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WO2002086170A1 - Tankyrase h, compositions impliquees dans le cycle cellulaire et methodes d'utilisation - Google Patents

Tankyrase h, compositions impliquees dans le cycle cellulaire et methodes d'utilisation Download PDF

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Publication number
WO2002086170A1
WO2002086170A1 PCT/US2002/013185 US0213185W WO02086170A1 WO 2002086170 A1 WO2002086170 A1 WO 2002086170A1 US 0213185 W US0213185 W US 0213185W WO 02086170 A1 WO02086170 A1 WO 02086170A1
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WIPO (PCT)
Prior art keywords
cell cycle
protein
cell
nucleic acid
taho
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2002/013185
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English (en)
Inventor
Ying Luo
Eva Chan
Xiang Xu
Betty Huang
Valeria Ossovskaya
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rigel Pharmaceuticals Inc
Original Assignee
Rigel Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/843,159 external-priority patent/US6887675B1/en
Application filed by Rigel Pharmaceuticals Inc filed Critical Rigel Pharmaceuticals Inc
Publication of WO2002086170A1 publication Critical patent/WO2002086170A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • telomeres The synthesis of telomeres involves unique DNA replication mechanisms. These mechanisms act to extend telomeres prior to cell division, and are critical to the determination of telomere length in daughter cells.
  • telomere synthesis Several molecules involved in telomere synthesis have been identified, including the proteins telomerase, TRF-1 and tankyrase. These and other molecules involved in telomere synthesis provide unique targets for intervention strategies designed to modulate cell proliferation.
  • the present invention provides a method for screening for agents capable of interfering with the binding of the TaHo cell cycle protein and a p21 protein.
  • a screening method comprises combining TaHo protein, a candidate bioactive agent and a p21 protein, and determining the binding of the TaHo protein and the p21 protein in the presence and absence of candidate bioactive agent.
  • the cell cycle protein and the p21 protein are combined first.
  • Figure 10 shows cell cycle analysis of A549 tumor cells and HeLa cells transfected with T11 TaHo antisense oligonucleotide and cotransfected with FITC-labeled random oligonucleotide. Cell cycle determination was done on the top 5% of GFP-expressing cells using Hoechst dye.
  • Figure 15 shows a dose response inhibition of TaHo PARP activity by the human PARP inhibitor phenanthridinone.
  • the cell cycle protein is termed "tankyrase homolog", sometimes referred to herein as “tankyrase h” or “TaHo".
  • the amino acid sequence is shown in Figure 3 and Figure 4, and the nucleic acid sequence is shown in Figure 1 and Figure 2.
  • the amino acid sequence of tankyrase H bears homology to tankyrase, but preferably, less than 80%.
  • Tankyrase is an enzyme which binds to TRF1 and which has been indicated as having a role in maintaining telomere length. Smith, et al., Science, 282(5393): 1484-7 (1998). More particularly, tankyrase has homology to ankyrins and binds to the telomeric protein TRF1 , a negative regulator of telomere length maintenance.
  • sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleosides, frameshifts, unknown nucleosides, or other types of sequencing errors in any of the sequences; however, the correct sequences will fall within the homology and stringency definitions herein.
  • TaHo cell cycle proteins of the present invention may be shorter or longer than the amino acid sequence encoded by the nucleic acid shown in the Figures.
  • included within the definition of cell cycle proteins are portions or fragments of the amino acid sequence encoded by the nucleic acid sequence provided herein.
  • dominant negative TaHo protein isoforms are capable of inhibiting wildtype TaHo protein activity in vivo. Accordingly, the present invention provides antagonists of wildtype TaHo activity, which include dominant negative isoforms of TaHo.
  • the overall sequence identity of the nucleic acid sequence is commensurate with amino acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence.
  • sequence identity of the nucleic acid sequence as compared to the nucleic acid sequence of the Figures is preferably greater than 75%, more preferably greater than about 80%, particularly greater than about 85% and most preferably greater than 90%. In some embodiments the sequence identity will be as high as about 93 to 95 or 98%.
  • TaHo nucleic acid sequence fragments that differ significantly from the sequence of tankyrase may be of use in the specific antisense targeting of TaHo.
  • TaHo nucleic acid sequence fragments having high identity to tankyrase nucleic acid sequence fragments may be used to target both tankyrase and TaHo by antisense oligonucleotides.
  • nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides.
  • the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids.
  • Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half life of such molecules in physiological environments.
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.
  • the depiction of a single strand also defines the sequence of the other strand (“Crick"); thus the sequences depicted in the Figures also include the complement of the sequence.
  • recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature.
  • substitutions deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the cell cycle protein are desired, substitutions are generally made in accordance with the following chart:
  • Another type of covalent modification of cell cycle comprises linking the cell cycle polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters may be either naturally occurring promoters or hybrid promoters.
  • Hybrid promoters which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.
  • Suitable selection genes include genes which render-the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
  • Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
  • Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.
  • the cell cycle proteins and nucleic acids are useful in a number of applications.
  • the isolation of mRNA comprises isolating total cellular RNA by disrupting a cell and performing differential centrifugation. Once the total RNA is isolated, mRNA is isolated by making use of the adenine nucleotide residues known to those skilled in the art as a poly (A) tail found on virtually every eukaryotic mRNA molecule at the 3'end thereof. Oligonucleotides composed of only deoxythymidine [olgo(dT)] are linked to cellulose and the oligo(dT)-cellulose packed into small columns.
  • transgenic animals comprising a gain of cell cycle protein function exhibit an increase in the rate of cell proliferation, an increase in the potential for proliferation, an increase in the rate of progression through a stage of the cell cycle, an increase in the number of cells, or an alteration in apoptosis.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • Preferred cell types for use in the invention include, but are not limited to, mammalian cells, including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colonrectal, leukemia, brain, etc.
  • mammalian cells including animal (rodents, including mice, rats, hamsters and gerbils), primates, and human cells, particularly including tumor cells of all types, including breast, skin, lung, cervix, colonrectal, leukemia, brain, etc.
  • the methods comprise assaying one or more of several different cell parameters, including, but not limited to, cell viability, cell proliferation, and cell phase. Other parameters include assaying telomere length.
  • Preferred exclusion dyes include, but are not limited to, ethidium bromide; ethidium homodimer-1; propidium iodine; SYTOX green nucleic acid stain; Calcein AM, BCECF AM; fluorescein diacetate; TOTO® and TO-PROTM (from Molecular Probes; supra, see chapter 16) and others known in the art.
  • differential expression refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among the cells.
  • a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus apoptotic cell. That is, genes may be turned on or turned off in a particular state, relative to another state. As is apparent to the skilled artisan, any comparison of two or more states can be made. Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both.
  • cells are contacted with from one to many antibodies to the cell cycle protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected. In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label. In another method the primary antibody to the cell cycle protein(s) contains a detectable label. In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label.
  • the label may be detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used in this method.
  • FACS fluorescence activated cell sorter
  • Oligonucleotides complementary to the TaHo nucleic acid sequence fragment GTGGAACAGAGGGTGCTTCC were transfected into A549 cells and Hela cells. These dominant negative oligonucleotides inhibited cell proliferation in both cell types, as depicted in Figures 9). Further, an increase in the amount of such TaHo antisense oligonucleotide was inversely correlated with the amount of TaHo mRNA detected in these cells, and was further correlated with the degree of proliferation inhibition observed (Figure 9).

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne de nouveaux polypeptides, acides nucléiques et molécules associées présentant un effet sur ou étant impliqués dans le cycle cellulaire. L'invention concerne également des vecteurs et des cellules hôtes comprenant ces séquences d'acides nucléiques, des molécules polypeptidiques chimériques comprenant les polypepides de la présente invention fusionnés avec des séquences polypeptidiques hétérologues, des anticorps de liaison aux polypeptides de l'invention et des méthodes de production des polypeptides de l'invention. L'invention concerne en outre des méthodes d'identification de nouvelles compositions impliquées dans la bioactivité du cycle cellulaire, et l'utilisation de ces compositions dans le diagnostic et le traitement de maladies.
PCT/US2002/013185 2001-04-25 2002-04-25 Tankyrase h, compositions impliquees dans le cycle cellulaire et methodes d'utilisation Ceased WO2002086170A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US09/843,159 US6887675B1 (en) 1999-10-25 2001-04-25 Tankyrase H, compositions involved in the cell cycle and methods of use
US09/843,159 2001-04-25

Publications (1)

Publication Number Publication Date
WO2002086170A1 true WO2002086170A1 (fr) 2002-10-31

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6387902B1 (en) * 1998-12-31 2002-05-14 Guilford Pharmaceuticals, Inc. Phenazine compounds, methods and pharmaceutical compositions for inhibiting PARP

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6387902B1 (en) * 1998-12-31 2002-05-14 Guilford Pharmaceuticals, Inc. Phenazine compounds, methods and pharmaceutical compositions for inhibiting PARP

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SMITH S. ET AL.: "Tankyrase, a poly(ADP-ribose) polymerase at human telomeres", SCIENCE, vol. 282, 20 November 1998 (1998-11-20), pages 1484 - 1487, XP002118903 *

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