WO2007113683A2 - Modulation de fonction de tc10 et procédé de traitement de troubles du métabolisme du glucose - Google Patents

Modulation de fonction de tc10 et procédé de traitement de troubles du métabolisme du glucose Download PDF

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WO2007113683A2
WO2007113683A2 PCT/IB2007/001658 IB2007001658W WO2007113683A2 WO 2007113683 A2 WO2007113683 A2 WO 2007113683A2 IB 2007001658 W IB2007001658 W IB 2007001658W WO 2007113683 A2 WO2007113683 A2 WO 2007113683A2
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ariadne
modulator
cell
molecule
tclo
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PCT/IB2007/001658
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WO2007113683A3 (fr
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Siew Hwa Ong
Jeffrey E. Pessin
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Agency For Science, Technology And Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to methods of treating hyperglycemia and diseases related to hyperglycemia, including diabetes and obesity.
  • TC 10 is a Rho GTPase involved in mediating the translocation of the GLUT4 glucose transporter to the cell surface. GLUT4 facilitates glucose uptake into muscle and adipose cells stimulated by insulin.
  • a recent report indicates that insulin- induced TClO activation is impaired in tissues of heart muscle and adipose of obese and diabetic mouse models (Gupta, A., and Mora, S., (2006, in press), BBRC).
  • a method of increasing glucose uptake of a cell comprising modulating activity of Ariadne- 1.
  • the activity may be E3 ligase activity of Ariadne- 1.
  • the modulating may comprise exposing the cell to a modulator of Ariadne- 1.
  • the modulator may be a small molecule modulator or a polypeptide modulator.
  • the modulating may comprise administering to the cell a nucleic acid molecule encoding the polypeptide modulator.
  • the polypeptide modulator may be a TClO mutant having a mutation of Glyl8 or Thr23.
  • the TC 10 mutant may be a Gl 8V mutant or a T23N mutant.
  • the modulating may comprise inhibiting Ariadne- 1 gene expression in the cell.
  • the modulating may comprise exposing the cell to a DNA enzyme that targets an Ariadne- 1 gene transcript.
  • the modulating may comprise exposing the cell to a small interfering RNA molecule that targets an Ariadne- 1 gene transcript.
  • a method of treating hyperglycemia or a disorder related to hyperglycemia in a patient comprising administering to the patient a modulator of Ariadne- 1 activity in a cell of the patient, wherein the cell is associated with the hyperglycemia or the disorder related to hyperglycemia.
  • the activity may be E3 ligase activity of Ariadne- 1.
  • the patient may be a human.
  • the disorder may be diabetes or obesity.
  • the modulator may be a small molecule modulator or a polypeptide modulator.
  • the method may comprise administering to the cell a nucleic acid molecule encoding the polypeptide modulator.
  • the polypeptide modulator may be a TClO mutant having a mutation of GIy 18 or Thr23.
  • the TClO mutant may be a G18V mutant or a T23N mutant.
  • the modulator may be capable of modulating Ariadne- 1 gene expression in the cell.
  • the modulator may be a DNA enzyme that targets an Ariadne- 1 gene transcript.
  • the modulator may be a small interfering RNA molecule that targets an Ariadne- 1 gene transcript. J
  • a molecule capable of modulating activity of Ariadne- 1 may be a polypeptide modulator of Ariadne- 1.
  • the polypeptide modulator may be a TClO mutant having a mutation of GIy 18 or Thr23.
  • the TClO mutant may be a G18V mutant or a T23N mutant.
  • the molecule may inhibit Ariadne- 1 gene expression.
  • the molecule may be a DNA enzyme that targets a Ariadne- 1 gene transcript.
  • the molecule may be a small interfering RNA that targets an Ariadne- 1 gene transcript.
  • a pharmaceutical composition comprising a modulator of Ariadne- 1 activity and a pharmaceutically acceptable diluent.
  • a method of identifying a modulator of Ariadne- 1 comprising contacting TClO with Ariadne- 1 in the presence of a test compound, and determining the effect of the test compound on the binding of TClO with Ariadne- 1.
  • the test compound may be a small molecule or a polypeptide.
  • a genetically modified non-human animal having an altered level of Ariadne- 1 activity as compared to said animal in non-genetically modified form.
  • the transgenic non-human animal may have substantially decreased or no expression of functional Ariadne- 1.
  • the genetically modified animal may comprise genetic modification of a nucleic acid encoding Ariadne- 1 such that at least part of a open reading frame encoding Ariadne- 1 is deleted, replace or interrupted, such that substantially less or no gene product, no stable gene product or no functional gene product is expressed.
  • the genetically modified non-human animal may comprise a nucleic acid molecule that interferes with or inhibits the transcription or translation of an Ariadne- 1 coding sequence in said animal.
  • Figure 1 is a schematic diagram depicting an insulin-triggered cascade in which TClO participates
  • Figure 2 is a schematic depicting regulation of TClO
  • Figure 3 is a schematic depicting protein interactions of TClO
  • Figures 4 to 7, 8A, 8B, 9A, 9B, and 10 are images showing immunoblotting results
  • Figures 11 to 13 show images of tested cells
  • Figure 14 shows test results of cells coexpressing Ariadne- 1 and GLUT4 upon insulin treatment
  • Figure 15 shows a prediction of ubiquinated lysine residues in TClO
  • Figure 16 to 18 are images showing immunoblotting results
  • Figure 19 is an image of an SDS gel
  • Figures 20 to 21 are images showing immunoblotting results
  • Figures 22 and 23 are computer screenshots showing data for gene structures of human and mouse Ariadne- 1 respectively.
  • TClO is involved in mediating translocation of GLUT4, a glucose transporter molecule, to the cell surface, in response to insulin signaling.
  • the insulin- triggered cascade in which TClO participates is depicted in Figure 1.
  • the inventors have discovered that the RING-type E3 ligase Ariadne- 1 associates specifically with TClO and ubiquitinates and induces proteasomal degradation of TClO.
  • the inventors have shown that Ariadne- 1, through ubiquitination of TClO leading to subsequent degradation of TClO, can abrogate insulin-induced GLUT4 expression on the plasma membrane of adipocytes.
  • a recent report (Gupta and Mora, (2006) BBRC) indicates that TClO activation is impaired in obesity and insulin deficiency.
  • El is an enzyme that activates ubiquitin. Once activated, ubiquitin is transferred to a ubiquitin-carrier protein, E2. E2 transfers activated ubiquitin to a protein substrate that is to be targeted for proteasomal degradation, either directly or by formation of an additional thiol-ester intermediate on the E3 ligase. Multiple ubiquitin moieties are conjugated on the target protein, forming a poly-ubiquitin tagged protein, which serves as the binding site and degradation signal for the 26S proteasome. The proteasome degrades the poly-ubiquitin protein to short peptides. Mono-ubiquitin moieties are released by de-ubiquitinating enzymes, thus becoming available for ligation on a subsequent protein substrate by an E3 ligase.
  • the present invention relates to the model that blocking the E3 ligase activity of Ariadne- 1 provides a novel method to promote blood glucose clearance via uptake by responsive cells (adipocyctes and muscle cells).
  • Such a method may be used to treat glucose metabolic disorders such as hyperglycemia, diabetes and obesity, and to complement current treatments of hyperglycemia and diseases or disorders associated with hyperglycemia, including diabetes and obesity. That is, in insulin deficient/resistant states such as diabetes and obesity, the prolongation of GLUT4 expression on the cell surface is a novel therapy.
  • the present methods relate to modulation of the function of Ariadne- 1 , to ultimately effect up-regulation of glucose uptake in cells by increasing the levels of TClO activity in cells, particularly in cells associated with hyperglycemia and hyperglycemia-related disorders.
  • a method of increasing glucose uptake in a cell involving modulation of the E3-ligase activity of Ariadne- 1.
  • a method for inhibiting ubiquitination of TClO in a cell comprising modulating activity of Ariadne- 1.
  • Ariadne- 1 refers to any Ariadne- 1 protein.
  • Ariadne- 1 is an E3 ligase that includes a RING domain, and which binds to and ubiquitinates TClO.
  • an E3 ligase is a protein involved in the ubiquitination pathway that interacts with an E2 ubiquitination-conjugating enzyme to receive ubiquitin moieties, and functions to transfer such ubiquitin moieties onto a protein substrate.
  • TClO is the protein substrate and Ariadne- 1 functions to transfer ubiquitin moieties onto TClO at the ubiquitination sites of TClO.
  • Ariadne- 1 as used herein includes homologs, fragments, derivatives or variants of Ariadne- 1 that possess the E3 ligase activity of Ariadne- 1.
  • a polynucleotide sequence or polypeptide sequence is a "homolog” of, or is “homologous” to another sequence if the two sequences have substantial identity over a specified region and the functional activity of the sequences is conserved (as used herein, the term “homologous” does not imply evolutionary relatedness).
  • Two polynucleotide sequences or polypeptide sequences are considered to have substantial identity if, when optimally aligned (with gaps permitted), they share at least approximately 50% sequence identity, or if the sequences share defined functional motifs.
  • optimally aligned sequences may be considered to be substantially identical (i.e.
  • identity refers to sequence similarity between two peptides or two polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the ClustalW program, available at http ://clustalw. genome. ad. j p, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J MoI. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.).
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. MoI. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis are available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.govA).
  • homologous amino acid sequence includes any polypeptide which is encoded, in whole or in part, by a nucleic acid sequence which hybridizes at 25-35°C below critical melting temperature (Tm), to any portion of a nucleic acid sequence encoding Ariadne- 1, including a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOS.: 1 to 10, set out below.
  • Tm critical melting temperature
  • a variant or derivative of Ariadne- 1 refers to an Ariadne- 1 or a fragment thereof, which retains the E3 ligase activity of Ariadne- 1, or an Ariadne- 1 that has been mutated at one or more amino acids, including point, insertion or deletion mutations, but still retains the E3 ligase activity of Ariadne- 1.
  • a variant or derivative therefore includes deletions, including truncations and fragments; insertions and additions, for example conservative substitutions, site-directed mutants and allelic variants; and modifications, including peptoids having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications.
  • conservative substitutions site-directed mutants and allelic variants
  • modifications including peptoids having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications.
  • the term “conserved amino acid substitutions” or “conservative substitutions” refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • the Ariadne- 1 may be human, chimpanzee, rat, mouse, cat, cow, chicken, Drosophila, Xenopus or zebrafish Ariadne- 1.
  • the Ariadne- 1 may have the sequence as set out in the following SEQ ID NOS.: 1 to 10.
  • KYSEQPSVNSNFQCEIRKPSMDVIV YVPKSNL YLKKLCGLVTKVP AVSVTVSV
  • VDDNTVMRLITDSKVKLKYQHLITNSFVECNRLLKWCP APDCHHVVKVQYP
  • E3 ligase activity of Ariadne- 1 refers to the ability of Ariadne- 1 to ligate ubiquitin onto TClO, resulting in poly-ubiquination of TClO. Such E3 ligase activity has the effect of targeting TClO for degradation by the 26S proteasome.
  • the term "cell” refers to a single cell, a plurality of cells or a population of cells, unless otherwise indicated herein.
  • the cell may be any cell in which glucose uptake is desired to be increased, including a cell that has decreased glucose uptake in response to insulin signaling.
  • the cell may be a cell in culture or it may be a cell within a patient.
  • glucose uptake of or by a cell refers to the process of transporting glucose across the cell membrane into the cell, including by the glucose transporter GLUT4, located on the surface of the cell.
  • the cell may be derived from any organism that expresses an Ariadne- 1 homolog, and in particular embodiments is a mammalian cell, including a mouse cell, a rat cell, a chimpanzee cell, a cow cell, a cat cell, a rabbit cell or a human cell.
  • the term "increasing glucose uptake” in a cell or “increase in glucose uptake” of a cell includes increasing the concentration or levels of TClO in the cell, increasing the half-life of TClO in the cell, and increasing the amount of GLUT4 on the cell surface. Increasing glucose uptake may be performed in vitro or in vivo.
  • Modulating or “modulation of, the E3 ligase activity of Ariadne- 1 refers to any mechanism of disrupting, interrupting, reducing, limiting, blocking or preventing the ability of Ariadne- 1 to perform its biological function or activity, including binding to TClO, ligating ubiquitin onto TClO and poly-ubiquitinating TClO, thereby resulting in an decrease in the rate of degradation of TClO, and thus ultimately leading to an increase in GLUT4 on the cell surface.
  • Modulation includes physical alteration of Ariadne- 1 , for example by post- translational modification, loss or lack of necessary post-translational modification, mutation of the amino acid sequence, including deletion, insertion and substitution mutation, or protein digestion.
  • Modulation also includes de-stabilizing, antagonizing, inhibiting, or competing with, the interaction between Ariadne- 1 and TClO, and which Ariadne- 1 interacts with TClO to effect transport or location of GLUT4 to the cell surface.
  • Modulation also includes genetic modification that results in substantially decreased or no expression of functional Ariadne- 1.
  • “Substantially decreased” and “substantially less” refers to levels of expression of Ariadne- 1 or functional Ariadne- 1 that are, for example, approximately 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% of the levels of expression of functional Ariadne- 1 that would occur in an unmodulated cell.
  • Such genetic modification includes a modification of a nucleic acid encoding Ariadne- 1, including an Ariadne- 1 gene, such that part, or all, of the open reading frame has been deleted, replaced or interrupted such that substantially less or no gene product, no stable gene product, or no functional gene product is expressed.
  • Such genetic modification also includes modification of a nucleic acid encoding Ariadne- 1, including an Ariadne- 1 gene, such that part, or all, of the Ariadne- 1 gene regulatory region has been deleted, replaced, interrupted or inhibited, resulting in substantially less or no protein being expressed from the gene encoding Ariadne- 1.
  • Functional Ariadne- 1 protein is Ariadne- 1, or a fragment of Ariadne- 1, that is translated, folded, post-translationally modified and localized within the cell, and which possesses the biological function or activity of Ariadne- 1, which may include the E3 ligase function of Ariadne- 1.
  • Such genetic modification further includes modifying the cell to transcribe an antisense RNA transcript that is complementary to at least a fragment of the mRNA molecule that is transcribed from the Ariadne- 1 gene, resulting in no translation, or reduced translation of the Ariadne- 1 transcript.
  • Such genetic modification further includes modifying the cell to transcribe or express a small interfering RNA (siRNA) molecule that targets an Ariadne- 1 gene transcript, resulting in no translation, or reduced translation of the Ariadne- 1 transcript.
  • siRNA small interfering RNA
  • the E3 ligase activity of Ariadne- 1 is modulated by a modulator molecule.
  • the modulator may be any molecule that competes with or mimics the binding of Ariadne- 1 to TClO, thereby blocking or reducing the ability of Ariadne- 1 to bind to TClO.
  • the modulator may be capable of being delivered internally to a cell, for example by active or passive transport into the cell, or by diffusion into the cell.
  • the modulator may also be modified to include a transport tag that will facilitate its transport into a cell.
  • Specific transport tags may be used in order to direct the modulator to be taken up by specific target cells.
  • the modulator may be modified to include a galactose residue to increase uptake of the modulator by hepatocytes, as is described in US 6,844,319, which is herein fully incorporated by reference.
  • the modulator may be included in a biomaterial which increases or induces uptake of the modulator by the cell, for example, by encapsulating the modulator in a liposome preparation.
  • a biomaterial which increases or induces uptake of the modulator by the cell
  • liposome preparation for example, by encapsulating the modulator in a liposome preparation.
  • the modulator is a polypeptide comprising part or all of TClO, including mutants or variants of TClO.
  • the polypeptide may comprise or consist of a mutated TClO, including human TClO having a mutation at GIy 18 or Thr23.
  • the polypeptide modulator is a TClO mutant having the mutation G18V or T23N.
  • the polypeptide may comprise or consist of the region of TC 10 that interacts with Ariadne- 1.
  • the polypeptide modulator may be a polypeptide containing a sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the region of TClO that interacts with Ariadne- 1.
  • some modifications and changes can be made in the structure of a polypeptide without substantially altering the biological activity of that peptide, to obtain a functionally equivalent polypeptide.
  • the present invention extends to biologically equivalent polypeptides that differ from a portion of the amino acid sequence of Ariadne- 1 interacting region of TClO and biologically active or immunogenic fragments thereof by conservative amino acid substitutions.
  • conservative amino acid substitution refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, VaI, Leu, lie, Phe, Trp, Pro, Met; acidic: Asp, GIu; basic: Lys, Arg, His; neutral: GIy, Ser, Thr, Cys, Asn, GIn, Tyr.
  • Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid.
  • Naturally- occurring non-genetically encoded amino acids include beta-alanine, 3 -amino- propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino- butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t- butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4- chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4- fluorophenylalanine, penicillamine, l,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienyla
  • the modulator peptide further comprises a sequence such as a membrane-translocating sequence that allows the peptide in which it is included to transported into a cell, for example the penetratin sequence derived from the Drosophila melanogaster antennapedia homeodomain protein, for example having the sequence ERQIKIWFQNRRMKWKK [SEQ ID NO. 11].
  • a sequence such as a membrane-translocating sequence that allows the peptide in which it is included to transported into a cell, for example the penetratin sequence derived from the Drosophila melanogaster antennapedia homeodomain protein, for example having the sequence ERQIKIWFQNRRMKWKK [SEQ ID NO. 11].
  • the polypeptide modulator may be incorporated into a larger fusion protein in order to increase the stability of the protein and to assist in delivery to a target cell.
  • the fusion protein may be designed to incorporate a specific protease cleavage site for recognition by a protease expressed in the target cell so that the polypeptide modulator is released from the fusion protein upon entry into the target cell.
  • a polypeptide modulator can be synthesized using standard protein synthesis techniques as are known in the art, for example using chemical peptide ligation methods, including solid phase peptide synthesis, to synthesize the peptide in the C-terminal to N-terminal direction, including using an automated peptide synthesizer.
  • molecular biology techniques may be used to design an expression cassette that will encode the polypeptide modulator, using standard molecular biology techniques known in the art, and described for example in Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual, 3 rd ed., Cold Spring Harbour Laboratory Press).
  • the expression cassette can be used in a suitable expression system.
  • the cassette may be contained in a bacterial plasmid and may be expressed in a bacterial cell, from which the polypeptide modulator can be isolated and purified.
  • the expression cassette will contain an open reading frame encoding the polypeptide modulator, optionally as a complete peptide or as part of a chimeric or fusion peptide or protein, from which the peptide may be released, for example by protease digestion.
  • the expression cassette will also contain suitable regulatory regions operably linked to the open reading frame, for example a promoter region, which may be an inducible promoter region.
  • modulation of protein-protein interaction is achieved by exposure of the cell to the modulator, allowing for uptake of the modulator by the cell, allowing the modulator to interact with Ariadne- 1, thereby reducing or blocking the ability of the Ariadne- 1 protein to effect its activity, such as its E3 ligase activity in the cell.
  • the modulator is a polypeptide
  • inclusion of the penetratin sequence, or any sequence which allows for polypeptide transport into a cell facilitates the uptake of the modulator by the cell.
  • the polypeptide modulator may also be encoded in a nucleic acid vector, which is delivered to the cell.
  • a nucleic acid vector which is delivered to the cell.
  • Such vectors and methods of making such vectors are known and include plasmids and viruses that have been genetically engineered to express the polypeptide modulator within the cell to which the modulator is to be delivered.
  • standard molecular biology techniques to prepare such vectors are known in the art, for example as described in Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual, 3 rd ed., Cold Spring Harbour Laboratory Press).
  • the modulator is a molecule capable of inhibiting expression of nucleic acid encoding Ariadne- 1, including an Ariadne- 1 gene.
  • the molecule capable of inhibiting expression of nucleic acid encoding Ariadne- 1, including an Ariadne- 1 gene may be a DNA enzyme that targets the transcript of a gene encoding Ariadne- 1.
  • a DNA enzyme is a magnesium-dependent catalytic nucleic acid composed of DNA that can selectively bind to an RNA substrate by Watson-Crick base-pairing and potentially cleave a phosphodiester bond of the backbone of the RNA substrate at any purine-pyrimidine junction (Santiago, F. S., et al., (1999) Nat Med 5: 1264-1269).
  • a DNA enzyme is composed of two distinct functional domains: a 15-nucleotide catalytic core that carries out phosphodiester bond cleavage, and two hybridization arms flanking the catalytic core; the sequence identity of the arms can be tailored to achieve complementary base-pairing with target RNA substrates.
  • the DNA enzyme will therefore have complementary regions that can anneal with regions on the transcript of an Ariadne- 1 gene flanking a purine- pyrimidine junction such that the catalytic core of the DNA enzyme is able to cleave the transcript at the junction, rendering the transcript unable to be translated to produce a functional Ariadne- 1 protein.
  • the DNA enzyme is designed to cleave the Ariadne- 1 transcript between the A and the U residues of the AUG start codon.
  • the DNA enzyme may be synthesized using standard techniques known in the art, for example, standard phosphoramidite chemical ligation methods may be used to synthesize the DNA molecule in the 3' to 5' direction on a solid support, including using an automated nucleic acid synthesizer.
  • the DNA enzyme may be synthesized by transcribing a nucleic acid molecule encoding the DNA enzyme.
  • the nucleic acid molecule may be contained within a DNA or RNA vector, for delivery into a cellular expression system, for example, a viral vector. Suitable viral vectors include vaccinia viral vectors and adenoviral vectors.
  • the modulation is achieved by exposing the cell to the DNA enzyme so that the DNA enzyme is taken up by the cell, and is able to target and cleave an Ariadne- 1 transcript in the cell, resulting in decreased or no expression of functional Ariadne- 1 protein in the cell.
  • Exposure may include transfection techniques, as are known in the art, or by microinjection techniques in which the DNA is directly injected into the cell.
  • Exposure may also include exposing the cell to the naked DNA enzyme, as cells may take up naked DNA in vivo.
  • the DNA enzyme is included in a nucleic acid vector, such as a viral vector, the cell may be infected with the viral vector.
  • the nucleic acid modulator capable of inhibiting expression of nucleic acid encoding Ariadne- 1, including an Ariadne- 1 gene may be an antisense RNA molecule or a small interfering RNA (siRNA) molecule.
  • siRNA small interfering RNA
  • the antisense RNA molecule will contain a sequence that is complementary to at least a fragment of an RNA transcript of an Ariadne- 1 gene, and which can bind to the Ariadne- 1 transcript, thereby reducing or preventing the expression of the Ariadne- 1 gene in vivo.
  • the antisense RNA molecule will have a sufficient degree of complementarity to the target mRNA to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, such as under physiological conditions.
  • the siRNA molecule may be any double-stranded RNA molecule, including a self-complementary single-stranded molecule that can fold back on itself to form the double-stranded siRNA, which induces gene-specific RNA interference in a cell, leading to decreased or no expression of the Ariadne- 1 gene in vivo.
  • An siRNA typically targets a 19-23 base nucleotide sequence in a target mRNA, as described in Elbashir, et al. (2001) EMBO J 20: 6877-6888, the contents of which is incorporated herein by reference.
  • the cell is exposed to the antisense RNA, a nucleotide encoding the antisense RNA, the siRNA or a nucleotide encoding the siRNA, for example a nucleic acid vector containing a nucleic acid molecule which allows for transcription of an antisense transcript or a single-stranded, self- complementary siRNA molecule capable of forming a double-stranded siRNA.
  • a nucleic acid vector containing a nucleic acid molecule which allows for transcription of an antisense transcript or a single-stranded, self- complementary siRNA molecule capable of forming a double-stranded siRNA.
  • Such an antisense molecule, siRNA molecule or vector may be synthesized using nucleic acid chemical synthesis methods and standard molecular biology cloning techniques as described above.
  • the method involves modulating the activity of Ariadne- 1, including the E3 ligase activity of Ariadne- 1, in a cell of the patient, wherein the cell is associated with hyperglycemia or the disorder related to hyperglycemia.
  • a "disorder related to hyperglycemia” is a disease or disorder which is characterized by the presence of hyperglycemia, includes hyperglycemia as a symptom, is caused by hyperglycemia, or is linked to the presence or occurrence of hyperglycemia in a patient.
  • a disorder related to hyperglycemia includes diseases or disorders in which a cell of a patient has reduced uptake of glucose in response to insulin signaling, which in a healthy individual would exhibit normal glucose uptake in response to insulin signaling. Such disorders include hyperglycemia, diabetes and obesity.
  • a cell is associated with hyperglycemia or a disorder related to hyperglycemia if that cell is a cell that has reduced glucose uptake, including as a result of reduced levels of GLUT4 on the surface of the cell, so as to result in the disorder in the patient, or if the disorder is characterized by the presence of such a cell.
  • treating hyperglycemia or a disorder related to hyperglycemia refers to an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, amelioration or palliation of the disease state, and remission (whether partial or total).
  • Treating can also mean prolonging survival of a patient beyond that expected in the absence of treatment.
  • “Treating” can also mean inhibiting the progression of disease, slowing the progression of disease temporarily, although more preferably, it involves halting the progression of the disease permanently.
  • the patient is any animal in need of treatment of hyperglycemia or a disorder related to hyperglycemia, including a mammal, including a mouse, rat, rabbit or human.
  • the therapeutic effect is achieved by administering to the patient modulator of Ariadne- 1 activity.
  • the modulator is a molecule capable of modulating the E3 ligase activity of Ariadne- 1.
  • the modulator of Ariadne- 1 activity is any molecule that may be used to effect modulation in a cell as described above, including a modulator that mimics the binding of TClO to Ariadne- 1, thereby binding to Ariadne- 1 in such a manner to block or reduce the ability of Ariadne- 1 to bind to endogenous TClO in the cell.
  • Such a modulator may be for example a small molecule or a polypeptide, as described above.
  • the modulator of Ariadne- 1 may be a DNA enzyme, as described above, including a DNA enzyme molecule, or a nucleic acid molecule encoding a transcribable DNA enzyme.
  • the molecule capable of modulating the activity of Ariadne- 1 may be a nucleic acid molecule encoding a transcribable antisense mRNA that contains a sequence that is complementary to an Ariadne- 1 mRNA transcript, or a molecule encoding an siRNA molecule that targets an Ariadne- 1 mRNA transcript.
  • an effective amount of the modulator is administered to the patient.
  • effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to treat the hyperglycemia or the specific disorder related to hyperglycemia.
  • the molecule is administered to the patient using standard techniques known in the art.
  • the molecule may be administered systemically, or may be administered directly at the site at which the proliferating cell that is associated with the proliferative disorder is located. Delivery to the site includes topical administration, injection to the site, or surgical implantation, for example at a site of a tumour.
  • the concentration and amount of the modulator of activity of Ariadne-1 to be administered will vary, depending on the proliferative disorder to be treated, the type of cell associated with the proliferative disorder, the type of molecule that is administered, the mode of administration, and the age and health of the patient.
  • the modulator of Ariadne-1 activity may be formulated as an ingredient in a pharmaceutical composition. Therefore, in a further embodiment, there is provided a pharmaceutical composition comprising a modulator of Ariadne-1, and optionally a pharmaceutically acceptable diluent.
  • the invention in one aspect therefore also includes such pharmaceutical compositions for use in treating hyperglycemia or a disorder related to hyperglycemia.
  • the compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives and various compatible carriers.
  • the modulator of Ariadne-1 may be formulated in a physiological salt solution.
  • peptides and proteins may be unstable upon administration, where the modulator of Ariadne-1 is a peptide or a protein, it may be desirable to include the peptide or the protein in a liposome or other biomaterial useful for protecting and/or preserving the peptide or protein until it is delivered to the target cell.
  • Solutions of modulator of Ariadne- 1 may be prepared in a physiologically suitable buffer. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms, and that will maintain the function of the modulator of Ariadne- 1.
  • suitable formulations Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
  • compositions may additionally contain other therapeutic agents useful for treating the hyperglycemia or the particular disorder related to hyperglycemia.
  • the proportion and identity of the pharmaceutically acceptable diluent is determined by chosen route of administration, compatibility with live cells, and standard pharmaceutical practice. Generally, the pharmaceutical composition will be formulated with components that will not kill or significantly impair the biological properties of the modulator of Ariadne- 1.
  • the pharmaceutical composition can be prepared by known methods for the preparation of pharmaceutically acceptable compositions suitable for administration to patients, such that an effective quantity of the modulator of Ariadne- 1 , and any additional active substance or substances, is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the pharmaceutical compositions include, albeit not exclusively, solutions of the modulator of Ariadne- 1, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffer solutions with a suitable pH and iso-osmotic with physiological fluids.
  • the pharmaceutical composition may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
  • the composition of the invention may be administered topically, surgically or by injection to a desired site.
  • the dose of the pharmaceutical composition that is to be used depends on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and other similar factors that are within the knowledge and expertise of the health practitioner. These factors are known to those of skill in the art and can be addressed with minimal routine experimentation.
  • modulators of Ariadne- 1 as described herein including a polypeptide modulator of Ariadne- 1, a DNA enzyme that targets an Ariadne- 1 transcript, an antisense RNA that is complementary to at least a portion or fragment of an Ariadne- 1 transcript or a small interfering RNA molecule that targets an Ariadne- 1 transcript.
  • the molecule is a polypeptide comprising a sequence of TClO, or a TClO mutant including a TClO having a mutation at GIy 18 or Thr23, including a TClO having a G18V or T23N mutation.
  • a method for identifying or screening for modulators of Ariadne- 1, including small molecule or peptide modulators, is also provided.
  • Test molecules are used to compete with the binding of TClO with Ariadne- 1.
  • Ariadne- 1 is contacted with TClO in the presence of the test compound, and the effect of the test compound on the binding between Ariadne- 1 and TClO is monitored.
  • competition assays, and methods of measuring interruption of protein-protein interactions are known, and include chromatography assays, immunoassays, immobilization assays, immunoprecipitation techniques, gel retardation assays.
  • the methods of screening for modulators of Ariadne- 1 are well suited for screening combinatorial libraries of compounds.
  • transgenic non-human animals such as for example mice, in which the gene for Ariadne- 1 has been deleted, knock out, or the expression of the gene has been interrupted or in which Ariadne- 1 is over-expressed in specific tissues for use as model systems to study glucose metabolic disorders, including hyperglycemia or a disorder related to hyperglycemia.
  • Methods of generating transgenic animals are known in the art.
  • nucleic acid molecule that is designed to interrupt or disrupt expression of endogenous Ariadne- 1 or to overexpress Ariadne- 1 in the resulting animal is introduced into a ⁇ progenitor cell of the animal, the cell is allowed to develop into an animal, and the animal is genetically screened to determine if the nucleic acid molecule has been integrated into the chromosomal material of the animal.
  • Animals in which the nucleic acid molecule has been chromosomally integrated will be heterozygous for the transgene, and may be crossbred with each other to produce a homozygous transgenic animal, if desired.
  • EXAMPLE 1 Interaction between Ariadne- 1 and TClO in vitro
  • HEK293T cells were transfected with a plasmid encoding flag-tagged Ariadne- 1. Following expression of Ariadne- 1, the cells were lysed in protein lysis buffer. Meanwhile, samples of bacterially produced GST-TClO wild type (WT) were coupled to glutathione beads. Aliquots of beads labeled with or without GDP ⁇ S and GTPpS, as indicated, were incubated with cell Iy sates containing Ariadne- 1 for 1 hour by rotating in microtubes at 4°C.
  • HEK293T cells were transfected with a plasmid encoding flag-tagged Ariadne- 1 C terminal construct. Following expression of Ariadne- 1, the cells were lysed in protein lysis buffer. Meanwhile, samples of bacterially produced GST-TClO wild type (WT) were coupled to glutathione beads. Aliquots of beads labeled with or without GDP ⁇ S and GTP ⁇ S, as indicated, were incubated with cell lysates for Ariadne-1 C terminal for 1 hour by rotating in microtubes at 4°C.
  • EXAMPLE 2 Interaction between Ariadne-1 and TClO in vivo
  • HEK293T cells were co-transfected with plasmids encoding flag-tagged Ariadne-1 construct and various HA-tagged TClO mutants, as indicated, and lysed in protein lysis buffer.
  • Cell lysates were subjected to anti-HA immunoprecipitation (IP) followed by immunoblotting with anti-Flag to detect associated Ariadne-1.
  • IP anti-HA immunoprecipitation
  • Reciprocally, cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting with anti-HA to detect associated TClO. Total protein expression was confirmed by immunoblotting as shown in Figure 6.
  • T41A and Y46C both are mutations that result in effector binding defects; T23N, a dominant negative mutant; Q67L, a mutant that is constitutively active; Gl 8 V, an inactive mutant; and double mutants G18V/T41A and G18V/Y46C.
  • EXAMPLE 3 Ariadne-1 targets TClO for ubiquitination and degradation through ubiquitin-dependent proteasome pathway
  • TClO stability Expression of Ariadne-1 decreases the steady- state level of TClO: HEK293T cells were transiently transfected with the indicated combinations of plasmids encoding flag-tagged Ariadne-1, and/or myc-tagged ubiquitin, and HA-tagged TClO wild type or mutants. 16 hours after transfection, cells were incubated with or without 20 ⁇ M MGl 32 for another 4 hours and then lysed in protein lysis buffer. Steady state protein levels were determined by immunoblotting total cell lysates with anti-HA antibody.
  • HEK293T cells were transfected with the indicated combinations of plasmids encoding flag-tagged Ariadne- 1, and/or myc- tagged ubiquitin, and HA-tagged TClO wild type. After 24 hours, cells were directly lysed in lysis buffer containing 1% SDS by boiling for 10 minutes. Direct lysis of cells by boiling in SDS lysis buffer prevents degradation of poly-ubiquitinated substrate by proteases after cell lysis. Most protein-protein interactions are dissociated while poly-ubiquitin chains remain attached to the substrate after the boiling because they are covalently bound. A poly-ubiquitin ladder (myc-tagged) was detected by immunoblot, seen in Figures 9A and 9B. Pull-down of TClO using GST- PBD revealed the polyubiquitinated TClO.
  • Figure 20 demonstrates the results of degradation of TClO in the presence of the insulin receptor.
  • EXAMPLE 5 Translocation of GLUT4 And Insulin Treatment of
  • a cell-based system was set up to analyse the GLUT4 glucose transporter in insulin signaling and glucose metabolic disorders. Techniques involved include differentiation of 3T3-L1 preadipocytes to adipocytes; electroporation protocol for differentiated 3T3-L1 adipocytes; GLUT4 translocation assays in 3T3-L1 adipocytes.
  • GFP-tagged GLUT4 transporter in 3T3-L1 adipocytes resulted in approx. 50% reduction in plasma membrane translocation of GLUT4 stimulated by insulin treatment.
  • EXAMPLE 6 Mapping of Ubiquinitated Residues in TClO
  • Figures 15- 20 shown data relating to ubiquination of specific residues in TClO.

Abstract

L'invention concerne une méthode pour augmenter l'absorption de glucose d'une cellule consistant à moduler l'activité d'Ariadne-1. L'invention concerne également une méthode de traitement de l'hyperglycémie ou d'un trouble lié à l'hyperglycémie chez un patient, consistant à administrer au patient un modulateur de l'activité d'Ariadne-1 dans une cellule du patient, la cellule étant associée à l'hyperglycémie ou au trouble lié à l'hyperglycémie.
PCT/IB2007/001658 2006-03-31 2007-03-30 Modulation de fonction de tc10 et procédé de traitement de troubles du métabolisme du glucose WO2007113683A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2449417A1 (fr) * 2002-12-04 2004-06-04 Claus-Jens Walter Doersen Methodes d'identification de composes pour le traitement de dysfonctionnements cardiaques

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Publication number Priority date Publication date Assignee Title
CA2449417A1 (fr) * 2002-12-04 2004-06-04 Claus-Jens Walter Doersen Methodes d'identification de composes pour le traitement de dysfonctionnements cardiaques

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CHIANG S.-H. ET AL.: 'Cloning and Functional Characterization of Related TC10 Isoforms, a Subfamily of Rho Proteins Involved in Insulin-stimulated Glucose Transport' J. BIOL. CHEM. vol. 277, no. 15, 2002, pages 13067 - 13073 *
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