • the reaction products are separated from unreacted components by any of a number of techniques, including but not limited to differential centrifugation (for example, Rivas et al., 1993, Trends Biochem. Sci. 18:284-7), chromatography (gel filtration chromatography, ion- exchange chromatography), electrophoresis (e.g., Ausubel et al., eds. Current Protocols in Molecular Biology. 1999, J. Wiley: New York.), and immunoprecipitation (for example, Ausubel et al., eds., 1999, Current Protocols in Molecular Biology, J. Wiley: New York).
  • differential centrifugation for example, Rivas et al., 1993, Trends Biochem. Sci. 18:284-7
  • chromatography gel filtration chromatography, ion- exchange chromatography
  • electrophoresis e.g., Ausubel et al.
  • the assay includes contacting a FoxOl or CsI binding partner or biologically active portion thereof with a known compound that binds to the binding partner to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the binding partner, wherein determining the ability of the test compound to interact with the binding partner is determined by assaying the ability of the test compound to preferentially bind to the binding partner or biologically active portion thereof as compared to the known compound.
  • FoxOl stabilizes the Notch-IC/Csl complex. Accordingly, assays can be performed that determine the ability of a test compound to dissociate FoxOl from a Notch- IC/Csl complex. Alternatively, a test compound can be assayed for the ability to prevent a FoxOl polypeptide from binding to such a complex.
  • the invention provides methods for determining the ability of the test compound to modulate the activity of a FoxOl polypeptide through modulation of the expression or activity of a downstream gene or gene product such as Hesl .
  • the ability of a test compound to inhibit or promote an activity regulated by a FoxOl /CsI interaction is assayed.
  • assays include assays in which a myoblast is contacted with a test compound and the effect of the test compound on Myogenesis is analyzed. In general, this type of assay is used after identification of a candidate compound that can bind FoxOl or a candidate compound that can modulate the interaction between a FoxOl polypeptide and a CsI polypeptide.
  • test compounds that disrupt preformed complexes e.g., compounds with higher binding constants that displace one of the components from the complex
  • test compounds that disrupt preformed complexes can be tested by adding the test compound to the reaction mixture after complexes have been formed. Examples of the various formats are briefly described below.
  • either of the binding partners is anchored onto a solid surface (e.g., a microtiter plate), while the non-anchored species is labeled, either directly or indirectly.
  • the anchored species can be immobilized by non-covalent or covalent attachments.
  • an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.
  • a homogeneous assay can be used.
  • a preformed complex of the binding partners is prepared in which one of the binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496).
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances that disrupt binding partner interaction can be identified.
  • FoxOl and CsI polypeptides can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al., 1993, Cell 72:223-232; Madura et al., 1993, J. Biol. Chem. 268:12046-12054; Bartel et al., 1993, Biotechniques 14:920-924; Iwabuchi et al., 1993, Oncogene 8:1693-1696; Licitra et al., 1996, Proc. Nat. Acad. Sci.
  • test polypeptide test compounds that bind to or interact with one or both of the binding partners to modulate their interaction.
  • test polypeptides can be activators or inhibitors of signaling affected by the interaction between FoxOl and CsI, e.g., Notch signaling.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a compound that can modulate the interaction between a FoxOl polypeptide and a CsI polypeptide can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of Notch signaling can be confirmed in vivo, e.g., in an animal model suitable for examining Notch-pathway differentiation or a disorder related to a Notch signaling pathway.
  • This invention further pertains to novel agents identified by the screening assays described herein. Accordingly, it is within the scope of this invention to further use a modulating agent identified as described herein (e.g., an agent that can increase the interaction or activity of a FoxOl/Csl complex or an agent that can inhibit the interaction or activity of a FoxOl/Csl complex) in an appropriate animal model to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, novel agents identified by the above-described screening assays can be used for treatments as described herein.
  • a modulating agent identified as described herein e.g., an agent that can increase the interaction or activity of a FoxOl/Csl complex or an agent that can inhibit the interaction or activity of a FoxOl/Csl complex
  • Candidate compounds that affect the interaction between FoxOl and CsI can be tested for their ability to modulate disorders related to the interaction between FoxOl and CsI (e.g., disorders that may be ameliorated by interfering with the interaction between FoxO 1 and CsI).
  • disorders related to the interaction between FoxOl and CsI e.g., disorders that may be ameliorated by interfering with the interaction between FoxO 1 and CsI.
  • animal models suitable for such testing include, without limitation, animal models of diabetes (e.g., Accili et al., 1996, Nat. Genet. 12:106-109), muscular dystrophy (e.g., Guo, 2005, Dan. Med. Bull. 52:117), neurodegenerative disorders such as Parkinson's disease (e.g., Gonzales et al., 2006, Brain Res.
  • Candidate compounds are tested by administering various dosages of the compound that the animal model and determine whether features of the targeted disease are modulated.
  • Tested features can include, for example, changes in RNA expression, changes in protein expression, changes in all metabolism or structural features, or changes disease symptoms demonstrated by the animal.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone that are resistant to enzymatic degradation but that nevertheless remain bioactive; see, e.g., Zuckermann et al. (1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997 Anticancer Drug Des. 12:145).
  • the compound can interact with CsI and is derived from a naturally occurring CsI binding protein other than a CsI polypeptide or a FoxOl polypeptide.
  • naturally occurring CsI binding proteins include Kaposi's sarcoma-associated herpes virus RTA protein, Epstein-Barr virus proteins (the EBNA-2 and -3 proteins of Epstein-Barr Virus (EBV) (Grossman et al. 1994; Hsieh and Hayward 1995; Johannsen et al. 1996)) and the 13S isoform of adenovirus ElA
  • Toxicity and therapeutic efficacy of compounds identified as described herein can be determined using known pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5O/ED5O.
  • Compounds that exhibit high therapeutic indices are generally used. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a myoblast is treated by contacting the myoblast with a compound that that decreases the expression or activity of FoxOl.
  • the myoblast can initiate differentiation.
  • treatments are useful for, e.g., muscle regeneration in damaged tissues, for use in conjunction with muscle transplants, and in myodegenerative diseases such as muscular dystrophy, in which it is desirable to stimulate differentiation or in conjunction with gene therapy methods in which muscle progenitor cells are transplanted into a subject and stimulated using a compound as described herein to promote muscle differentiation.
  • Compounds that can inhibit the interaction between FoxOl and CsI are also useful for treatment of disorders in which it is desirable to promote one or more features of neurogenesis, e.g., neurite sprouting.
  • disorders include nerve damage or neurodegenerative disorders, including, without limitation, neurodegenerative disorders such as Parkinson's disease, Huntington's disease, multiple sclerosis, and trauma of the spinal cord.
  • a compound that enhances the interaction between FoxOl and CsI is useful for treating a disorder in which it is desirable to inhibit one or more features of neuronal development such as sprouting.
  • such compounds may be useful in vitro to drive differentiation of neural progenitors into specific neuronal sub-types, e.g., for use in the preparation of neuronal cells for therapy of neuromuscular disorders and spinal cord injury.
  • Compounds that affect FoxOl/Csl interaction are useful for treatment of disorders associated with FoxOl and CsI.
  • such compounds can be employed to treat diabetes by one or more of the following; reducing FoxO activity at the promoters of genes controlling liver glucose production and liver lipid synthesis, storage, and release; increasing the differentiation of endocrine stem cells into insulin-producing beta cells in the pancreas; decreasing the output of neuropeptides that influence appetite and satiety from the hypothalamus; treating vascular complications of diabetes by way of compound-eluting stents applied to arteries of diabetic individuals (for example, during angioplasty); decreasing the number or size of fat cells and thus decreasing body fat content.
  • Compounds that affect the interaction between FoxO and CsI can also be useful to prevent or slow the growth of Notch-dependent cancers.
  • at least a subset of the compounds identified as described herein can be Notch antagonists and thus can be used to inhibit cellular dedifferentiation in cancers associated with Notch activity, e.g., breast or prostate cancer.
  • compositions typically include the active compound and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal, and rectal administration; or oral administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability 'exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the selected particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • one or more isotonic agents are included, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be effected by including in the composition one or more agents that delay absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the specified amount in an appropriate solvent with one or a combination of ingredients enumerated above, as needed, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients selected from those enumerated above or others known in the art.
  • the methods of preparation are known in the art and include, for example, vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • an active compound is prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations are known to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the selected pharmaceutical carrier.
  • a therapeutically effective amount of an active compound that is a protein or peptide ranges from about 0.001 to about 30 mg/kg body weight, about 0.01 to about 25 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 1 to about 10 mg/kg body weight, about 2 to about 9 mg/kg body weight, about 3 to about 8 mg/kg body weight, about 4 to about 7 mg/kg body weight, or about 5 to about 6 mg/kg body weight.
  • the active compound can be administered one time per week for between about 1 to about 10 weeks, between about 2 to about 8 weeks, between about 3 to about 7 weeks, or for about 4 weeks, about 5 weeks, or about 6 weeks.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or can include a series of treatments.
  • the dosage is generally about 0.1 mg/kg of body weight (generally about 10 mg/kg to about 20 mg/kg). If the antibody is to act in the brain, a dosage of about 50 mg/kg to about 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. (1997, J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).
  • the present invention encompasses agents that can, for example, be small molecules.
  • small molecules include, but are not limited to, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, non-nucleic acid organic compounds, or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • peptides e.g., peptoids
  • amino acids amino acid analogs
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated.
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • An active agent that is a nucleic acid molecule can be inserted into a vector and used as gene therapy vectors.
  • An active agent that is a protein or peptide can also be provided to a subject by inserting a nucleic acid sequence encoding the protein or polypeptide into a vector, which is used as a gene therapy vector.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. USA 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • Notch/FoxO cooperation integrates environmental cues through Notch with metabolic cues through FoxOl to regulate progenitor cell maintenance and differentiation.
  • This provides a general mechanism for committed progenitor cells to avoid differentiation in response to developmental cues transmitted by Notch signaling when FoxOl is active (i.e., in the absence of growth factors).
  • Such cells can persist in a dormant state in adult tissues in which they can undergo terminal differentiation in response to a combination of Notch ligand and hormonal/nutritional cues that lead to FoxOl inhibition.
  • diabetes is associated with excessive angiogenesis in the eye and in the coronary arteries.
  • Foxol gain-of-function 3-5 phenocopies Notchl activation (13, 17, 22, 23) in every cellular differentiation context.
  • Foxol ablation (24) phenocopies Notchl ablation (25) in mice.
  • Foxo and Notch signal through two seemingly distinct mechanisms, the phosphatidylinositol-3-kinase pathway (Foxo), and the Hes/Hey pathway (Notch).
  • Foxo physically and functionally interacts with Notch by promoting co-repressor clearance from CsI, thus controlling the myogenic program.
  • Myogenic precursors arise from mesodermal stem cells (26) and are converted into myotubes by a multi-step process culminating in the expression of myogenic transcription factors of the MRF family (MyoD, Myogenin, MRF4 and Myf5) (27).
  • Myogenic transcription factors heterodimerize with E proteins and promote expression of muscle-specific genes, acting in close coordination with myocyte-specific MEF2 enhancer factors (28).
  • Type I fibers express primarily slow-twitch MyHC
  • type II fibers express fast-twitch MyHC (29).
  • the process of fiber-type specification is controlled at multiple steps. First, there appears to be heterogeneity among myogenic precursor cells, and evidence from avian embryo cross-transplantation experiments indicates that early precursors contribute primarily to slow muscle fibers, and later precursors to fast fibers (29). Post-natally, fiber type specification is also affected by cell autonomous factors, including innervation and endocrine/nutritional cues (28).
  • the Foxo co-activator Pgcl ⁇ plays a critical role in promoting the formation of slow-twitch fibers (30), and recent data have also implicated the Foxo deacetylase Sirtl in this process (31).
  • conditional mutagenesis in mice, we show that Foxol 's role in suppressing MyoD-dependent myogenesis in C2C12 cells is mirrored by an increase of MyoD-containing myofibers in Foxol -deficient skeletal muscle, consistent with a key function in myoblast lineage specification.
  • Foxo transcription factors govern metabolism and cellular differentiation. Unlike Foxo-dependent metabolic pathways and target genes, the mechanisms by which these proteins regulate differentiation have not been explored. Activation of Notch signaling mimics the effects of Foxo gain-of-function on cellular differentiation.
  • Activation of Notch signaling mimics the effects of Foxo gain-of-function on cellular differentiation.
  • muscle differentiation as a model system, we show that Foxo physically and functionally interacts with Notch by promoting co-repressor clearance from the Notch effector CsI, leading to activation of Notch target genes. Inhibition of myoblast differentiation by constitutively active Foxol is partly rescued by inhibition of Notch signaling, while Foxol loss-of- function precludes Notch inhibition of myogenesis and increases MyoD expression.
  • conditional Foxol ablation in skeletal muscle results in increased formation of MyoD-containing (fast-twitch) muscle fibers and altered fiber type distribution at the expense of Myogenin-containing (slow-twitch) fibers.
  • Notch/Foxol cooperation may integrate environmental cues through Notch with metabolic cues through Foxol to regulate progenitor cell maintenance and differentiation.
  • mice [0130] Myogenin-cre (49) and Foxol ⁇ ox mice have been described (9). The wild-type, null and Foxol flox alleles were detected using PCR with primers 5'-GCT TAG AGC AGA GAT GTT CTC ACA TT-3', 5'-CCA GAG TCT TTG TAT CAG GCA AAT AA-3' and 5'-CAA GTC CAT TAA TTC AGC ACA TTG A-3'. Prior to the treadmill performance test, mice were trained for 2 days (Columbus Instruments). The test was performed at 15 m/min for the first 30 min, followed by lm/min increases at 10 min intervals until exhaustion.
  • FoxOl-ADA, Notchl-IC, Jagged 1, CsI, and Notch-decoy adenoviral and mammalian expression vectors were as described in Nakae et al. (2001, J. Clin. Invest. 108:1359-1367) and Das et al. (2004, J. Biol. Chem. 279:30771-30780).
  • Retroviruses expressing FoxOl-ADA and Notchl-IC were generated using the pQCXIH vector (BD Biosciences/Clontech, Palo Alto, CA).
  • pAdlox Notch IECD- Fc the extracellular domain of Notch l(bp 241-4229, GenBank Accession No.
  • Retroviral supernatant was produced from cells transiently co-transfected with pVSV-G (BD Biosciences/Clontech) vector and designated pQCXIH vector into GP2-293 cells (BD Biosciences, Palo Alto, CA). FoxOl and GFP siRNAs were from Santa Cruz Biotechnology, Inc. To generate the DNA binding-deficient Foxol, N208 and H212 were replaced with alanine and arginine, respectively, using QuikChange Mutagenesis Kit (Stratagene). The mutations were then cloned in the backbone of the Foxol -ADA mutant.
  • C 2 C] 2 cells (a murine myoblast cell line) and 3T2-F442A cells (a pre-adipocyte cell line) were differentiated as described in Nakae et al. (2003, Dev. Cell 4:119-129) and Hribal et al. (2003, J. Cell Biol. 162:535-541).
  • PC12 cells American Type Culture Collection; ATCC
  • ATCC American Type Culture Collection
  • NGF nerve growth factor
  • RNA isolation, northern blotting, semiquantitative reverse trancriptase-polymerase chain reaction (RT-PCR), and real-time RT-PCR were performed using techniques known in the art. Primer sequences were designed and tested using methods known in the art.
  • RT-PCR semiquantitative reverse trancriptase-polymerase chain reaction
  • HEK293 cells were transfected with murine Hesl- reporter sequence (base pairs -194 to 160 relative to the transcription start of murine Hesl (HESl/pGL2 basic).
  • murine Hesl- reporter sequence base pairs -194 to 160 relative to the transcription start of murine Hesl (HESl/pGL2 basic).
  • Synthetic Hesl- reporter sequence containing a four CsI binding sites operatively linked to the Hesl- reporter sequence, 4x Csl/pGL2 basic
  • Csl-luciferase -1536 to 22 Csl/pGL2 basic reporter sequence along with pCMV5, pCMV5-FoxOl-ADA, pQNC-Notchl-IC, pHyTc Notch decoy or FoxOl SiRNA.
  • Plasmid pRSW- ⁇ -galactosidase was used as a control for transfection efficiency (Das et al., 2004, J. Biol. Chem. 279:30771-80).
  • Notch 1 was expressed in C 2 Ci 2 CeIIs and Jaggedl or LacZ in HEK293 cells by transfection.
  • HEK293 cells were harvested and seeded on C 2 Ci 2 cells. After incubation for one hour, the co-cultured cells were used in experiments.
  • dimethylpyrimilidate (DMP from Pierce Biotechnology, Inc., Rockford, IL) was used to cross-link antibodies to Protein A beads and avoid IgG contamination of eluted protein complexes (Chi et al., 2004, Methods Enzymol. 377:299-316).
  • ChIP assay in C 2 Ci 2 cells was performed as described in Nakae et al. (2003, Dev. Cell 4:1 19-129) and in co-cultured cells as described by Fryer et al. (2004, MoI. Cell 16:509- 520).
  • the primer pairs employed to amplify the CsI binding site of the Hesl promoter were 5'-GCA AAG CCC AGA GGA AAG AGT TAG-3' and 5'-AGG AGA GAG GTA GAC AGG GGA TTC-3 1 .
  • siRNA-specific siRNA sequence was AC GGA GGA TTG AAC CAG TAT A.
  • the CsI specific siRNA sequence was TAG GGA AGC TAT GCG AAA TTA.
  • siRNA was transfected using LipofectamineTM-Plus reagent (Invitrogen Corp., Carlsbad, WA).
  • siRNA-resistant FoxOl was generated by replacing three residues (underlined) in the sequence AC GGC GGTCTG AAC CAG TAT A.
  • N208 and H212 of FoxO 1 were replaced to be alanine and arginine residues, respectively using a QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA). The mutations were then introduced in the backbone of the FoxOl -ADA mutant.
  • GST-FLAG-CsI encompassing amino acids 1-527, 1-279, 1-172 and 279-527 fragments were generated by cloning into pGEX6P-l .
  • GST-FoxOl constructs have been described (Puigserver et al., 2003, Nature 423:550-555). Following bacterial culture and IPTG induction, GST fusion proteins were purified and incubated together. Thereafter, GST-FLAG/Csl was isolated by immunoprecipitation with anti-FLAG antibody, the immune pellets were washed extensively, and immunoblots were performed using anti- FoxOl antiserum.
  • FIG. IA and IB show that FoxOl inhibition by siRNA did not affect these processes.
  • the FoxOl siRNA effectively suppressed expression of both transfected FLAG-FoxOl (FIG. 1C) and endogenous FoxOl (FIG. ID) in a dose-dependent manner, without affecting control proteins or other FoxO isoforms (FIG. IH).
  • Myoblast differentiation was inhibited by constitutively active Notchl-lC, encoding a truncated intracellular form of Notch 1 (FIG. IA 5 FIG. IB).
  • Neither FoxOl -ADA nor Notchl-1 affected C 2 C 2 proliferation (FIG. II).
  • the decoy did not affect the ability of the C 2 Cj 2 cells to undergo differentiation and activate myosin expression in response to growth factor withdrawal (FIG. IA, FIG. IB).
  • a presenilin inhibitor (PSE) As an alternative to block Notch signaling, a presenilin inhibitor (PSE), compound E (Pan et al., 2004, Dev. Cell 7:731-743) was used. This reagent also rescued FoxOl -ADA inhibition of myoblast differentiation (FIG. IA).
  • FoxOl siRNA and Notch- 1C were co-transfected into cells. FoxOl siRNA rescued inhibition of myoblast differentiation and myosin expression by Notch 1-Ic (FIG. IA, FIG.
  • FIG. IB A quantitative analysis of these data is demonstrated in FIG. IB, showing that Foxol and Notch 1-IC decreased myosin levels by >80%, while Notch decoy and Foxol siRNA restored them to -70% of fully differentiated cells. Similar data was obtained by performing a morphometric analysis of Myosin-positive cells (FIG. IK). These data indicate that Foxol is required for the effect of Notch on myoblast differentiation.
  • Example 3 Altered Pattern of Muscle Differentiation Markers in FoxO* Embryos
  • MyoD and Myf5 expression were assayed in FoxO J ⁇ ' embryos and controls using real time RT-PCR.
  • MyoD expression increased by about threefold in FoxOV 1 " embryos and E8.5 and E9.5, whereas Myfi was unaffected (FIG. IF).
  • Notch-IC has been reported to promote expression of Notch ligands and receptors (Shawber et al, 2003, Ann. N. Y. Acad. Sci. 995:162-70). Consistent with this finding, it was observed that Notch 1 -IC increased expression of the Notch ligand Jagged 1, and of the Notch 1 and Notch2 receptors.
  • FIG. ID a truncated Notchl receptor lacking the transmembrane anchor and intracellular domain was used to act as a decoy receptor by binding Notch ligands (U.S. Patent Application 20060030694).
  • This mutant (termed “Notch decoy") partially rescued FoxOl-ADA as well as Notch 1 -IC inhibition of myoblast differentiation (FIG. IA) and restored myosin expression to about 30% of control values (FIG. IB).
  • the partial ability of the Notch decoy to rescue differentiation is consistent with Notch acting on a ligand synthesized in response to Notchl -IC and FoxOl-ADA.
  • FoxOl expression was inhibited in C 2 C 12 cells using small interfering RNA (siRNA).
  • siRNA effectively suppressed expression of both transfected FLAG-FoxOl (FIG. IE) and endogenous FoxOl (FIG. IF) in a dose-dependent manner without affecting control proteins or other FoxO isoforms (FIG. IG).
  • Fox Ol siRNA partially rescued inhibition of myoblast differentiation by Notchl-IC (FIG. IA), while the control SiRNA had no effect on myoblast inhibition.
  • Notchl-IC binds to and co-activates CsI to promote Hesl and Heyl expression (Lai, 2002, EMBO Rep 3:840-5).
  • C 2 Ci 2 cells expressing Notchl receptor were co-cultured with HEK293 cells expressing the Notch ligand Jagged 1 (denoted by the "+” sign), or LacZ as a negative control (denoted by the "-” sign).
  • Several methods were used to investigate whether FoxOl and CsI interact in the cultured cells.
  • pull down assays were carried out using affinity- purified GST-FoxOl that was produced in bacteria and FLAG-CsI expressed in HEK293 cells. CsI associated with full-length and N-terminal FoxOl (amino acids 1-300), but not with C-terminal FoxOl (amino acids 290-655) or GST (FIG. 2H).
  • This region contains the CsI corepressor-binding domain (Hsieh et al., Science, 1995, 268:560-563; Kao et al., 1998, Genes Dev. 12:2269-2277) (FIG. 2J, diagram). Interestingly, this domain is required for DNA and corepressor binding, but does not contribute to Notch binding (38, 39).
  • CsI binds to a consensus sequence in the Hesl promoter (Tun et al., 1994, Nucleic Acids Res. 22:965-971), which thus provides a useful readout assay of the FoxOl/Csl interaction. If the latter were required to regulate C 2 C 12 differentiation, three predictions should be met: (a) FoxOl should be detected in chromatin immunoprecipitation assays (ChIP) spanning the CsI element in the Hesl promoter, (b) the interaction should be differentiation-dependent and (c) inhibition of differentiation by FoxOl-ADA should be accompanied by constitutive binding to the CsI element in the Hesl promoter.
  • FIG. 2K demonstrates that all predictions are fulfilled.
  • ChIPs were performed using primers spanning the CsI binding site of Hesl in differentiating C 2 Ci 2 cells. Endogenous FoxOl, Notch 1, and CsI were detected in immunoprecipitates from undifferentiated cells (FIG. 2K, Endog. lanes, Day 0). As the PCR-amplified sequence contains no forkhead binding sites, it was concluded that FoxOl binds to this DNA fragment via CsI. Moreover, binding of both FoxOl and Notch 1 decreased as cells became differentiated (day 1 and day 2). When cells were transduced with constitutively nuclear FoxOl -ADA, differentiation was inhibited FIG. IA and mutant FoxOl was persistently bound to the Hesl promoter, as were both CsI and Notchl (FIG. 2K, FoxOl -ADA lanes).
  • Notch decoy restored neurite outgrowth to 50% of control levels in cells transduced with FoxOl -ADA and induced with NGF.
  • FoxOl siRNA restored neurite outgrowth to 53% of control in cells transduced with Notchl-IC (FIG. 5A and FIG. 5D).
  • FoxO/Notch interaction was also studied in an angiogenesis model.
  • collagen sandwich assays were performed with human umbilical vein endothelial cells (HUVEC). Formation of a capillary-like network was inhibited to a similar extent by either FoxOl-ADA or Notchl-IC.
  • Notch inhibition by PSI provided partial rescue of FoxOl -ADA inhibition (FIG. 5C and FIG. 5F). From these data, it can be concluded that the FoxO/Notch interaction is a general mechanism to control cell differentiation.
  • Example 8 Altered fiber type composition in skeletal muscle lacking Foxol
  • Foxo isoforms There are three Foxo isoforms in mice: Foxol, 3, and 4 (8, 9). The latter is predominant in most muscle types (45) except soleus, where Foxol is the most abundant (FIG. 6A). Coincidentally, soleus is also physiologically enriched in slow-twitch fibers, and thus readily allowed to test the hypothesis. Foxol expression was inactivated in skeletal muscle by crossing mice homozygous for a floxed Foxol allele with Myogenin-cre transgenics. mRNA analysis indicated that the knockout occurred as planned (data not shown).
  • MyoD expression was determined in Foxol (24) and Notchl knockout (25) embryos at E9.5.
  • the increase in MyoD expression observed in vivo is consistent with the physical and functional interactions between Foxol and Notch at this key signaling nexus in myoblast differentiation.
  • the fiber-type switch in Myog-Foxol mice is purportedly the result of accelerated differentiation of MyoD-containing myoblasts during embryonic development.
  • the EXAMPLES above provide biochemical, cellular and genetic evidence that Foxo and Notch pathways cooperate in the regulation of muscle differentiation.
  • the data reveal a novel mode of Foxo 1 action to promote corepressor exchange at the Hesl promoter via direct binding to the CsI NTD region (FIG. 4F).
  • Foxol binding to this domain stabilizes the Notch/Csl complex and promotes corepressor clearance and Mamll recruitment, consistent with the proposed role of NTD from structural studies (37).
  • the findings also provide a mechanism by which two major biochemical pathways, the phosphoinositol-3 -kinase/ Akt pathway and the Notch/Hes pathway, converge in a synergistic manner to control cellular differentiation in vivo.
  • This two-tiered mechanism allows committed progenitor cells in various tissues to avoid differentiation in response to developmental cues (Notch) when Foxol is active, i.e., in the absence of growth factors. These cells would then persist in a dormant state in adult tissues, where they can terminally differentiate in response to a combination of Notch ligand and hormonal/nutritional cues leading to Foxol inhibition.
  • This interpretation is consistent with the fiber-type switch observed in Foxo 1 -deficient muscle, an observation that appears to position Foxol as a fate decider within the myogenic lineage, as opposed to an inducer of the myogenic program. It remains to be seen whether other Foxo and Notch isoforms also interact and how they contribute to this process.
  • the lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway. Genes Dev 16:1977- 1989.
  • the forkhead transcription factor Foxol links insulin signaling to Pdxl regulation of pancreatic beta cell growth. J Clin Invest 1 10:1839-1847.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés d'identification de composés utiles lors de la régulation de la différenciation par des interactions avec du FoxO1.
PCT/US2007/014537 2006-06-22 2007-06-22 Modulation de la différenciation et des fonctions d'une cellule par fox01 et signalisation notch WO2007149550A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81605306P 2006-06-22 2006-06-22
US60/816,053 2006-06-22

Publications (2)

Publication Number Publication Date
WO2007149550A2 true WO2007149550A2 (fr) 2007-12-27
WO2007149550A3 WO2007149550A3 (fr) 2008-03-27

Family

ID=38834138

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/014537 WO2007149550A2 (fr) 2006-06-22 2007-06-22 Modulation de la différenciation et des fonctions d'une cellule par fox01 et signalisation notch

Country Status (1)

Country Link
WO (1) WO2007149550A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9457079B2 (en) 2010-05-12 2016-10-04 The Trustees Of Columbia University In The City Of New York Methods for producing enteroendocrine cells that make and secrete insulin
US10487314B2 (en) 2014-06-26 2019-11-26 The Trustees Of Columbia University In The City Of New York Inhibition of serotonin expression in gut enteroendocrine cells results in conversion to insulin-positive cells
CN113301908A (zh) * 2019-01-03 2021-08-24 纽约市哥伦比亚大学理事会 共同给药抑制剂以生成产生胰岛素的肠道细胞

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040097459A1 (en) * 2002-09-26 2004-05-20 Dobie Kenneth W Modulation of forkhead box O1A expression
US20060069049A1 (en) * 2003-12-29 2006-03-30 President And Fellows Of Harvard College Methods and reagents related to foxo
WO2007008982A2 (fr) * 2005-07-11 2007-01-18 Irm Llc Methodes et composition modulant l'activite du foxo1 et la signalisation par l'insuline

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040097459A1 (en) * 2002-09-26 2004-05-20 Dobie Kenneth W Modulation of forkhead box O1A expression
US20060069049A1 (en) * 2003-12-29 2006-03-30 President And Fellows Of Harvard College Methods and reagents related to foxo
WO2007008982A2 (fr) * 2005-07-11 2007-01-18 Irm Llc Methodes et composition modulant l'activite du foxo1 et la signalisation par l'insuline

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KITAMURA ET AL.: 'A Foxo/Notch Pathway Controls Myogenic Differentiation and Fiber Type Specification' J. CLIN. INVEST. vol. 117, no. 9, September 2007, pages 2477 - 2485 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9457079B2 (en) 2010-05-12 2016-10-04 The Trustees Of Columbia University In The City Of New York Methods for producing enteroendocrine cells that make and secrete insulin
US10544415B2 (en) 2010-05-12 2020-01-28 The Trustees Of Columbia University In The City Of New York Methods for producing enteroendocrine cells that make and secrete insulin
US10487314B2 (en) 2014-06-26 2019-11-26 The Trustees Of Columbia University In The City Of New York Inhibition of serotonin expression in gut enteroendocrine cells results in conversion to insulin-positive cells
US11060063B2 (en) 2014-06-26 2021-07-13 The Trustees Of Columbia University In The City Of New York Inhibition of serotonin expression in gut enteroendocrine cells results in conversion to insulin-positive cells
CN113301908A (zh) * 2019-01-03 2021-08-24 纽约市哥伦比亚大学理事会 共同给药抑制剂以生成产生胰岛素的肠道细胞
EP3906041A4 (fr) * 2019-01-03 2022-11-30 The Trustees of Columbia University in the City of New York Co-administration d'inhibiteurs pour produire des cellules intestinales produisant de l'insuline

Also Published As

Publication number Publication date
WO2007149550A3 (fr) 2008-03-27

Similar Documents

Publication Publication Date Title
Webster et al. O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development
Santoro et al. Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction
Avram et al. Isolation of a novel family of C2H2 zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors
van Tetering et al. Metalloprotease ADAM10 is required for Notch1 site 2 cleavage
Ul-Hussain et al. Internal ribosomal entry site (IRES) activity generates endogenous carboxyl-terminal domains of Cx43 and is responsive to hypoxic conditions
Sessoms-Sikes et al. CaMKIIα enhances the desensitization of NR2B-containing NMDA receptors by an autophosphorylation-dependent mechanism
US20050136429A1 (en) SIRT1 modulation of adipogenesis and adipose function
US6596473B1 (en) Active survival domains of IGF-IR and methods of use
Qin et al. Cooperation of tyrosine kinases p72syk and p53/56lyn regulates calcium mobilization in chicken B cell oxidant stress signaling
US20050130232A1 (en) Methods of screening for compounds that modulate hormone receptor activity
Li et al. A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43
US20100003256A1 (en) Use of TGF-Beta Antagonists in Treatment of Parathyroid-Related Disorders
WO2007149550A2 (fr) Modulation de la différenciation et des fonctions d'une cellule par fox01 et signalisation notch
Ballardin et al. Comparing Two Neurodevelopmental Disorders Linked to CK2: Okur-Chung Neurodevelopmental Syndrome and Poirier-Bienvenu Neurodevelopmental Syndrome—Two Sides of the Same Coin?
Wu et al. CD45 recruits adapter protein DOK-1 and negatively regulates JAK–STAT signaling in hematopoietic cells
US20080019909A1 (en) Modulation of Programmed Necrosis
Schaff et al. SLIC‐1/sorting nexin 20: A novel sorting nexin that directs subcellular distribution of PSGL‐1
EP1544295A1 (fr) Kinases 2 induites par des sels et leur utilisation
US20080069773A1 (en) Novel Sodium Channel
US7083935B2 (en) Androgen receptor complex-associated protein
US7892755B2 (en) Screening method
US20050142634A1 (en) Novel modulator of non-genomic activity of nuclear receptors (mnar) and uses thereof
WO2006021892A2 (fr) Hm74 et hm74a associes a l'inflammation dans les cellules endotheliales cuboides
US20070128667A1 (en) Secreted neural apoptosis inhibiting proteins
Si et al. RETRACTED: The retinoic acid receptor/CaMKII interaction: Pharmacologic inhibition of CaMKII enhances the differentiation of myeloid leukemia cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07796340

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

NENP Non-entry into the national phase in:

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07796340

Country of ref document: EP

Kind code of ref document: A2