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WO2003024997A1 - Peptides inhibiteurs de la telomerase et leurs utilisations - Google Patents

Peptides inhibiteurs de la telomerase et leurs utilisations Download PDF

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Publication number
WO2003024997A1
WO2003024997A1 PCT/AU2002/001263 AU0201263W WO03024997A1 WO 2003024997 A1 WO2003024997 A1 WO 2003024997A1 AU 0201263 W AU0201263 W AU 0201263W WO 03024997 A1 WO03024997 A1 WO 03024997A1
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Prior art keywords
peptide
telomerase
teipp
seq
teipp1
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PCT/AU2002/001263
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English (en)
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Jun-Ping Liu
He Li
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Jun-Ping Liu
He Li
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Publication of WO2003024997A1 publication Critical patent/WO2003024997A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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 to peptides that inhibit telomerase, and uses of these peptides, particularly in a method of treating cancer.
  • Telomerase a specialized ribonucleoprotein complex that stabilizes and extends telomeres of eukaryotic chromosomes, plays an important role in cell survival and replicative lifespan.
  • telomerase In many human somatic tissues, telomerase is repressed, but it is activated in the early stages of tumor progression in most human cancers.
  • Telomeres are critical in maintaining genome integrity and in controlling cell lifespan. Cancer cells obtain immortality and proliferate indefinitely through the maintenance of telomere structures by telomerase.
  • telomere The activity of telomerase is repressed in many somatic tissues, whereas the enzyme is activated during tumor progression in most cancers.
  • the enzyme telomerase becomes present in most of all type cancers and its presence is fundamental to cancer development.
  • telomerase activity has a role in cancer and other proliferative disorders, effective inhibitors of telomerase and methods of inhibiting telomerase have yet to be elucidated.
  • the present invention provides a peptide sequence encoding a telomerase inhibitory peptide (TEIPP) having an amino acid sequence of GARTFRRXKRAXRLTSRVK (SEQ ID NO:1), a functional equivalent, variant or fragment thereof and wherein X is E or Q.
  • TEIPP telomerase inhibitory peptide
  • the present invention provides a peptide sequence encoding a telomerase inhibitory peptide (TEIPP) having an amino acid sequence of GARTFRREKRAERLTSRVK (SEQ ID NO:2) or GARTFRRQKRAQRLTSRVK (SEQ ID NO:3), a functional equivalent, variant or fragment thereof.
  • TEIPP telomerase inhibitory peptide
  • the TEIPP amino acid sequence corresponds to amino acids 641 to 659 of a native TERT amino acid sequence.
  • the present invention also includes as another embodiment a nucleotide sequence which encodes a telomerase inhibitory peptide (TEIPP) said sequence selected from the group including: a) a nucleotide sequence which encodes a peptide having an amino acid sequence of: GARTFRRXKRAXRLTSRVK (SEQ ID NO:1) a functional equivalent, variant or fragment thereof and wherein X is E or Q; b) a nucleotide sequence which encodes a peptide having an amino acid sequence of:
  • GARTFRREKRAERLTSRVK (SEQ ID NO:2); c) a nucleotide sequence which encodes a peptide having an amino acid sequence of:
  • GARTFRRQKRAQRLTSRVK (SEQ ID NO:3) d) a nucleotide sequences capable of hybridising to a sequence of a), b) or c); e) a nucleotide sequences degenerate, as a result of the genetic code, from a nucleotide sequence of a), b) or c); and f) functional equivalents, variants or fragments of any one of sequences a) to e).
  • a composition for inhibiting telomerase comprising a telomerase inhibitory peptide (TEIPP) including an amino acid sequence of GARTFRRXKRAXRLTSRVK (SEQ ID NO:1), a functional equivalent, variant or fragment thereof and wherein X is E or Q, GARTFRREKRAERLTSRVK (SEQ ID NO:2) or GARTFRRQKRAQRLTSRVK (SEQ ID NO:3), a functional equivalent, variant or fragment thereof and a carrier.
  • TEIPP telomerase inhibitory peptide
  • composition for inhibiting telomerase further includes another telomerase inhibitory peptide, telomerase- inhibitory polypeptide 1 (TEIPP1) a functional equivalent, variant or fragment thereof.
  • TEIPP1 telomerase- inhibitory polypeptide 1
  • a method of inhibiting cell growth in a cell culture comprising administering an effective amount of a telomerase inhibitory peptide selected from TEIPP, TEIPP1 , or a combination thereof, functional equivalents, variants or fragments thereof to a cell culture.
  • a method of inducing cell death comprising administering an effective amount of a telomerase inhibitory peptide selected from TEIPP, TEIPP1 , or a combination thereof, functional equivalents, variants or fragments thereof.
  • the present invention provides a method of treating cancer or other proliferative disorder in a subject, the method including: administering an effective amount of a telomerase inhibitory peptide to the subject, wherein the telomerase inhibitory peptide includes at least one peptide selected from the group including TEIPP and TEIPP1 or a functionally equivalent variant thereof.
  • the present invention provides a vaccine for treating or preventing cancer or other proliferative disorder wherein the vaccine includes a telomerase inhibitory peptide, wherein the telomerase inhibitory peptide includes at least one peptide selected from the group including TEIPP and TEIPP1.
  • the present invention provides a method of inducing cell proliferation by inhibiting production in the cell of a telomerase inhibitory peptide, wherein the telomerase inhibitory peptide is at least one of TEIPP or TEIPP1.
  • FIG. 1 shows a schematic presentation of human telomerase reverse transcriptase (hTERT) and the origin from which peptide TEIPP is produced. Five peptides from hTERT were tested, their locations are indicated from A to E. The sequence and putative -helix structure of TEIPP are presented with positively and negatively charged residues on each side indicated.
  • hTERT human telomerase reverse transcriptase
  • Figure 2 shows a peptide from telomerase catalytic subunit, termed TEIPP, inhibit telomerase activity in both sequence- and enzyme/substrate concentration- dependent manners.
  • TEIPP a peptide from telomerase catalytic subunit
  • FIG. 1 An autoradiograph showing TEIPP inhibition of telomerase activity reflected by the quantities of de novo synthesized telomere ladders spaced in six nucleotides in each track from track 1 to track 10.
  • Nuclear telomerase extracts from human breast cancer cells (0.4 ⁇ g) were incubated in a typical telomerase activity assay in the presence or absence of different concentrations of TEIPP or scrambled control peptide at 30 °C for 30 min and the synthesized telomeric DNA resolved by electrophoresis.
  • telomere ladders the control non-telomeric DNA synthesized, TEIPP at different concentrations and the scrambled peptide are indicated.
  • N negative control in the presence of RNase A to eliminate telomerase activity.
  • B Effect of increasing concentrations of telomerase extracts on TEIPP inhibition of telomerase activity. Nuclear telomerase extracts at different concentrations indicated were used in telomerase activity analysis in the presence or absence of TEIPP. Telomerase activity is shown as the levels of telomeres in each lane.
  • C Effect of increasing concentrations of telomerase substrate (TS) on TEIPP inhibition of telomerase activity. Different concentrations of TS were included in telomerase activity analysis in the presence or absence of TEIPP as indicated. Telomerase activity is shown as the levels of telomeres in each lane. All results are from one of at least two similar experiments.
  • Figure 3 shows TEIPP and TEIPP1 inhibit telomerase activity synergistically.
  • A Effect of combinations of TEIPP1 and TEIPP scrambled peptide on telomerase activity was determined by incubating telomerase extract in a telomerase activity assay in the presence or absence of TEIPP1 (0.2-0.8 ⁇ M) plus or minus TEIPP scrambled control peptide (0.2-0.8 ⁇ M).
  • ⁇ Effect of combinations of TEIPP1 and TEIPP. Results are means ⁇ SD from three similar experiments.
  • Figure 4 shows effects of TEIPP on telomerase activity and cell survival in cultured human breast cancer cells.
  • A Effects of TEIPP1 and TEIPP on telomerase activity in intact cells.
  • Cultured MCF-7 cells were incubated with penetratin-coupled TEIPP1 (lane 7), TEIPP (lane 6), TEIPP1 plus TEIPP (lane 5), TEIPP1 scrambled peptide (S1 , lane 4), TEIPP scrambled (S2, lane 3) or S1 plus S2 (lane 2) for 15 hours and telomerase activity was measured in the nuclear lysates.
  • N negative controls of telomerase activity, ( ⁇ ,C).
  • TEIPP1 and TEIPP induce rapid cell death.
  • Phase contrast microphotographs show massive dead floating cells after 24 hours of treatments with TEIPP1 or TEIPP as indicated ( ⁇ ) and markedly reduced cell numbers after 48 hours in MCF-7 cell cultures (C). Results are representatives of three separate experiments.
  • Figure 5 shows effects of amino acid deletion from TEIPP1 and TEIPP on TEIPP inhibition of telomerase activity.
  • A Deletions of either N- or C-terminal regions of TEIPP eliminate the TEIPP inhibitory activity. Five peptides including TEIPP and its truncated forms as indicated were examined for their effects on telomerase activity. Ctl, control in the presence of RNase A. TE, in the absence of synthetic peptides. Results shown are one of three similar experiments, ( ⁇ ). Various peptides derived from hTERT and inhibitory effects of various TEIPP derivatives on telomerase activity. (C).
  • FIG. 6 shows phosphorylation of TEIPP by different protein kinases.
  • PKC protein kinase C ⁇
  • CDK1 protein kinase C ⁇
  • TEIPP and its scrambled control peptide were incubated with different protein kinases as indicated under appropriate protein phosphorylation conditions (See examples for details),
  • ⁇ Phosphorylation of TEIPP by different protein kinases differentially regulates TEIPP inhibition of telomerase activity.
  • TEIPP Phosphorylated (P, lanes 4 and 6) and non-phosphorylated (N, lanes 3 and 5) TEIPP by various protein kinases as indicated were tested for their activity in telomerase inhibition.
  • C control in the presence of RNase A.
  • T (lane 2), telomerase activity in the absence of any forms of TEIPP.
  • C Single amino acid mutations of TEIPP to mimic protein phosphorylation eliminate the inhibitory activity of TEIPP. Effects of TEIPP, scrambled control peptide (A), TEIPP mutant carrying T644D mutation (B) and TEIPP mutant with T655D mutation (C) on telomerase activity were determined. At three concentrations as indicated, both T644D and T655D completely block TEIPP inhibition of telomerase activity. Results are representative of three separate similar experiments.
  • GARTFRRXKRAXRLTSRVK (SEQ ID NO: 1) a functional equivalent, variant or fragment thereof and wherein X is E or Q.
  • a peptide sequence encoding a telomerase inhibitory peptide having an amino acid sequence of GARTFRREKRAERLTSRV (SEQ ID NO: 2) or
  • GARTFRRQKRAQRLTSRVK (SEQ ID NO: 3), a functional equivalent, variant or fragment thereof.
  • the amino acid sequence corresponds to amino acids 641 to 659 of a native TERT amino acid sequence.
  • Applicants have identified peptides that represent inhibitory elements of the telomerase complex. Application of the peptides in cancer cell cultures results in specific inhibition of telomerase activity in vivo, and induces significant apoptosis of cultured cancer cells.
  • the inhibitory actions of the peptides can be further modulated by altering the charged residues and by protein phosphorylation. Accordingly, the present invention provides telomerase and cancer inhibitory reagents useful in developing a therapeutic modality to treat conditions such as malignancy.
  • Human telomerase is composed of an RNA subunit and several protein components.
  • the RNA subunit (hTR) contains the template for telomeric DNA reverse transcription, whereas the protein components include human telomerase reverse transcriptase (hTERT) and the telomerase associated protein 1 (hTEP1) that co-purifies with telomerase.
  • hTR and hTEP1 are widely expressed in normal human tissues, but hTERT is rate-limiting and expressed concomitantly with telomerase activity during cell immortalization. Reconstitution experiments suggest that hTERT and hTR constitute a minimum core structure for the telomerase holoenzyme.
  • telomere elongation and permanent cell growth suggest hTERT as rate limiting for telomerase activity, whereas inhibition of telomerase results in erosion of telomeres, compromise of growth capacity and apoptosis of highly proliferative cells.
  • Both hTERT and hTEP1 have been shown to be phosphoproteins, phosphorylated by protein kinase C ⁇ and dephosphorylated by protein phosphatase 2A. Phosphorylation and dephosphorylation reversibly increase and decrease telomerase activity.
  • hTERT and hTEP1 are large proteins and their enzymatic activity is controlled by inter- and intra-molecular interactions between and within telomerase protein subunits.
  • telomerase, and telomerase inhibitory peptide may be from any species, including human, bovine, ovine, porcine, murine, equine, and is preferably mammalian.
  • the telomerase is inhibited in a tumour.
  • a "telomerase inhibitory peptide " as used herein includes a peptide that decreases telomerase activity and/or interaction with telomeres.
  • telomerase activity is decreased by the peptide competing with structures of telomerase components. Most preferably, the decreased activity results in inhibition of the telomerase activity.
  • inhibitors or “inhibitory” as used herein means substantial inhibition where the activity is substantially ceased.
  • TEIPP telomerase inhibitory peptide derived from the sequence of TERT. Most preferably, the TEIPP is derived from human TERT.
  • Distal as used herein includes peptides that substantially correspond to regions of TERT, and includes functionally active variants.
  • fragment relates to a portion of the peptide sequence that is less than that of a full length sequence but functions in a similar manner to the full length sequence of the peptide.
  • TEIPP may be produced using any techniques well known to those of skill in the art, including, but not limited to, synthesis using commercially available peptide synthesis apparatus or TEIPP may be isolated and purified from natural sources or followed by fragmentation to obtain the desired peptide.
  • the synthetic TEIPP peptide may also be obtained from commercial sources such as Auspep International Pty. Ltd. (Victoria, Australia) or Chiron Technologies Pty. Ltd. (Victoria, Australia).
  • the present invention also includes as another aspect a nucleotide sequence which encodes a telomerase inhibitory peptide (TEIPP) said sequence selected from the group including: a) a nucleotide sequence which encodes a peptide having an amino acid sequence of:
  • TEIPP telomerase inhibitory peptide
  • GARTFRRXKRAXRLTSRVK (SEQ ID NO: 1) a functional equivalent, variant or fragment thereof and wherein X is E or Q; b) a nucleotide sequence which encodes a peptide having an amino acid sequence of:
  • GARTFRREKRAERLTSRVK (SEQ ID NO: 2); c) a nucleotide sequence which encodes a peptide having an amino acid sequence of:
  • GARTFRRQKRAQRLTSRVK (SEQ ID NO: 3); d) a nucleotide sequences capable of hybridising to a sequence of a), b) or c); e) a nucleotide sequences degenerate, as a result of the genetic code, from a nucleotide sequence of a), b) or c); and f) functional equivalents, variants or fragments of any one of sequences a) to e).
  • the peptide sequence of the present invention may therefore be produced recombinantly by expressing the nucleotide sequence which encodes the TEIPP.
  • fragment relates to a nucleic acid sequence it means that it is less than that of full length but is capable of hybridising to a full length nucleotide capable of encoding an amino acid sequence of a telomerase inhibitory peptide as herein described. Where these terms relate to a changed nucleic acid molecule, the change does not result in a change in the reading frame of a peptide coding region and preferably encodes a peptide having no change, or only a minor reduction in biological function.
  • nucleic acid sequences which encode the same or functionally equivalent amino acid sequence are included within the scope of the current invention.
  • Such alterations of the nucleotide sequence may include substitutions of different nucleotides resulting in the same or a functionally equivalent gene product.
  • the TEIPP peptide may be derived from analysis of sequence hydrophilicity and charge characteristics to identify amino acid sequences involved in inter- and/or intra-molecular interactions within or between telomerase components. Regions of native TERT corresponding to these characteristics may be selected from the group including CRAVRSLLRSHYREV (SEQ ID NO: 4), GPPSTSRPPRPWDTPC (SEQ ID NO: 5), CAREKPQGSVAAPEEEDTD (SEQ ID NO: 6), GARTFRREKRAERLTSRVK (SEQ ID NO: 2), GARTFRRQKRAQRLTSRVK (SEQ ID NO: 3) and ANPALSSDFKTILD (SEQ ID NO: 7) or a functionally equivalent variant thereof. More preferably the regions correspond to sequences selected from the group including 7 CRAVRSLLRSHYREV 21 (SEQ ID NO: 4), 306 GPPSTSRPPRPWDTPC 321 (SEQ ID NO: 5),
  • TEIPP is 641 GARTFRREKRAERLTSRVK 659 (SEQ ID NO: 2) or 641 GARTFRRQKRAQRLTSRVK 659 (SEQ ID NO: 3) or a functional equivalent, variant or fragment thereof.
  • Other functional equivalents may be provided by varying charged residues. Applicants have found that charged residues play an important role in the interaction between telomerase inhibitory peptides and the telomerase holoenzyme.
  • TEIPP has a molecular mass of approximately 2421 Dalton. More preferably, the telomerase inhibitory peptide is substantially smaller than the telomerase components TERT and TEP1 (-130,000 and 300,000 Dalton, respectively).
  • the TEIPP peptide forms an ⁇ -helix with positively charged amino acid residues on one side of the helix and negatively charged/potentially negatively charged (phosphorylation sites) on another. More preferably the TEIPP helix interacts with an electrochemically polarized groove on the surface of telomerase through mutual electrostatic attractions that are regulated by protein phosphorylation (adding a phosphate moiety). Therefore, without being limited by theory, the telomerase inhibitors of the present invention may cause cancer cell death by a mechanism involving competing internal sequences of telomerase through interactions involving electrostatic force and thereby inhibiting telomerase interactions with telomeres.
  • TEIPP interacts with the telomerase holoenzyme. More preferably, TEIPP forms an ⁇ -helix with positively charged amino acid residues on one side of the helix and negatively charged/potentially negatively charged (phosphorylation sites) on another. Most preferably, the TEIPP helix interacts with an electrochemically polarized groove on the surface of the telomerase holoenzyme through mutual electrostatic attractions. These mutual electrostatic attractions may be regulated by protein phosphorylation.
  • Phosphorylation, and phosphorylation mimicking mutations of putative phosphorylation sites may inhibit the inhibitory function of the peptide of the invention. Therefore, regulation of phosphorylation mechanisms, for example reducing or inhibiting phosphorylation by protein kinase C, may enhance inhibition of telomerase by the peptides of the invention. Examples of phosphorylating mechanisms include, but are not limited to, mechanisms involving protein kinase C ⁇ , ERK1 and cdc2 protein kinase.
  • Preferably regulation of phosphorylation includes, but is not limited to phosphorylation of threonine or serine residues. Most preferably inhibition of telomerase is increased by dephosphorylation of at least one or more of these potentially phosphorylated residues.
  • compositions for inhibiting telomerase comprising a telomerase inhibitory peptide (TEIPP) including an amino acid sequence of GARTFRREKRAERLTSRVK (SEQ ID NO: 2) or GARTFRRQKRAQRLTSRVK (SEQ ID NO: 3), a functional equivalent, variant or fragment thereof and a carrier.
  • TEIPP telomerase inhibitory peptide
  • composition for inhibiting telomerase further includes another telomerase inhibitory peptide, telomerase- inhibitory polypeptide 1 (TEIPP1) a functional equivalent, variant or fragment thereof.
  • TEIPP1 telomerase- inhibitory polypeptide 1
  • the TEIPP1 and TEIPP may be administered in combination with each other, to provide a synergistic effect, and/or another factor as part of a complex.
  • Suitable complexes may include a radioisotope or toxin.
  • Preferably low concentrations of TEIPP are used in combination with TEIPP1 to inhibit telomerase.
  • TEIPP1 and TEIPP may exert their actions at sites that are distinct but coordinated by a trans mechanism.
  • the TEIPP1 peptide is selected from the group including HRAKRHPRRPPRSPG (SEQ ID NO: 8), TRNEKNRPRRRFLC (SEQ ID NO: 9), WGVTEEETRRNRQLEVC (SEQ ID NO: 10) and DSEPTPHLKTRQRR (SEQ ID NO: 11) or functionally equivalent or variants thereof. More preferably the peptide is selected from the group including 385 HRAKRHPRRPPRSPG 399 (SEQ ID NO: 8) 599 TRNEKNRPRRRFLC 612 (SEQ ID NO: 9)
  • TEIPP1 peptide is 385 HRAKRHPRRPPRSPG 399 (SEQ ID NO: 8) or a functional equivalent or variant thereof.
  • micro molar doses of telomerase inhibitory peptide are administered. More preferably, 0.1-100 ⁇ M of telomerase inhibitory peptide is administered. Most preferably the TEIPP peptide has a half-maximal inhibitory concentration of approximately 1.8 ⁇ M and near complete inhibition at approximately 4.2 ⁇ M.
  • compositions of the present invention may be formulated for administration to a subject orally, rectally, parenterally (i.e. intravenously, intramuscularly, or sub-cutaneously), intracisternally, intravaginally, mtraperitoneally, topically (as by powders, ointments, or drops), transdermally, bucally, or as an oral or nasal spray.
  • composition of the present invention may also include a suitable carrier to facilitate uptake of the peptide, or a pharmaceutically acceptable excipient.
  • suitable carriers may be added to the peptide to facilitate delivery and include those well known to those of skill in the art including, but not limited to, the Drosophila Atennapedia homeodomain-derived carrier peptide, penetratin (C- RQIKIWFQNRRMKWKK) (SEQ ID NO: 12).
  • the carrier and the telomerase inhibitory peptide are prepared in an equimolar ratio.
  • compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents.
  • adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and gly
  • the dosage form may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
  • the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.
  • the active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifier
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Dosage forms for topical administration of a compound of this invention include powders, sprays, ointments and inhalants.
  • the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.
  • the TEIPP1 or TEIPP peptide in the composition may also be conjugated or linked to a further molecule such as an antigen, antibody, ligand or cytotoxic agent such as a toxin or radioisotope.
  • a further molecule such as an antigen, antibody, ligand or cytotoxic agent such as a toxin or radioisotope.
  • a method of inhibiting cell growth in a cell culture comprising administering an effective amount of a telomerase inhibitory peptide selected from TEIPP1, TEIPP, or a combination thereof, functional equivalents, variants or fragments thereof to a cell culture.
  • “Inhibiting cell growth” as used herein includes, but is not limited to, inducing cell death, substantially decreasing cell proliferation, or inducing senescence in the cells. Preferably inhibition of cell growth is associated with apoptosis in the cells.
  • a cell culture includes, but is not limited to, primary cultures, transformed cell lines, cancer cells, immortalized cell lines or explant cultures.
  • the cell culture is breast cancer cell cultures. More preferably, the cell culture is a PMC42 or MCF-7 cell culture.
  • micro molar doses of telomerase inhibitory peptide are administered. More preferably, 0.1-100 ⁇ M of telomerase inhibitory peptide is administered. Most preferably the TEIPP1 and TEIPP peptide have a half- maximal inhibitory concentration of approximately 0.4 ⁇ M and 1.8 ⁇ M, and near complete inhibition at approximately 1.O ⁇ M and 4.2 ⁇ M, respectively.
  • the TEIPP1 and TEIPP may be administered in combination with each other, to provide a synergistic effect, and/or another factor as part of a complex.
  • Suitable complexes may include a radioisotope or toxin.
  • Preferably low concentrations of TEIPP are used in combination with TEIPP1 to inhibit telomerase.
  • TEIPP1 and TEIPP may exert their actions at sites that are distinct but coordinated by a trans mechanism.
  • a method of inducing cell death comprising administering an effective amount of a telomerase inhibitory peptide selected from TEIPP1 , TEIPP, or a combination thereof, functional equivalents, variants or fragments thereof.
  • cell death includes but is not limited to, apoptosis, necrosis or lysis. Preferably cell death is associated with apoptosis. Cell death can be characterised by, but not limited to, plasma membrane blebbing, cell volume loss, nuclear condensation, and endonucleolytic degradation of DNA at nucleosome intervals.
  • the present invention provides a method of treating cancer or other proliferative disorder in a subject, the method including: administering an effective amount of a telomerase inhibitory peptide to the subject, wherein the telomerase inhibitory peptide includes at least one peptide selected from the group including TEIPP and TEIPP1 or a functionally equivalent variant thereof.
  • the TEIPP and TEIPP1 may be administered in combination with each other and/or another factor as part of a complex. Suitable complexes may include a radioisotope or toxin.
  • Preferably low concentrations of TEIPP are used in combination with TEIPP1 to inhibit telomerase.
  • TEIPP and TEIPP1 may exert their actions at sites that are distinct but coordinated by a trans mechanism.
  • Cancer as used herein includes malignant tumours and their cells, and includes both cancerous and pre-cancerous tissues and cells.
  • the subject may be from any species, including human, bovine, ovine, porcine, murine, equine, and is preferably mammalian.
  • the cancer is a tumour of epithelial tissues such as carcinomas of breast, prostate, kidney, thyroid or lung, sarcomas derived from mesoderm, or leukemia from blood.
  • pre-cancerous cells refers to cells that exhibit histologic changes that are associated with an increased risk of cancer development. Examples include adenomatous polyps of the colon, dysplastic nevi of the skin and atypical hyperplasia of the mammary glands. Certain syndromes that commonly display pre-cancerous cells are also referred to by the term “precancerous” including dysplastic nevus syndrome and the colonic polyposis syndromes. “Pre-cancerous” refers to these cells or syndromes of various tissues whether or not the cells are clinically identifiable.
  • the proliferative disorders of the present invention include, but are not limited to, disorders correlated with telomerase activity or increased telomerase activity relative to the normal state.
  • Peptides are shorter in amino acid sequence than proteins that are building blocks of our body, and peptides are much easier to be absorbed as nutrients and to be delivered as drugs into the body than proteins.
  • telomerase inhibitory peptides of the present invention may be formulated for administration to a subject orally, rectally, parenterally (e.g. intravenously, intramuscularly, or sub-cutaneously), intracisternally, intravaginally, mtraperitoneally, topically (as by powders, ointments, or drops), transdermally, bucally, as an oral or nasal spray, or intra-tumour injection with or without endoscopies.
  • the telomerase inhibitory peptide may be administered at any time.
  • the telomerase inhibitory peptide is administered after diagnosis of the cancer or proliferative disorder.
  • the inhibition micro molar doses are administered. More preferably, 0.1-100 ⁇ M of telomerase inhibitory peptide is administered.
  • the telomerase inhibitory peptide may be administered in conjunction with a carrier to facilitate uptake of the peptide.
  • Suitable carriers include those well known to those of skill in the art including, but not limited to, the Drosophila Atennapedia homeodomain-derived carrier peptide, penetratin (C- RQIKIWFQNRRMKWKK) (SEQ ID NO: 12).
  • the carrier and the telomerase inhibitory peptide are prepared in an equimolar ratio.
  • the TEIPP peptide forms a ⁇ -helix with positively charged amino acid residues on one side of the helix and negatively charged/potentially negatively charged (phosphorylation sites) on another. More preferably the TEIPP helix interacts with an electrochemically polarized groove on the surface of telomerase through mutual electrostatic attractions that are regulated by protein phosphorylation (adding a phosphate moiety).
  • an effective amount refers to an amount that inhibits cancer growth or spread, or alleviates the proliferative disorder or induces cell death.
  • treatment of a subject with a telomerase inhibitory peptide results in death of the cancer cell or proliferative disorder cell. More preferably the telomerase inhibitory peptide causes cell death by a mechanism involving competing internal sequence of telomerase through interactions involving electrostatic force and thereby inhibiting telomerase interactions with telomeres. Most preferably treatment with the telomerase inhibitory peptide results in cell death associated with apoptosis.
  • the present invention provides a vaccine for treating or preventing cancer or other proliferative disorder wherein the vaccine includes a telomerase inhibitory peptide, wherein the telomerase inhibitory peptide includes at least one peptide selected from the group including TEIPP and TEIPP1.
  • the present invention provides a method of inducing cell proliferation by inhibiting production in the cell of a telomerase inhibitory peptide, wherein the telomerase inhibitory peptide is at least one of TEIPP or TEIPP1.
  • Example 1 Competitive inhibition of telomerase activity by telomerase inhibitory peptides
  • ATP ATP
  • Taq DNA polymerase dNTP
  • T4 gene 32 protein was from Boehringer Mannheim Australia Pty. Ltd. (New South Wales, Australia)
  • [ ⁇ - 32 P]ATP and [ ⁇ - 32 P]ATP were from Amersham Australian Pty. Ltd. (New South Wales, Australia).
  • Gel electrophoresis reagents were from Bio-Rad. All other chemicals were from Sigma Chemical.
  • Protein kinase B ⁇ (Akt1), extracellular signal-regulated kinase 1 (ERK1), and cdc2 protein kinase (cyclin-dependent protein kinase 1, CDK1) were from Upstate Biotechnology Inc.
  • Protein kinase C ⁇ was from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA).
  • Casein kinase I, casein kinase II, and DNA-dependent protein kinase were from Promega Corporation (Madison, Wl).
  • An analysis of sequence hydrophilicity and charge characteristics was employed to identify potential amino acid sequences involved in inter- and intramolecular interactions within or between telomerase components.
  • Peptides corresponding to highly amphiphilic regions of human telomerase-associated protein 1 (hTEP1) and human telomerase reverse transcriptase (hTERT) were synthesized and purified to > 90% purity by Auspep International Pty. Ltd. (Victoria, Australia) or Chiron Technologies Pty. Ltd. (Victoria, Australia).
  • telomere reverse transcriptase hTERT
  • hTEP1 human telomerase-associated protein 1
  • the four peptides from hTEP1 were 385 HRAKRHPRRPPRSPG 399 (TEIPP1) (SEQ ID NO:
  • telomerase activity assay was used.
  • a TRAP assay performed essentially as described in Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L, Coviello, G. M., Wright, W. E., Weinrich, S. L, and Shay, J. W. (1994) Science 266, 2011-2015 was employed to determine the effects of various peptides on telomerase activity.
  • telomeres were incubated in the presence or absence of different synthetic peptides as indicated in individual experiments.
  • de novo synthesized telomeres were amplified by PCR and the resultant 32 P-labeled products resolved by polyacrylamide slab gel electrophoresis followed by autoradiography.
  • de novo synthesized telomeres were extracted with phenol and chloroform post-incubation, with telomerase and the isolated telomeres used in PCR amplification.
  • an internal control was used to monitor for non-specific PCR effects by including additional primers NT (ATCGCTTCTCGGCCTTTT) (SEQ ID NO: 13) and TSNT (AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT) (SEQ ID NO: 14).
  • Negative controls containing either added RNase-A, alkaline phosphatase or sample heated to 80°C to inactivate telomerase were included in each experiment assessing telomerase activity.
  • TEIPP1 corresponding to the region of hTEP1 385"399 , inhibited telomerase activity with a half maximal inhibitory concentration of 0.4 ⁇ M and near complete inhibition at 1 ⁇ M (Li, H., Cao, Y., Berndt, M. C, Funder, J. W., and Liu, J. P. (1999) Oncogene 18, 6785-6794).
  • TEIPP corresponding to hTERT 641"659 (Fig. 1) showed a half-maximal inhibitory concentration of 1.8 ⁇ M and near complete inhibition at 4.2 ⁇ M (Fig.
  • telomerase activity was analyzed in the presence or absence of various concentrations of nuclear telomerase extract or TS, plus or minus TEIPP1 or TEIPP.
  • Increasing amounts of nuclear telomerase extracts produced increases in basal telomerase activity, with inhibition of telomerase by TEIPP1 (not shown) and TEIPP (Fig. 1 ⁇ ) overcome in a dose-dependent manner. This effect was not due to peptide degradation, since increasing amounts of telomerase extract produced increases in basal telomerase activity (Fig.
  • TEIPP inhibits telomerase activity by competing with certain structures of telomerase components but not with telomerase substrate DNA.
  • TEIPP1 and TEIPP are net positively charged and arginine-rich, whether or not the two peptides might interact with the same site on telomerase holoenzyme was determined by examining the effects of TEIPP1 plus TEIPP at various concentrations on telomerase activity. While scrambled peptide of TEIPP at different concentrations (1-4 ⁇ M) produced no effect on TEIPP1 inhibition of telomerase activity (Fig. 3>A), TEIPP at low concentrations enhanced the inhibitory effect of TEIPP1 on telomerase activity although these doses of TEIPP alone had minimal effect on basal telomerase activity (Fig. 3 ⁇ ). These data suggest that TEIPP1 and TEIPP exert their actions at the sites that are distinct but coordinated by a trans mechanism.
  • Example 2 Effects of TEIPP on telomerase activity and cell viability in cell cultures
  • telomerase inhibitory peptides were cultured and treated with or without TEIPP1 , TEIPP, the combination of both, or scrambled control peptides for telomerase activity and cell biology.
  • TEIPP1 , TEIPP and their respective scrambled peptides were either unmodified or cross-linked via an N-terminal Cys-Cys bond to penetratin, the Drosophila Antennapedia homeodomain-derived carrier peptide (C-RQIKIWFQNRRMKWKK) (SEQ ID NO: 12) as described in Schwarze, S. R., and Dowdy, S. F. (2000) Trends Pharmacol Sci 21 , 45-48.
  • Peptides were introduced into cultured human breast cancer MCF-7 cells as carrier-peptide conjugates, with carrier-carrier and carrier-scrambled peptides as controls (see figures).
  • penetratin or telomerase inhibitory peptides 600 ⁇ M were treated separately with tris-(2- carboxyethyl)phosphine hydrochloride (TCEP, Molecular Probes) in an equimolar ratio.
  • TCEP tris-(2- carboxyethyl)phosphine hydrochloride
  • Equimolar penetratin and TEIPPs were mixed and incubated at 37°C for 1 hour, before use with a final applied concentration of TEIPP at ⁇ 100 ⁇ M in cell cultures. Cell morphology and count were examined after 24h, 48h and 72h of the treatment.
  • TUNEL TdT-mediated dUTP-biotin nick end labeling
  • telomerase inhibitory peptides amino acid residues at the N- and C-terminal regions of TEIPP were deleted and the truncated TEIPP tested in telomerase activity assay. As shown in Fig. 5, removal of either N- or C-terminal regions of TEIPP abolished TEIPP inhibition of telomerase activity (Fig. 5>A & ⁇ ). Mutations of the acidic residues in TEIPP enhance the telomerase inhibitory activity of TEIPP, suggesting that the negatively charged residues play an inhibitory role in the peptide inhibition of telomerase activity and that potent telomerase inhibitory peptides can be developed with retained specificity by modifications of these residues.
  • hTEP1 and hTERT are phosphoproteins with phosphorylation being important for telomerase activity and since TEIPP contains putative serine/threonine phosphorylation sites, we determined if the inhibitory effect of TEIPP might be reversibly regulated through protein phosphorylation.
  • telomerase inhibitory polypeptide 1 TEIPP1
  • TEIPP telomerase inhibitory polypeptide 2
  • phosphorylation buffer 50 ⁇ l
  • ATP -40 ⁇ M, 3 ⁇ Ci [ ⁇ - 32 P]ATP
  • Mg 2+ 1 mM
  • other kinase-specific activators 40 ⁇ M, 3 ⁇ Ci [ ⁇ - 32 P]ATP
  • the reaction was allowed to proceed for 10 min at 32 °C before being terminated by acidification and cooling the reactions.
  • Purified protein kinases included casein kinase I (10 units), casein kinase II (10 units), protein kinase B ⁇ (Akt1 , 500 ng), protein kinase C ⁇ (25 ng), extracellular signal-regulated protein kinase (ERK1 , 200 ng), cdc2 protein kinase (15 ng) and DNA-dependent protein kinase (15 units).
  • protein kinase B was activated by PI(3,4,5)P 3 -dependent protein kinase 1 (PDK1 , 5 ng) in an appropriate buffer (20 ⁇ l) containing ATP (100 ⁇ M), Mg 2+ (10 mM), phosphatidylserine (80 ⁇ g/ml) and diolein (8 mg/ml) for 30 min at 30 °C.
  • PDK1 PI(3,4,5)P 3 -dependent protein kinase 1
  • PDK1 PI(3,4,5)P 3 -dependent protein kinase 1
  • ATP 100 ⁇ M
  • Mg 2+ 10 mM
  • phosphatidylserine 80 ⁇ g/ml
  • diolein 8 mg/ml
  • Protein kinase C ⁇ was activated by phosphatidylserine (40 ⁇ g/ml), diolein (4 mg/ml) and Ca 2+ (200 ⁇ M), and DNA
  • crostide, MARCKS peptide and myelin basic protein (MBP) were phosphorylated in parallel by protein kinase B, protein kinase C ⁇ and ERK1 respectively.
  • Phosphorylated peptides were spotted onto P81 chromatography paper which was then washed in 75-mM phosphoric acid to remove free 32 P-ATP and counted in a ⁇ counter.
  • TEIPP and control peptides were phosphorylated by various protein kinases in the presence of cold ATP for 30 min at 32°C and the reaction terminated by heating to 80°C for 2 min. The phosphorylation mixtures were then used in telomerase activity analysis.
  • TEIPP TEIPP phosphorylation with known substrates of protein kinase B, protein kinase C ⁇ and ERK1 showed that TEIPP was phosphorylated by protein kinase C ⁇ or ERK1 to higher levels than that of the protein kinase C substrate MARCKS peptide and ERK1 substrate myelin basic protein (MBP).
  • MARCKS peptide and ERK1 myelin basic protein
  • TEIPP was stoichiometrically phosphorylated by protein kinase C ⁇ , ERK1 , cdc2 protein kinase respectively and the phosphorylated peptides then tested for effects on telomerase activity. While the peptide incubated with protein kinase B or casein kinase II showed no effect on TEIPP inhibition of telomerase activity, peptides phosphorylated by either protein kinase C ⁇ , cdc2 protein kinase (Fig. 6 ⁇ ) or ERK1 (not shown) showed decreased inhibitory activity.
  • TEIPP is phosphorylated by protein kinase C ⁇ , ERK1 and cdc2 protein kinase, but not by casein kinase I, casein kinase II, protein kinase B or DNA-dependent protein kinase. Phosphorylation, and phosphorylation-mimicking mutations of the putative phosphorylation sites to aspartic acid, inhibits the inhibitory function of TEIPP, suggesting that the sequence of TEIPP serves as an inhibitory element that can be switched off by phosphorylation of the threonine or serine residue.
  • FaxNumber EmailAddress : ⁇ 110> LastName : Jun-Ping ⁇ 110> FirstName : Liu ⁇ 110> Middlelnitial : ⁇ 110> Suffix :
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString : GARTFRRXKR AXRLTSRVK 19
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString : GARTFRREKR AERLTSRVK 19
  • SequenceName SEQ ID NO:2
  • SequenceDescription Sequence
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName Homo sapiens
  • OrganismName Homo sapiens
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName Homo sapiens ⁇ 400> PreSequenceString :
  • OrganismName drosophila ⁇ 400> PreSequenceString :
  • OrganismName Artificial ⁇ 400> PreSequenceString : atcgcttctc ggcctttt 18 ⁇ 212> Type : DNA ⁇ 211> Length : 18
  • OrganismName Artificial ⁇ 400> PreSequenceString : aatccgtcga gcagagttaa aaggccgaga agcgat 36 ⁇ 212> Type : DNA ⁇ 211> Length : 36

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Abstract

L'invention concerne des peptides inhibant la télomérase ainsi que leurs utilisations, en particulier dans une méthode de traitement du cancer. L'invention a pour objet des peptides inhibiteurs de la télomérase présentant une séquence d'acides aminés de GARTFRRXKRAXRLTSRVK (SEQ ID NO : 1) (X équivalant à E ou Q) ou des équivalents fonctionnels ou des fragments de ces derniers. L'invention concerne également une séquence de nucléotides qui code le peptide inhibiteur de la télomérase (TEIPP).
PCT/AU2002/001263 2001-09-14 2002-09-13 Peptides inhibiteurs de la telomerase et leurs utilisations WO2003024997A1 (fr)

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WO2007105954A1 (fr) * 2006-03-16 2007-09-20 Leiden University Medical Center Procédés d'identification d'épitopes de lymphocytes t associés à une compromission de la transformation de peptides et applications des épitopes identifiés

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WO2014046490A1 (fr) * 2012-09-19 2014-03-27 주식회사 카엘젬백스 Peptide de pénétration cellulaire, conjugué le comprenant, et composition comprenant le conjugué
CN108129553B (zh) * 2018-01-05 2020-12-08 杭州端丽生物技术有限公司 防治端粒功能异常相关疾病的多肽及医药用途
CN114920802B (zh) * 2022-05-16 2023-05-23 中国地质大学（武汉） 调节端粒酶限速蛋白运动的多肽探针及其复合物和方法

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GB2317891A (en) * 1996-10-01 1998-04-08 Geron Corp hTRT, the reverse transcriptase subunit of human telomerase
WO1998021343A1 (fr) * 1996-11-15 1998-05-22 Amgen Inc. Genes codant des proteines de telomerase

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WO1998007838A1 (fr) * 1996-08-21 1998-02-26 Mitsubishi Chemical Corporation Proteine de telomerase d'animaux superieurs et gene codant pour elle
GB2317891A (en) * 1996-10-01 1998-04-08 Geron Corp hTRT, the reverse transcriptase subunit of human telomerase
WO1998021343A1 (fr) * 1996-11-15 1998-05-22 Amgen Inc. Genes codant des proteines de telomerase

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LI H. ET AL.: "Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro", ONCOGENE, vol. 18, 1999, pages 6785 - 6794 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007105954A1 (fr) * 2006-03-16 2007-09-20 Leiden University Medical Center Procédés d'identification d'épitopes de lymphocytes t associés à une compromission de la transformation de peptides et applications des épitopes identifiés

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