WO2000008135A1 - Methodes et compositions d'inhibition ou de stimulation d'un assemblage de telomerase - Google Patents

Methodes et compositions d'inhibition ou de stimulation d'un assemblage de telomerase Download PDF

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WO2000008135A1
WO2000008135A1 PCT/US1999/017724 US9917724W WO0008135A1 WO 2000008135 A1 WO2000008135 A1 WO 2000008135A1 US 9917724 W US9917724 W US 9917724W WO 0008135 A1 WO0008135 A1 WO 0008135A1
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telomerase
chaparonin
compound
cell
activity
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PCT/US1999/017724
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English (en)
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Michael A. White
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Board Of Regents, The University Of Texas System
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Priority to AU53381/99A priority Critical patent/AU5338199A/en
Publication of WO2000008135A1 publication Critical patent/WO2000008135A1/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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • 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 concerns assembly of the multi-component enzyme telomerase.
  • this invention relates to methods of identifying or screening for telomerase activity modulators, and to methods and compositions for modulating telomerase activity in a composition or cell (be it in culture or in vivo) comprising a telomerase catalytic protein subunit and a telomerase RNA component.
  • this material relates to telomerase, a ribonucleoprotein (RNP) complex which functions in cells to maintain the integrity of chromosome ends of eukaryotic cells.
  • RNP ribonucleoprotein
  • Chromosomes are replicated by DNA polymerases as a necessary prerequisite for almost all eukaryotic cell division.
  • the DNA polymerases responsible for chromosomal replication synthesize DNA in a 5 'to 3' direction and require a primer to initiate synthesis. Because of this, the "lagging strand" does not replicate to the very ends of linear chromosomes. The chromosome is thus shortened with every cell division.
  • the ends of chromosomes are called telomeres, and in vertebrates are typically composed of long sequences which contain hundreds of thousands of repeats of short, specific nucleotide sequences. For example, in humans the telomeric repeat consists of the hexanucleotide 5'-TTAGGG-3'. The telomeric repeat sequences for many other organisms are also known. See PCT WO 93/23572, for example.
  • Telomerase activity in a cell is associated with the cell's proliferative capacity. See U.S. patent nos 5,646,245 and 5,645,986. All normal diploid, somatic vertebrate cells lack telomerase activity and have a limited capacity to proliferate, a phenomenon known as the Hayflick limit or replicative senescence ( . e. , loss of the ability of a cell to replicate in the presence of normally appropriate replicative signals). For example, in human fibroblasts, this limit occurs after 50-100 population doublings, after which the cells remain in a viable but non-dividing senescent state for many months. Typically, about 50-200 or more nucleotides are lost from the ends of telomeres during each cell division.
  • telomere shortening occurs both in vivo and in vitro. This contrasts with the behavior of germline and stem cells (and the cells of many lower eukaryotes, e.g., yeast), which have long telomeres and active telomerase. It has also been observed that neoplastic cells (e.g., cancer cells), which have escaped from the controls limiting their proliferative capacity, are immortal. These cells have regained telomerase activity and thus can maintain telomere length, although the telomeres of such cells are typically much shorter than those of corresponding embryonic, germline, or stem cells.
  • neoplastic cells e.g., cancer cells
  • telomerase activity has been detected in a diverse set of tumor tissues and in immortal cell lines, but is not detected in assays of most normal somatic cells. See, Shay and Bacchetti, 1997, Eur. J. Cancer, 33:111-191, and Morin, 1989, Cell 59:521. Moreover, increasing levels of active telomerase in telomerase- negative cells results in dramatically increased proliferative capacity. Bodnar, et al, 1998, Science 279:349-352.
  • telomere acts as a specialized reverse transcriptase to synthesize telomeric repeat sequences at the 3' end of the telomere, thus extending the DNA to prevent loss of telomeric sequence, thereby preventing telomere shortening.
  • the telomerase RNP includes a catalytic protein subunit referred to as "TERT” (for telomerase reverse transcriptase) and a telomerase RNA component (referred to herein as "TR").
  • TR telomerase RNA component acts as a reverse transcriptase template for the catalytic protein. TERTs and TRs from multiple species have been cloned and characterized.
  • RNA component of telomerases have also been characterized in several species, as described in, e.g., U.S. Patent No.
  • telomere-associated proteins have been reported, some of which may have a structural or regulatory function (see, e.g., Harrington, et al, 1997, Science 275:973-977; Collins, et al, 1995, Cell, 81:677; Nakayama, et al, 1997, Ce/7 88:1-20; U.S. Pat. No.
  • telomerase activity can be generated in cell-free systems. In vitro transcription and translation of human TERT (“hTERT”), when co-synthesized or mixed with the human telomerase RNA (“hTR”), results in activity characteristic of native human telomerase. See, e.g., Weinrich, et al, 1997, Nature Genetics 17:498-502. Because telomerase plays a role in controlling cell proliferation, telomerase is an ideal target for diagnosing and treating diseases relating to cellular hyperproliferation, of which cancer is an example. Indeed, telomerasec activity has been detected in over 98% of immortal cell lines and in more than 85% of malignant tumor biopsies.
  • telomere activation of telomerase is an exciting approach to diseases and conditions associated with senescence, including aging and age-related processes.
  • This invention addresses this need by providing methods and compositions for identifying molecules that assist and/or participate in the formation of biologically active telomerase holoenzyme complexes, as well as compounds that interact with such molecules.
  • RNA component refers to the RNA molecule which is incorporated into the telomerase RNP and required for biological activity.
  • the RNA component used is from the same species as the telomerase catalytic protein component, e.g., the human telomerase component is used in conjunction with the human telomerase catalytic protein component. It is also preferred to use a telomerase component comprising a nucleotide sequence which has the same nucleotide as the naturally occurring version of that RNA.
  • the telomerase RNA component employed may contain at least one nucleotide substitution, insertion, or deletion as compared to the corresponding naturally occurring RNA, be it inside and/or outside of the telomeric repeat template region of the RNA.
  • any RNA component which serves as a template for synthesis of a telomeric repeat once assembled into an active telomerase RNP complex can be used in accordance with the present invention.
  • such RNAs can readily be identified by incorporating the i 4 same into a telomerase RNA and testing, preferably in vitro, whether telomerase RNP complexes containing the same exhibit telomerase activity.
  • telomerase activity refers specifically to telomerase activity, preferably the processive catalytic activity of telomerase as described in WO 98/14593.
  • Other activities of telomerase which may be screened include non- processive catalytic activity, reverse transcriptase activity, nucleolytic activity, primerr or substrate binding activity, RNA (i.e., TR) binding activity, and protein binding activity.
  • Telomerase processive catalytic activity is characterized by the ability of the telomerase RNP to extend a DNA primer that functions as a telomerase substrate by adding multiple (i.e., at least two) repeats of a sequence (e.g., 5'-TTAGGG-3') encoded by the template nucleic acid (e.g., hTR).
  • This activity can be assayed, e.g., as described in Morin, 1989, Cell 59:521 ("conventional assay") or using the TRAP assay disclosed in U.S. Patent No. 5,629,154.
  • telomerase assembly refers to the formation of a ribonucleoprotein complex including the telomerase catalytic subunit protein (TERT) and a template RNA (e.g., TR) and having telomerase activity.
  • TERT telomerase catalytic subunit protein
  • TR template RNA
  • in vitro assembly refers to the generation of telomerase activity in vitro by combining (e.g., by coexpression, mixing, or a combination thereof) a TERT polypeptide, preferably a recombinant form thereof, with a synthetic or recombinantly produced telomerase RNA component (e.g., hTR) or a functionally equivalent template RNA.
  • a TERT polypeptide preferably a recombinant form thereof
  • a synthetic or recombinantly produced telomerase RNA component e.g., hTR
  • in vitro assembly is described generally in PCT Publication WO 98/14593, and in Weinrich et al., supra. Also see, Beattie et al, 1998, Curr. Biol 8:177-180.
  • in vitro assembly is accomplished by coexpression of the TERT polypeptide and a telomerase RNA in a cell-free transcription-translation system (e.g., a wheat germ or rabbit reticulocyte lysate).
  • a cell-free transcription-translation system e.g., a wheat germ or rabbit reticulocyte lysate.
  • TERT and the RNA component are separately expressed (and preferably one or both are partially or completely purified) and then mixed to achieve assembly of an active telomerase RNP.
  • telomerase assembly related proteins or "telomerase chaparonins” are proteins that facilitate the association of TERT and TR in assembly of the telomerase RNP.
  • a telomerase chaparonin is a protein that binds to or is associated with unfolded or partially folded telomerase protein component to maintain their unfolded or partially folded state to ensure proper folding or assembly into multi-subunit complexes, e.g. telomerase.
  • These "chaparonins” or “assembly related proteins” include, but are not limited to, phosphoprotein 23, heat shock proteins 40, 70, and 90, and HOP (also called p60).
  • telomere chaparonins from non-human species (e.g., mice and yeast) are publicly available and/or can be obtained by one of ordinary skill using routine techniques. It should also be recoginized that compounds other than proteins may exhibit telomerase chaparonin-like activity. For example, as part of this invention, it is been determined that molybdate can substitute for one or more of the telomerase chaparonins required assembly of the telomerase RNP.
  • telomerase chaparonin non-polypeptide compounds which stabilize the same conformational positions as a polypeptide telomerase chaparonin will be considered a telomerase chaparonin for purposes of this invention.
  • Such compounds can be identified using techniques known in the art, for example, by screening various compounds, preferably in a high throughput format, to determine whether they enhance or inihibit functional telomerase RNP assembly in an in vitro assembly reaction.
  • p23 refers to an approximately 23 kDa phosphoprotein.
  • human p23 protein is provided in GenBank Accession No. 1362904 and is encoded by a gene having the sequence of GenBank Accession No. Z69891.
  • the bacterial expression and purification of human p23 has been described (see, e.g., Johnson et al, 1994, J. Biol. Chem. 269:24989).
  • the soluble fraction of bacterial lysate i 6 derived from bacterial cells engineered to express the protein was fractionated by DEAE- cellulose column chromatography, followed by phenyl-sepharose (hpl660) FPLC, dialyzed into 10 mM Tris-HC 1 , 1 mM DTT and 1 mM EDTA, pH 7.5, and stored at 70°C.
  • the preparation was greater than 99% pure as assessed by densitometry of SDS- PAGE gels. Protein concentration can be determined by amino acid analysis.
  • hsp90 refers to an abundant heat shock phosphoprotein found in the cytosols of animal and plant cells and having an approximate molecular weight of 90 kDa; the sequence of human hsp90 ⁇ protein is provided in GenBank Accession No. J04988 and is encoded by a gene having the sequence of GenBank Accession No. XI 5183 .
  • GenBank Accession No. J04988 The bacterial expression and purification of human hsp90 has been described (see Sullivan, et al, J.Biol.Chem. 272:8007). Briefly, human HSP90 ⁇ was overexpressed in SF9 cells.
  • the protein was purified from cytosol extracts to greater than 99%o purity by chromatography on columns of DEAE-cellulose, heparin-agarose, and mono Q. Purified hsp90 was stored at -70°C in 10 mM Tris-HC 1, 100 mM KC1, 1 mM EDTA and 10% glycerol, pH 7.4.
  • hsp40 refers to an approximately 40 kDa phosphoprotein.
  • the sequence of human hsp40 protein is provided in GenBank Accession No. 1816452, and is encoded by a gene having the sequence of GenBank Accession No. 085492.
  • the bacterial expression and purification of Ydjlp, the yeast homolog of human hsp40, was performed by fractionating bacterial cell lysates engineered to overexpress the protein by DEAE-cellulose chromatography followed by hydroxylapatite column chromatography. The preparation was approximately 80% pure as assessed by densitometry of SDS-PAGE. Protein concentration can be determined by amino acid analysis.
  • hsp70 refers to an approximately 70 kDa phosphoprotein.
  • human hsp70 protein is provided in GenBank Accession No. 292160, and is encoded by a gene having the sequence of GenBank Accession No. L12723 .
  • the bacterial expression of human hsp70 employed the same system as used for expression of p23. Briefly, hsp70 was prepared by the overexpression in SF9 cells. The protein was 1 then purified by fractionating cell lysates by DEAE-cellulose column chromatography followed by ATP-agarose column chromatography. Protein was precipitated using ammonium sulfate (75% saturation), and the redissolved hsp70 was fractionated by 16/60 Superdex 200 FPLC. Only the monomer peak of hsp70 was retained. The preparation was approximately 97% pure as assessed by densitometry of SDS-PAGE gels. Protein concentration can be determined by amino acid analysis.
  • HSP Organizing Protein CHOP or p60 refers to an abundant, stress-induced protein found in certain cells.
  • the sequence of human HOP protein is provided in GenBank Accession No. 306890 and is encoded by a gene having the sequence of GenBank Accession No. M34664.
  • the bacterial expression and purification of human HOP has been described by Schumacher et al. 1994, J Biol Chem. 269:9493-9499. Briefly, human Hop was expressed in bacteria. Bacterial lysates were then fractionated by DEAE-cellulose chromatography followed by hydroxylapatite column chromatography.
  • modulate in the context of modulating telomerase activity or assembly in a cell, cell lysate, in vitro composition, or cell (in culture or in vivo) refers to the inhibition or stimulation of telomerase activity or assembly.
  • modulator refers to any synthetic or natural compound or composition that can modulate telomerase assembly or activity.
  • a modulator can be any organic and inorganic compound, including, but not limited to, for example, small molecules, peptides, proteins, sugars, nucleic acids, fatty acids and the like.
  • the term "substantially pure,” or “substantially purified,” when referring to a composition comprising a specified reagent such as a telomerase assembly associated protein means that the specified reagent is at least about 75%, or at least about 90%), or at least about 95%, or at least about 99% or more of the composition (not including, e.g., solvent or buffer).
  • recombinant refers to a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide ("recombinant protein") encoded by a recombinant polynucleotide.
  • the objects of this invention include the provision of novel and non- obvious methods of screening for compounds that modulate telomerase activity, methods of assembling telomerase, as well as methods and compositions for modulating telomerase activity in vitro and in vivo.
  • telomerase activity modulators i.e., compounds which inhibit or stimulate telomerase activity
  • Telomerase activity is then measured and compared with the telomerase activity detected when telomerase is assembled in the absence of the test compound, and test compounds which increase or decrease telomerase activity are identified as telomerase activity modulators.
  • methods are provided for identifying chaparonins which facilitate assembly of active telomerase. In certain embodiments, these methods are performed in vitro, while in other embodiments, the methods are performed in vivo.
  • telomerase catalytic protein component and a telomerase RNA component are added to reaction mixture comprising at least one polypeptide, preferably at least one putative chaparonin, particularly a chaparonin selected from the group consisting of p23, hsp40, hsp70, hsp90, and HOP.
  • a chaparonin selected from the group consisting of p23, hsp40, hsp70, hsp90, and HOP.
  • one or more other chaperonins can also be included or substituted in such reactions, including, non- polypeptide-based chaperonins.
  • Telomerase activity of the reaction mixture is then compared to a reaction mixture control.
  • At least one, and preferably each, of the catalytic protein component, the telomerase RNA component, and polypeptide(s) are substantially purified, preferably from either a recombinant or natural source.
  • one or more of the catalytic protein component, the telomerase RNA component, and polypeptide(s), particularly the catalytic protein component and/or telomerase RNA component are also transcribed and/or translated, as the case may be, in the reaction mixture.
  • the catalytic protein component, the telomerase RNA component, and/or the chaparonin(s) are/is a human telomerase component or chaparonin, and can be derived from either a natural or recombinant source.
  • a related aspect of the invention concerns the assembly of telomerase in the presence of one or more chaparonins in vitro or in vivo.
  • the catalytic protein component and the RNA component of telomerase are added to a mixture comprising at least one chaparonin.
  • a chaparonin (or an expression vector encoding the same) is introduced into a cell which expresses the catalytic protein and RNA components of telomerase, thereby, for example, to increase or enhance the telomerase activity, preferably to extend its replicative capacity.
  • Another related aspect of the invention concerns in vitro and in vivo methods of screening for telomerase activity modulators which act by modulating chaparonin-mediated assembly of telomerase.
  • Certain preferred in vitro embodiments of this aspect relate to methods where the catalytic protein and RNA components of telomerase are assembled in a reaction mixture comprising at least one chaparonin in the presence or absence of a test compound.
  • High throughput screening method formats are well known in the art. Typically, a number of different compounds (from as few as about 10-100 to 10,000 or more) are individually, and frequently simulataneously, screened in separate reactions, are particularly preferred.
  • telomerase activity is then measured and compared with the telomerase activity in a control reaction mixture lacking the test compound, and test compounds which increase or decrease telomerase activity are identified as telomerase activity modulators.
  • test compounds are screened against cells known to possess telomerase activity, for example, an immortal cell line. Compounds found to confer mortality on such cells can then be identified. The use of such cell based assays is particualrly preferred to confirm modulating activity that has first been identified in an in vitro telomerase assembly screening assay.
  • the invention provides methods of enhancing telomerase activity in vitro or in vivo by providing at least one chaparonin to facilitate the assembly of TERT and TR into biologically active telomerase RNP complexes.
  • the chaparonin is selected from the group consisting of p23, hsp40, hsp70, hsp90, and HOP, with the chaparonin being a substantially purified natural or recombinant protein in particularly preferred embodiment.
  • the in vitro assembly of the catalyitc protein component and telomerase RNA component into biologically functional RNP complexes occurs in a cell extract-based translation system, e.g., a rabbit reticulocyte lysate or a wheat germ extract.
  • a cell extract-based translation system e.g., a rabbit reticulocyte lysate or a wheat germ extract.
  • RNA coding for one or more of the catalyitc protein component and chaparonin(s) are added to the translation system.
  • the translation system employed comprises only defined constituents, as opposed to being crude cell extracts. Yet other embodiments of this aspect may be conducted in vivo.
  • the invention provides a method of screening for telomerase assembly inhibitors by contacting (incubating) one or more telomerase chaparonins (e.g., p23, hsp90, hsp40, hsp70, and HOP) and TERT in the presence or absence of a test compound; comparing the association of the chaparonin(s) and TERT in the presence of the test compound with association in the absence of the test compound, and identifying a compound that decreases the association of the chaparonin(s) and TERT as a telomerase assembly inhibitor.
  • telomerase chaparonins e.g., p23, hsp90, hsp40, hsp70, and HOP
  • Yet another aspect of the invention concerns methods of modulating telomerase activity in a cell expressing a telomerase catalyitc protein component and a telomerase RNA component.
  • Certain preferred embodiments of this aspect concern decreasing the amount of a telomerase chaparonin in the cell; inhibiting association of a telomerase chaparonin with a telomerase catalytic protein component; inhibiting association of a telomerase chaparonin with a telomerase RNA component; stimulating association of a telomerase chaparonin with a telomerase catalytic protein component; stimulating association of a telomerase chaparonin with a telomerase RNA component; and increasing the amount of a telomerase chaparonin in the cell.
  • the cell to be treated is a vertebrate cell, preferably an animal cell, for example an avian, bovine, canine, equine, feline, ovine, or porcine cell.
  • animal cell for example an avian, bovine, canine, equine, feline, ovine, or porcine cell.
  • Particularly preferred cells are mammalian cells, including human cells, be they in culture or in vivo.
  • the compound, including the test compound is a small molecule (i.e., an organic molecule, other than nucleic acid or polypeptide, that has a molecular weight of less than about lOkD, preferably less than about 5kD, particularly less than 1.5kD), a nucleic acid (i.e., a synthetic or natural single or double-stranded polynucleotide comprising more than about six nucleotides), or a polypeptide (i.e., two or more amino acids joined by a peptide bond, including proteins, enzymes, and fragments thereof).
  • nucleic acids are expression vectors, antisense nucleic acids (DNA or RNA), oligonucleotides, and a ribozyme.
  • compositions which contain a therapeutically effective amount (e.g., the amount required to achieve the desired therapeutic effect, which will depend on the patient and condition to be treated, and will preferably be an optimized amount such that the desired modulating effect (inhibition or stimulation, as the case may be) is achieved without significant side-effects to the extent that those can be avoided, but to the extent they can not be or are otherwise tolerable, the minimum amount necessary to achieve the desired modulation) of a compound which modulates telomerase activity through modulation of the association between one or more telomerase chaparonins and the telomerase catalytic protein component and/or a telomerase RNA component of the telomerase holoenzyme.
  • a therapeutically effective amount e.g., the amount required to achieve the desired therapeutic effect, which will depend on the patient and condition to be treated, and will preferably be an optimized amount such that the desired modulating effect (inhibition or stimulation, as the case may be) is achieved without significant side-effects to the extent that those can
  • compositions may include one or more targeting elements.
  • Other compounds may also be included in the formulations of the invention, such as excipients, buffers, etc.
  • Such compositions include those which are in liquid or dry (e.g., freeze dried and lyophilized) form.
  • Such compositions may also be formulated as pills, such as for oral administration.
  • Other routes of administration of various embodiments include by parenteral (e.g., intravenous, intramuscular, and interperitoneal) injection, by inhalation, as well as other routes known in the art.
  • parenteral e.g., intravenous, intramuscular, and interperitoneal
  • the dosage and treatment regimen for a particular composition will depend on many factors, including the disease or condition to be treated, the age, weight, gender, and physical condition of the patient, etc.
  • Yet another aspect of the invention involves methods of modulating proliferation of cells by administering to cells which contain telomeres an amount of a compound which modulates telomerase activity through modulation of association between a telomerase chaparonin and a telomerase catalytic protein component and/or a telomerase RNA component.
  • the proliferative capacity of cells can be increased or decreased, depending upon the compound employed, and thus can be used to treat a wide variety of diseases, disorders, and conditions, for example, diseases associated with elevated levels of telomerase activity, e.g. cancer, leukemia, and infection with a eukaryotic pathogen, as well as states where telomerase activity is not detectable or is insufficient to prevent cellular senescence, for example, aging, expansion of somatic cells in culture, etc.
  • telomerase chaparonins e.g., phosphoprotein p23 (hereinafter “p23”) and the heat shock protein hsp90 (hereinafter “hsp90”)
  • p23 phosphoprotein p23
  • hsp90 heat shock protein
  • the instant invention provides novel methods and compositions for inhibiting or stimulating telomerase activity in a cell (in culture or in vivo) or in vitro composition to inihibit or stimulate, as the case may be, telomerase RNP assembly.
  • the present invention further provides screening methods for identifying novel compounds that inhibit or stimulate telomerase assembly and activity in cells or in vitro.
  • the invention further comprises methods of in vitro assembly of active telomerase.
  • such methods comprise combining TERT, TR, and p23, hsp40, hsp70, hsp90, and HOP proteins, particularly where one or more of the components of the reaction mixture are substantially purified, especially purified recombinant proteins.
  • telomere RNP telomerase RNA component
  • therapeutic e.g., anticancer
  • present invention provides an improved method for in vitro assembly of telomerase comprising combining TERT, a telomerase RNA component, and one or more of substantially purified or recombinant telomerase chaparonins p23, hsp40, hsp70, hsp90, and HOP protein.
  • the present invention encompasses several different ways of combining (or contacting) telomerase assembly associated proteins, TERT, and template RNA (e.g., hTR) to reconstitute active telomerase.
  • each of the components is separately produced and purified, and the purified components combined in a reaction mixture containing other constituents required for activity of the protein and/or nucleic acid components (e.g., typically in the presence of buffer, salt, protease inhibitors, RNAse inhibitors, ATP, Mg , and the like; see Dittmar, et al, 1996, J. Biol. Chem. 271:12833-12839, and Dittmar, et al, 1997, J. Biol.
  • one or more of the components is added in the form of a polynucleotide (e.g., RNA or DNA) comprising a sequence encoding the protein or RNA, which sequence is operably linked to appropriate regulatory elements such as a promoter, under conditions that permit the transcription and or translation of the protein or RNA.
  • expression vectors encoding one or more of the protein and RNA components is incubated in a cell-free expression system.
  • an expression vector encoding hTR e.g., U.S. Patent No. 5,583,016
  • hTR e.g., U.S. Patent No. 5,583,016
  • substantially purified hTERT, p23, hsp40, hsp70, hsp90 and HOP protein can be combined with substantially purified hTERT, p23, hsp40, hsp70, hsp90 and HOP protein to produce or enhance telomerase assembly and activity.
  • telomerase assembly by coexpression of TERT and TR in a rabbit reticulocyte lysate ("RRL") or other cell-free expression system (e.g., wheat germ) can be readily accomplished using art known methods.
  • Some such systems e.g., the RRL
  • Some such systems comprise a complex, undefined (or largely undefined) mixture of proteins and other molecules.
  • telomerase chaparonins or their functional equivalents
  • telomerase chaparonins or their functional equivalents
  • p23, hsp40, hsp70, hsp90 and HOP and possibly including polynucleotides encoding one or more of these proteins.
  • the present invention also contemplates the addition of one, several, or all of the telomerase assembly proteins, preferably in substantially purified, especially recombinant, form to a composition such as a rabbit reticulocyte lysate to increase or stimulate telomerase assembly and activity.
  • the invention provides methods of enhancing telomerase activity in vitro comprising adding at least one substantially purified or recombinant telomerase chaparonin protein, particularly one or more proteins selected from the group consisting of p23, hsp40, hsp70, hsp90, and HOP, to a composition (which may include components of a, e.g., reticulocyte lysate), comprising TERT and a telomerase RNA template.
  • a composition which may include components of a, e.g., reticulocyte lysate
  • the sequence of combination of the TERT, RNA, and telomerase chaparonin(s) may be selected based on the convenience of the practitioner. Thus, in some embodiments, for example, the TERT, RNA, and telomerase chaparonin(s) are combined at the same time. In other alternative embodiments, the TERT protein and telomerase chaparonin(s) are combined, and the RNA component is added subsequently.
  • the TERT, RNA, and chaparonin(s) are human proteins (i.e., they have the sequence of naturally occurring human proteins such as those described herein).
  • one or more of the chaparonins is a functionally active variant or fragment of the human proteins.
  • a variant of hsp90, hsp70, HOP or p23 is "functionally active” if it is able to convert the glucocorticoid receptor hormone binding domain from a non-steroid binding to a steroid binding confirmation, when combined with the other three components (see Dittmar, et al, 1996, J. Biol Chem.
  • the chaparonins are from species other than human, such as yeast, mouse, non-human primate, or plant, while in other embodiments, the chaperonins are non-polypeptide-based molecules that faciltate assembly of multi-subunit protein complexes and/or assumption and/or maintenance of a correctly folded three-dimensional conformation, i.e., a conformation that mimics the natural conformation of the protein, thereby enabling it to perform the appropriate biological function(s).
  • the in vitro reconstitution of telomerase includes the addition of additional substantially purified or recombinant proteins such as HIP (hsp70 interacting protein, also called p48; see Chen, et al, 1996, Mol Endocrinol, 10:682-693; Smith, et al, 1993, Mol Cell Biol. 13:869-876), other immunophilins (Pratt, et al, 1997, Endocrine Reviews 18:306-360; Freeman, et al, 1996, Science 274:1718-1720), and others.
  • HIP hsp70 interacting protein, also called p48
  • HIP hsp70 interacting protein, also called p48
  • p48 immunophilins
  • telomerase is reconstituted by combining hTERT, hTR, and substantially purified or recombinant telomerase assembly associated proteins (e.g., in a volume of 50 ⁇ l).
  • test compounds e.g., any synthetic or natural compound or composition, including small molecules, peptides, proteins, nucleic acids, sugars, nucleic acids, fatty acids
  • test compounds are preincubated in a total volume of 25 ⁇ L for 30 minutes at room temperature in the presence of 2.5 ⁇ L of a solution constituting the components required for telomerase assembly, 2.5% DMSO, and IX TRAP Buffer (20 mM Tris-HCl, pH 8.3, 1.5mM MgCl 2 , 63 mM KC1, 0.05%Tween20, 1.0 mM EGTA, 0.1 mg/ml Bovine serum albumin).
  • the TRAP assay reaction mixture is composed of IX TRAP buffer, 50 ⁇ L dNTP, 2.0 ⁇ g/ml primer ACX, 4 ⁇ g/ml primer U2, 0.8 attomol/ml TSU2, 2 units/50 ⁇ l Taq polymerase (Perkin Elmer), and 2 ⁇ g/ml [ P] 5 'end-labeled primer TS (3000Ci/mmol).
  • the reaction tubes are then placed in the PCR thermocycler (MJ Research) and PCR is performed as follows: 60 min at 30°C, 20 cycles of (30 sec at 94°C, 30 sec. at 60°C, 30 sec. at 72°C), 1 min at 72°C, cool down to 10°C.
  • the TRAP assay is described, as noted supra, in U.S. Patent No. 5,629,154.
  • the primers and substrate used have the sequences: TS Primer
  • telomerase activity in the presence of the test compound is measured by comparing the incorporation of label in reaction product to a parallel reaction lacking the agent.
  • telomerase assays known in the art may also be adapted for use in conjunction with the practice of this invention.
  • the telomerase assays preferred for use in accordance with this invention are those which are scalable and thereby useful in high throughput screening (e.g., to run in parallel from more than about 10 to about 10,000 or more separate reactions).
  • the present invention is based on the unexpected discovery that the proteins p23 and hsp90 interact with TERT in vitro and in vivo, and that this association results in enhanced assembly of the TERT/TR complex resulting in active telomerase.
  • One aspect of the present invention provides a method of screening for telomerase assembly inhibitors by incubating p23 (and/or other now known or later discovered telomerase chaparonins) and TERT with a test compound, comparing the association of p23 and TERT in the presence of the test compound, as compared to such association in the absence of the test compound, and identifying a compound that inhibits such association as a telomerase assembly inhibitor.
  • the invention provides methods of screening for telomerase assembly inhibitors by incubating hsp90 and TERT with test compound, comparing the association of hsp90 and TERT in the presence of the test compound with association in the absence of the test compound, and identifying a compound that inhibits the association of hsp90 and TERT as a telomerase assembly inhibitor.
  • association i.e., covalent or non-covalent binding or other intermolecular interaction
  • the association may be measured in vitro (e.g., using purified proteins in the presence or absence of the test compound), in cells (e.g., incubated in the presence or absence of the test compound), or in cell lysates (where the incubation with the test compound is carried out either in intact cells before lysis, or in the cell lysates themselves).
  • Any suitable method may be used in the present invention to measure the association between TERT telomerase chaparonins such as p23 or p90, including by gel filtration, gel-shift assays (also called electrophoretic/mobility shift assays), and matrix binding assays.
  • Matrix binding assays include several variations to a basic technique in which one protein (e.g., TERT) of a protein pair (e.g., TERT and p23, or TERT and p90) is bound (e.g., by UV cross-linking or by binding to an immobilized immunoglobulin) to a matrix (e.g., a nylon, nitrocellulose, or plastic matrix) and the second protein, which is detectably labeled or otherwise detectable, is added under conditions (e.g., pH, ionic strength, etc.) that permit the two proteins to associate. Residual unbound protein is removed by washing the membrane, and the amount of bound second protein is determined, e.g., by quantitation of the detectable signal.
  • TERT protein of a protein pair
  • a matrix e.g., a nylon, nitrocellulose, or plastic matrix
  • the second protein which is detectably labeled or otherwise detectable, is added under conditions (e.g., pH, ionic strength,
  • immunological assays such as a "two antibody sandwich assay” are used.
  • assays are well known in the art and are described, for example, in Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, COLD SPRING HARBOR LABORATORY, New York (1988), Chapter 14 (e.g., p. 583); and Ausubel et al., 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York.
  • an immobilized first antibody specific for one protein e.g., TERT
  • a heteromeric complex e.g., p23/hsp90/TERT
  • a second protein e.g., p23
  • numerous variations of these assays e.g., differences in the order of addition components, the use of a non-immobilized first antibody that is immobilized after complex formation, etc.
  • alternative assays and assay formats are well known in the art.
  • the present invention is based on the discovery that, unexpectedly, a native telomerase RNP complex in cells contains both p23 and hsp90.
  • the invention provides methods of detecting an active telomerase complex by detecting TERT associated with p23 or hsp90, or both. Assays such as those described herein can be used to detect such complexes in lysates of telomerase positive cells. Quantitation of the TERT/hsp90/p23 complexes can be used for diagnosis and prognosis of telomerase-associated diseases.
  • the present invention is based in part on the discovery that depletion of p23 from a reticulocyte lysate-based in vitro telomerase assembly reaction results in reduced telomerase activity in the reaction. Similarly, the inhibition of hsp90 function results in reduced telomerase activity.
  • the present invention provides methods and compositions for decreasing telomerase activity in a cell by decreasing the amount of one or more telomerase chaparonins, e.g., hsp90 or p23, in the cell and/or inhibiting the association between such proteins and TERT.
  • inhibition of telomerase activity in vitro or in vivo may be used to treat diseases and disorders associated with cell immortality, such as neoplasia and pathogenic eukaryotic organisms.
  • telomere activity by enhancing such association to increase telomerase activity and extend telomere length, and will thereby be useful in the treatment of conditions associated with cellular senescence (e.g., aging of tissues, organs, and organisms) or an increased rate of cell proliferation, such as is observed in cases of premature aging.
  • conditions associated with cellular senescence e.g., aging of tissues, organs, and organisms
  • an increased rate of cell proliferation such as is observed in cases of premature aging.
  • such stimulation would be useful to forestall and reverse cellular senescence, including but not limited to conditions associated with cellular senescence, e >.
  • cells with replicative capacity in the central nervous system including astrocytes, endothelial cells, and fibroblasts which play a role in such age-related diseases as Alzheimer's disease, Parkinson's disease, Huntington's disease, and stroke
  • cells with finite replicative capacity in the integument including fibroblasts, sebaceous gland cells, melanocytes, keratinocytes, Langerhan's cells, and hair follicle cells which may play a role in age- I
  • telomere assembly/activity in a cell is useful, ter alia, to change the proliferative capacity of the cell. For example, reduction of telomerase activity in an immortal cell, such as a malignant tumor cell, will reduce the ability of the cell to proliferate. Decreasing the proliferative capacity of tumor cells will alleviate the disease or reduce the aggressive nature of a cancer to a more manageable disease state (increasing the efficacy of traditional interventions). See, e.g., WO 98/14593.
  • cancers of any type which have an elevated level of telomerase activity (i.e., the absolute level of telomerase activity in the particular cell is elevated compared to normal cells in that individual, or compared to normal cells in other individuals not suffering from the condition), including solid tumors and leukemias (including those in which cells are immortalized, including: apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., b-cell, mixed-cell, null-cell,
  • telomeres in addition to neoplasias wherein the tumor cells have acquired an immortal phenotype through the inappropriate activation of telomerase, including various human and veterinary parasitic diseases, can also be treated in accordance with the instant methods, particularly where the parasites or pathogens which cause the disease themselves express telomerase, as is the case for many pathogens that are eukaryotes.
  • a representative sampling of some of these diseases include human protozoal pathogens such as; amebiasis from Entamoeba histolytica, amebic meningoencephalitis from the genus Naegleria or Acanthamoeba, malaria from Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum, Leishmaniasis from such protozoa as Leishmania donovani, Leishmania infantum, Leishmania chagasi, Leishmania tropica, Leishmania major, Leishmania aethiopica, Leishmania mexicana, and Leishmania braziliensis, Chagas' disease from the protozoan Trypanosoma cruzi, sleeping sickness from Trypanosoma brucei, Trypanosoma gambiense, and Trypanosoma rhodesiense, Toxoplasmosis from Toxoplasma gondii, giardias
  • Telomerase inhibitors would also be useful in treating certain helminthic infections including the species: Taenia solium, Taenia saginata, Diphyllobothrium lata, Echinococcus granulosus, Echinococcus multilocularis, Hymenolepis nana, Schistosoma mansomi, Schistosoma japonicum, Schistosoma hematobium, Clonorchis sinensis, Paragonimus westermani, Fasciola hepatica, Fasciolopsis buski, Heterophyes heterophyes, Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichinella spiralis, Wuchereria bancrofti, Onchocerca volvulus, Loa loa, Dracunculus medinensis, and fungal pathogen
  • veterinary protozoal pathogens such as: Babesia caballi, Babesia canis, Babesia equi, Babesia felis, Balantidium coli, Besnoitia darlingi, Eimeria acervulina, Eimeria adenoeides, Eimeria ahsata, Eimeria alabamensis, Eimeria auburnensis, Eimeria bovis, Eimeria brasiliensis, Eimeria brunetti, Eimeria canadensis, Eimeria cerdonis, Eimeria crandallis, Eimeria cylindrica, Eimeria debliecki, Eimeria despersa, Eimeria ellipsoidalis, Eimeria fauvei, Eimeria gallopavonis, Eimeria gilruthi, Eimeria granulosa, Eimeria hagani, Eimeria illinoisensis, Eimeria innoc
  • the quantity of hsp90 and/or p23 protein (or one or more other telomerase chaparonins) in a cell can be reduced by a variety of methods known in the art to result in a decrease in telomerase activity in a cell (and an associated decrease in telomere length and cell proliferative capacity).
  • the level of a protein in a cell is reduced by interfering with the expression of the gene encoding the protein (e.g., by reducing transcription, RNA processing or stability, or translation).
  • Exemplary methods include, but are not limited to, the use of antisense, triplex, or ribozyme polynucleotides (together referred to herein as "inhibitory nucleic acids”) to reduce expression of one or telomerase chaparonins, alone or in conjunction with reducing expression of HERT and/or TR.
  • antisense oligonucleotides or polynucleotides complementary to the nucleotide sequence of the target (e.g., human) p23 or hsp90 gene sequence (described elsewhere herein) can be expressed in, or administered to, cells to decrease hsp90/p23 levels (see, e.g., Milner, et al, 1997, Nature Biotechnology 15:537; Uhlmann et al., 1990, Chem. Reviews, 90:543-584). Without intending to be limited to any particular mechanism, it is believed that antisense oligonucleotides bind to, and interfere with the translation or stability of the target mRNA.
  • telomerase chaparonin-specific ribozymes for example, targeted to hsp90 and/or p23, comprising 5'- and 3'-terminal sequences complementary to target mRNA sequences can be engineered (see PCT publications WO 93/23572, WO 94/02595, and WO 93/23569) and administered to a cell.
  • nucleic acids including oligonucleotides, ribozymes, and expression vectors encoding one or more of such molecules (alone or in combination) to cells are known in the art (e.g., including introduction into the cell using liposomes, immunoliposomes, ballistics, direct uptake into cells, viral vectors, any of the foregoing of which may further comprise cell- or tissue-specific targeting molecules, and the like; see, e.g., U.S. Patent 5,272,065).
  • the present invention provides methods and compositions for decreasing telomerase activity in a cell by inhibiting the association between a telomerase chaparonin, e.g., p23 or hsp90, and a component of the telomerase holoenzyme (which comprises the telomerase catalytic protein component in association with the telomerase RNA component).
  • a telomerase chaparonin e.g., p23 or hsp90
  • a component of the telomerase holoenzyme which comprises the telomerase catalytic protein component in association with the telomerase RNA component.
  • the inhibitor is a molecule identified in a modulator screen as described herein.
  • the telomerase component/telomerase chaparonin interaction is inhibited by a molecule (e.g., a small, synthetic organic molecule or peptide) that interacts with the site in the amino terminal domain of TERT that binds p23 (e.g., a site within the amino-terminal 195 residues of hTERT).
  • a molecule e.g., a small, synthetic organic molecule or peptide
  • the HERT and/or telomerase chaparonin components useful in the practice of this invention include functionally active proteins and variants (e.g. , those having one or more amino acid changes as compared to the native amino acid sequence(s) then known for the corresponding polypeptide), inactive variants, and nucleic acids encoding the same.
  • nucleic acids used in practicing various aspects this invention need not be those of the corresponding naturally occurring genes, but instead can be any nucleic acid coding for an RNA or amino acid sequence, as the case may be, of the desired expression product.
  • a benzoquinone ansamysin such as geldanamycin, is used to inhibit telomerase assembly and activity.
  • compositions comprising one or more telomerase modulators according to the invention, alone or in combination with at least one other agent, such as a stabilizer, diluent, carrier, excipient, adjuvant or other ingredient not capable of eliciting the desired therapeutic effect.
  • the compounds of the invention are preferably delivered in any sterile, biopharmaceutical carrier, including, but not limited to saline, buffered saline, dextrose, and water, and can be delivered alone or in combination with other therapeutic agents, drugs, or hormones.
  • the therapeutically effective dose can be estimated initially from cell culture assays. Such information can be used to more accurately determine useful doses in organisms such as plants and animals, preferably mammals, and most preferably humans. Levels in plasma may be measured, for example, by HPLC or other means appropriate for detection of the particular compound.
  • the attending physician would know how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, or other systemic malady. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity).
  • the magnitude of an administered dose in the management of the disorder of interest will vary with the severity of the condition to be treated and the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above also may be used in veterinary applications of the invention.
  • Such agents may be formulated and administered systemically or locally, i.e., topically.
  • Techniques for formulation and administration may be found in Alfonso and Gennaro (1995). Suitable routes may include, for example, oral, rectal, transdermal, vaginal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, or intramedullary injections, as well as intrathecal, intravenous, or intraperitoneal injections.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention in particular those formulated as solutions, may be administered parenterally, such as by intravenous injection.
  • Appropriate compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Further details on formulation, compounding, and administration techniques can be found in the latest edition of "Remington's Pharmaceutical Sciences," Maack Publishing Co., Easton PA.
  • Compositions according to the invention include those in solution or dry formulations.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly. Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions, including those formulated for delayed release or only to be released when the pharmaceutical reaches the small or large intestine.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropyl
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to i
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 60%, preferably 80%), most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • Example 1 The Phosphoprotein p23 Interaction with hTERT
  • yeast two-hybrid system (Fields, et al, 1989, Nature 340:245-246) was used to screen for molecules that interact with the human telomerase reverse transcriptase (hTERT) protein using the LexA-dependent reporter strain L40 and a mouse embryo library (Vojtek et al., 1993, Cell 74:205-214).
  • the "bait" used in the screen was a gene encoding the N-terminal 195 residues of hTERT with codon usage optimized for expression in S. cerevisiae.
  • the gene was assembled from six oligonucleotide pairs (HHMI/Keck Oligonucleotide Facility, Yale Univ.), the ends of which were staggered by ten bases to create complementary sequences for ligation.
  • the oligonucleotides were gel purified. Each of the pairs was generated by annealing of gel purified oligonucleotides by heating to 100°C for 10', then at 90°C for 10', followed by rapid chilling.
  • the full-length construct (pJBTl) was built by sequential ligation of adjoining pairs with gel purification of ligated fragments prior to each subsequent ligation.
  • the final product was subcloned into pJBTO, a modified form of pBluescript II-SK+ (Stratagene).
  • the BamHI-Sall fragment of pJBTl was inserted into pBTM116 (Nojtek et al., supra) to create the bait encoding the first 195 amino acids of hTERT.
  • Transformants from the yeast reporter strain L40, selected to express LexA-hTERT (1-195) and VP16-p23 alone or together were tested for the ability to grow on media lacking histidine. Growth on the selective plate indicated a positive two-hybrid interaction (Johnson et al., 1994, Mol. Cell Bio. 14:1956-1963).
  • the screens of the mouse embryo library of cD ⁇ As expressed as fusions to the VP16 activation domain against a bait expressing the amino terminus of hTERT (amino acids 1-195) fused to the LexA D ⁇ A-binding domain resulted in the isolation of a clone encoding a 23 KDa phosphoprotein, p23.
  • Example 2 hTERT Associates with p23 and hsp90 In Vitro
  • p23 has been implicated in mediating formation of receptor/ligand complexes (Pratt et al., 1997, Endocrine Reviews 18:306-360) as well as a protein/RNA complex (Hu et al., 1997, EMBO 16:59-68).
  • yeast two-hybrid analysis played a role in the assembly of in vitro translated hTERT and the template RNA (hTR) into active complexes, the association of in vitro transcribed and translated hTERT with p23 and hsp90 was assayed.
  • hTERT was synthesized in the rabbit reticulocyte system (Promega) as previously described (Weinrich et al, 1997, Nature Gen. 17: 498-502) in the presence of 35 S-methionine.
  • hTERT cDNA was cloned into pcDNA3.1/HisC (Invitrogen) with the addition of a sequence encoding three copies of a carboxy-terminal hemagluttinen epitope (HA).
  • 35 S-labeled hTERT was incubated with normal mouse IgG, the monoclonal anti-p23 antibody JJ3 (Johnson et al., 1994, Mol. Cell. Bio.
  • hTERT 35 S-methionine labeled hTERT synthesized in rabbit reticulocyte lysate was immunoprecipitated with either anti-p23 or anti-hsp90 antibodies, demonstrating that full-length hTERT can associate with these molecules in vitro. This association did not require the presence of the telomerase template RNA.
  • Example 3 A Native Telomerase Complex in Cells Contains Both p23 and hsp90
  • telomerase activity was present in anti-p23 but not normal mouse IgG immunoprecipitates from HT1080 fibrosarcoma cells.
  • Hsp90 antibodies also coimmunopreciptated telomerase activity, demonstrating that a native telomerase complex in cells contains both p23 and hsp90.
  • HT1080 cells were cultured in 10%) serum on 15 cm plates. After reaching confluence, one plate of cells per immunoprecipitati ⁇ n was lysed by douncing in ice-cold PBS with protease inhibitors. Ten ⁇ l of antibody together with 30 ⁇ l protein A/G agarose were added to the lysates (1ml) and incubated at 4°C for 1 hour. Pellets were washed 4 times,
  • telomere activity assays can be used for various purposes, including for use in diagnosis and prognosis of telomerase-related diseases.
  • ⁇ 23 depleted RRL (p23 dep RRL) supplemented with 0 ng, 50 ng or 100 ng recombinant p23, or (4) a buffer comprising 10 mM Tris-HCl, 50 mM KC1, 5 mM MgCl 2 , 2 mM DTT, pH 7.5, ("-RRL").
  • the hTERT/RRL mixture was mixed with 0.5 ⁇ g of in vitro transcribed hTR. The mixture was incubated at 30°C for 90 minutes to allow assembly of hTERT and its hTR template. An aliquot was then removed and assayed for activity by TRAP.
  • telomere lysate Addition of reticulocyte lysate to the hTERT/l TR reconstitution step ("+RRL") resulted in a 50 to 100-fold increase in telomerase activity consistent with the observation that factors in rabbit reticulocyte lysate promote the efficient assembly of in vitro translated hTERT and the template RNA (hTR) into active complexes. This increased activity is independent of additional protein synthesis, as it was insensitive to addition of cyclohexamide (Weinrich et al., 1997, Nature Gen. 17: 498-502). However, immunodepletion of p23 from reticulocyte lysate significantly decreases its ability to stimulate generation of telomerase activity.
  • the benzoquinone ansamysin, geldanamycin inhibits some functions of hsp90 by binding to its ATP -binding site, and it also blocks ATP-dependent binding of p23 to hsp90 (Grenert et al., 1997, J. Biol. Chem. 272:23843-850; Prodromou et al., 1997, Cell 90:65-75).
  • Incubation of reticulocyte lysates with geldanamycin (Calbiochem) completely blocked the ability of reticulocyte lysates to enhance telomerase assembly (as assessed by the TRAP assay).
  • telomerase was assembled as described above, except that translated hTERT was diluted in RRL that had been incubated in the presence of geldanamycin (lOOug/ml in a final DMSO concentration of 10%) or DMSO only (10%) carrier for 30 minutes. Addition of geldanamycin (100 ⁇ g/ml) after the assembly step, but prior to the telomerase activity assay, had no effect on activity. Thus, thus geldanamycin did not inhibit association of hTERT/hTR with the substrate primer or inhibit enzymatic activity in vitro.
  • This linker hybridizes with a complementary biotinylated oligonucleotide, allowing for retrieval of ribonucleoprotein complexes with streptavidin magnetic beads.
  • 35 S-labeled hTERT was translated in RRL in the presence of 50 ⁇ g/ml geldanamycin or 5% DMSO (carrier).
  • Proteins bound to beads were analyzed by 7.5% SDS- PAGE and autoradiography. Northern analysis was used to confirm that linker-hTR and linker-U2 were captured in all reactions. An aliquot (10% of input) of each translation reaction was analyzed for comparison of amounts hTERT and luciferase present. The addition of geldanamycin had no effect on the association of hTERT and hTR.
  • telomere activity in human fibrosarcoma cells is downregulated when the cells become quiescent and is re-induced upon growth stimulation (Holt et al., 1997, Proc. Natl. Acad. Sci. USA 94:10687-692).
  • Serum-starved, quiescent HT1080 cells with low levels of telomerase activity were first treated with a range of concentrations of geldanmycin or the carrier DMSO, followed by addition of serum.
  • HT1080 cells were cultured as described previously (Holt et al., 1997, Proc.
  • telomere activity blocked induction of telomerase activity at concentrations that did not affect S-phase entry as determined by 3 H-thymidine incorporation assays (Holt et al., 1996, Mol. Cell. Biol. 16:2932-39. Short-term viability was also not affected. Effects on plating efficiency were assayed by cell counts before and after stimulation followed by replating. All samples had similar plating efficiencies of 70-80%) except for treatment with 40 ⁇ g/ml of CsA which reduced plating efficiencies to 60%.
  • Shock Proteins 40. 70 and 90 HOP, and p23.
  • telomere assembly assay 0.2 ⁇ l of in vztr ⁇ -transcribed and - translated hTERT and 0.5 ⁇ g of hTR were mixed together in a 4 ⁇ l assembly assay with or without additional fresh RRL. The reaction was mixed and incubated for 90 minutes at 30°C.
  • a 5 ⁇ L reconstitution is first prepared from the following components added in order:
  • Buffer for the reconstitution reaction is 10 mM Tris-HC 1, 50 mM KC1, 5 mM MgCl 2 and 2 mM dithiothreitol, pH 7.5.
  • telomerase activity To test for telomerase activity, the total volume is then brought up to 100 ⁇ L with CHAPS Lysis Buffer and 1 ⁇ L of this diluted sample replaces the usual 2 ⁇ L sample in a regular TRAP assay.

Abstract

L'invention concerne des méthodes et compositions d'assemblage de télomérase active in vitro et dans des cellules, en culture ou in vivo, ainsi que des méthodes d'inhibition ou de stimulation d'une activité de télomérase par modulation d'un assemblage de télomérase. Dans certains modes de réalisation préférés, l'invention concerne des méthodes d'assemblage in vitro d'un composant protéine de télomérase et d'un composant ARN de télomérase, les méthodes consistant à ajouter une ou plusieurs molécules de chaperonine, en particulier sensiblement purifiées ou des chaperonines de télomérase recombinées, y compris les protéines hsp40, hsp70, hsp90, p23 et HOP. Dans ces méthodes, on combine une ou plusieurs chaperonines de télomérase dans un mélange réactif contenant également la protéine catalytique et des composants ARN de télomérase. L'invention concerne en outre des méthodes de criblage permettant d'identifier un assemblage de télomérase et des inhibiteurs d'activité, ainsi que des méthodes de stimulation ou d'inhibition d'une activité et d'un assemblage de télomérase.
PCT/US1999/017724 1998-08-09 1999-08-05 Methodes et compositions d'inhibition ou de stimulation d'un assemblage de telomerase WO2000008135A1 (fr)

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WO2000068693A2 (fr) * 1999-05-07 2000-11-16 The European Molecular Biology Laboratory Methodes reposant sur la localisation d'hsp90 dans le centrosome
EP1254962A1 (fr) * 2001-04-30 2002-11-06 Roche Diagnostics GmbH Méthode d'expression de protéines dans un système de traduction in vitro avec co-expression de protéines d'aide au repliement
WO2010135247A1 (fr) 2009-05-18 2010-11-25 TA Therapeutics, Ltd. Compositions et procedes pour accroitre l'activite telomerase
US7846904B2 (en) 2003-06-23 2010-12-07 Geron Corporation Compositions and methods for increasing telomerase activity
CN1952663B (zh) * 2005-08-26 2011-05-18 中国科学院上海生命科学研究院 端粒酶结合蛋白p23的应用
US9248088B2 (en) 2003-06-25 2016-02-02 Telomerase Activation Sciences, Inc. Compositions and methods for skin conditioning

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000068693A2 (fr) * 1999-05-07 2000-11-16 The European Molecular Biology Laboratory Methodes reposant sur la localisation d'hsp90 dans le centrosome
WO2000068693A3 (fr) * 1999-05-07 2001-01-25 European Molecular Biology Lab Embl Methodes reposant sur la localisation d'hsp90 dans le centrosome
US6335157B1 (en) 1999-05-07 2002-01-01 The European Molecular Biology Laboratory Method based on localization of Hsp90 to the centrosome
EP1254962A1 (fr) * 2001-04-30 2002-11-06 Roche Diagnostics GmbH Méthode d'expression de protéines dans un système de traduction in vitro avec co-expression de protéines d'aide au repliement
JP2002335959A (ja) * 2001-04-30 2002-11-26 F Hoffmann La Roche Ag フォールディング補助タンパク質の共発現を伴うｉｎｖｉｔｒｏ翻訳系でのタンパク質の発現方法
US6929929B2 (en) 2001-04-30 2005-08-16 Roche Diagnostics Operations, Inc. Method for the expression of proteins in in vitro translation systems with coexpression of folding helper proteins
EP2548880A2 (fr) 2003-06-23 2013-01-23 Geron Corporation Compositions pour augmenter l'activité de la télomérase
US7846904B2 (en) 2003-06-23 2010-12-07 Geron Corporation Compositions and methods for increasing telomerase activity
US8759304B2 (en) 2003-06-23 2014-06-24 Telomerase Activation Science, Inc. Compositions and methods for increasing telomerase activity
US9248088B2 (en) 2003-06-25 2016-02-02 Telomerase Activation Sciences, Inc. Compositions and methods for skin conditioning
CN1952663B (zh) * 2005-08-26 2011-05-18 中国科学院上海生命科学研究院 端粒酶结合蛋白p23的应用
WO2010135247A1 (fr) 2009-05-18 2010-11-25 TA Therapeutics, Ltd. Compositions et procedes pour accroitre l'activite telomerase
US8481721B2 (en) 2009-05-18 2013-07-09 Telomerase Activation Sciences, Inc. Compositions and methods for increasing telomerase activity
US9403866B2 (en) 2009-05-18 2016-08-02 Telomerase Activation Sciences, Inc. Compositions and methods for increasing telomerase activity
US9913851B2 (en) 2009-05-18 2018-03-13 Telomerase Activation Sciences, Inc. Compositions and methods for increasing telomerase activity

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