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WO2003038078A2 - Enzymes et inhibition d'enzymes - Google Patents

Enzymes et inhibition d'enzymes Download PDF

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Publication number
WO2003038078A2
WO2003038078A2 PCT/GB2002/004940 GB0204940W WO03038078A2 WO 2003038078 A2 WO2003038078 A2 WO 2003038078A2 GB 0204940 W GB0204940 W GB 0204940W WO 03038078 A2 WO03038078 A2 WO 03038078A2
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WO
WIPO (PCT)
Prior art keywords
centrosome
nucleic acid
cell
leu
kinase
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/GB2002/004940
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English (en)
Other versions
WO2003038078A3 (fr
Inventor
David Stott
Seo Seung-Woon
Gavin M. Craig
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.)
University of Warwick
Original Assignee
University of Warwick
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Warwick filed Critical University of Warwick
Priority to AU2002339077A priority Critical patent/AU2002339077A1/en
Publication of WO2003038078A2 publication Critical patent/WO2003038078A2/fr
Publication of WO2003038078A3 publication Critical patent/WO2003038078A3/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.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the invention relates to centrosome associated kinases, and in particular, to the inhibition thereof.
  • Cell division in mammalian cells proceeds through a series of events which together make up the cell cycle. These events are normally regulated such that cells divide only when appropriate to the needs of the organism. This regulation is achieved by a combination of mechanisms reliant either on signals extrinsic to the cell, or on intrinsic molecular pathways.
  • the two essential components of the cell cycle in mammalian cells are replication of the genetic material and division of the cell to produce two daughter cells. These fundamental events are usually separated by growth periods during which the majority of protein synthesis occurs and are regulated in somatic cells such that successive divisions are separated by periods of chromosomal replication (Norbury and Nurse (1992), Annual Review of Biochemistry, 61, 441-470).
  • the replicated chromosomes are partitioned equally to the two daughter cells.
  • the process of cell division involves condensation of the genetic material to form mitotic chromosomes, dissolution of the nuclear membrane, assembly of a physical scaffold, the mitotic spindle, to which the chromosomes attach and movement of the chromosomes to opposite poles of the cell.
  • the mitotic spindle is composed of filaments known as microtubules.
  • Microtubules are composed of tubulins and associated proteins and are nucleated by a structure which is
  • centrosome detectable in the majority of cell types in higher animals and is known as the centrosome.
  • Post-mitotic cells contain a single centrosome and, in diploid organisms such as mammals, two copies of each chromosome. These cells are referred to as being in the Gl phase of the cell cycle and containing a 2n complement of chromosomes.
  • the centrosome is replicated, the chromosomes are replicated leading to a 4n chromosomal complement, and the two daughter centrosomes move to opposite poles of the cell and nucleate formation of a bipolar spindle at mitosis.
  • One centrosome becomes partitioned to each daughter cell at division, as does one set of chromosomes, resulting in return to the 2n state.
  • the centrosome can be regarded as an essential part of the mechanism by which cell division is accomplished in mammalian cells.
  • centrosome is intrinsically involved in the cell cycle.
  • Centrosome components include centrosome associated kinases, an example of which is Mak V, first reported by Korobko (Korobko et al ., (1997) Doklady Akademii Navk, 354, 554-556), and later renamed Hunk (Gardner et al., (2000) Genomics, 63, 46-59), although the function of the protein was not recognised at that time.
  • tumour cells including the embryonal carcinoma line PCC3 (Jakob, et al., (1973) Ann. Microbial (Paris), 124, 269-282) and the melanoma line B16F10 (Fidler, (1975) Cancer
  • centrosome (Zheng, et al, (1991) Cell, 65, 817-823).
  • mutated Mak N exhibits reduced localisation to the centrosomes, leading to an interruption of the cell cycle. Specifically, they have found that kinase activity, that is the ability to phosphorylate a substrate such as one or more arnino acids on the centrosome, is essential for cell cycle regulation.
  • an isolated centrosome associated kinase or a fragment thereof comprising a centrosome binding site, wherein the kinase has decreased or no kinase activity.
  • the centrosome associated kinase is preferably not Mak V (seq.DD No. 2).
  • the decrease in activity may be due to a naturally occurring - or artificially introduced - mutation in the centrosome-associated kinase.
  • centrosome binding site may be identified, for example, by labelling the polypeptide or fragment with, e.g. a fluorescent label, inducing the labelled polypeptide or fragment into a cell, and seeing if the polypeptide is localised at the centrosome in the cell.
  • labelling the polypeptide or fragment with, e.g. a fluorescent label, inducing the labelled polypeptide or fragment into a cell, and seeing if the polypeptide is localised at the centrosome in the cell.
  • the kinase may be 80%, 85%, 90%, 92%, 94%, 96%, 98% or 99% homologous to an amino acid molecule encoding the sequence shown in seq.ID No. 1.
  • Kinase activity is preferably measured against naturally-occurring non-mutated, e.g. wild type, centrosome associated kinase.
  • Wild ype Mak V is shown in Seq. ID 2.
  • kinase activity is compared with wild type Mak N activity.
  • the mutation is a deletion or a point mutation.
  • a deletion is where one or more amino acids have been deleted.
  • a point mutation is where one or more naturally occurring amino acids have been changed and replaced by different amino acids.
  • ATP binding sites may be identified by sequence homology with known ATP binding domains.
  • nucleic acid molecule encoding the centrosome associated kinase or fragment of the invention.
  • Nucleic acids complementary to such nucleic acid molecules are also provided.
  • the nucleic acid may be single or double stranded, DNA or RNA, naturally or non-naturally occurring.
  • the nucleic acid molecule is 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 100% homologous to the Mak V sequence shown in seq. ID 3.
  • the nucleic acid molecule may be capable of hybridising to the sequence shown in seq. ID 3 under high stringency conditions typically 50 mM sodium phosphate, 1% sodium dodecyl sulphate, pH 6.8, 65°C.
  • Vectors comprising nucleic acid molecules, according to the invention, are also provided.
  • Vectors are molecules which serve to transfer nucleic acids of interest into a cell.
  • Suitable vectors include, but are not limited to, bacterial or eukaryotic vectors such as
  • plasmids or cosmids phage vectors such as lambda phage
  • viral vectors such as adenoviral
  • vectors or baculoviral vectors and other vectors known in the art.
  • the vector preferably comprises suitable regulatory sequences to allow the nucleic acid molecule of the invention to be expressed in a suitable host cell to produce protein encoded by the nucleic acid molecule.
  • the vector comprises a suitable promoter and terminator sequences, or other sequences such as poly A sequences, operably linked to the nucleic acid molecule.
  • suitable regulatory sequences are well known in the art.
  • the vector may also comprise a gene to allow the vector to be selected within a cell, such as an antibiotic resistance gene or a nutritional gene.
  • a gene to allow the vector to be selected within a cell such as an antibiotic resistance gene or a nutritional gene.
  • Such genes are well known in the art.
  • the vector may comprise a reporter gene to allow the expression of the nucleic acid molecule into protein to be monitored. Preferably this is fused onto the nucleic acid molecule of the invention.
  • the promoter which is operably linked to the centrosome associated kinase, may be a regulatable promoter such as lactose inducible expression, available from Stratagene.
  • a system for regulating gene expression such as ecdysone-inducible expression (available from InVitrogen), tetracycline regulated expression (from Clontech)
  • the reporter gene is preferably Green Fluorescent Protein (GFP), which is known in the
  • a further reporter system which may be used is lacZ gene from E.coli. This encodes the
  • ⁇ -galactosidase enzyme This catalyses the hydrolysis of ⁇ -galactoside sugars such as
  • lactose The enzymatic activity in cell extracts can be assayed with various specialised
  • substrates for example X-gal, which allow enzyme activity quantitation using a
  • spectrophotometer fluorometer or a luminometer.
  • the reporter gene may be secreted alkaline phosphatase. This is a secreted enzyme which may be assayed from a supernatent by methods known in the art.
  • the cell may be bacterial, yeast or eukaryotic.
  • the invention further provides a method of identifying an inhibitor or activator of cell cycle progression comprising
  • the centrosome associated kinase may be as defined above, or may be Mak V, and may be
  • centrosome associated kinase is non-naturally occurring within the cell and/or is regulatable by means of an inducable or suppressible promoter.
  • the centrosome kinase may be provided within a vector as defined above.
  • an inhibitor, or activator of centrosome associated kinase identifiable or identified by the method of the invention is also provided.
  • the inhibitor or activator affects
  • the inhibitor or activator may be used in the manufacture of a medicament to treat uncontrolled cell cycle progression, such as cancer.
  • the invention also provides the use of such an inhibitor or activator to control cell cycle progression in vitro, for example in cultured cells and animal tissues.
  • Figure 1 shows Immunofluorescent labelling of B16F10 cells with anti-Mak (A) or anti-gamma tubulin (B), demonstrating Mak V localisation at the centrosome.
  • Figure 2 shows Green Fluorescent protein fluorescence in B16F10 melanoma cells
  • FIG. 1 shows fluorescent activated cell sorter analysis of cells expressing Mak V fusion
  • B16-F10 murine melanoma cells were cultured in RPMI 1640 medium supplemented with 5% fetal calf serum. After trypsinization in trypsin/EDTA (0.25% trypsin/lmM EDTA) for 5 min., cells were seeded onto glass coverslips in 12- well plates.
  • FIG. 1 shows the results of immunofluorescent staining of B16F10 cells with anti-Mak V
  • centrosome associated kinases The significance of the centrosome associated kinases was tested by producing variants of the protein using coding sequences harbouring point mutations designed to inhibit the kinase activity of the protein.
  • a lysine residue at position 91 in the Mak V polypeptide was mutated to arginine. This mutation was chosen because it has been shown, in a number of different kinases to render the kinase inactive.
  • the mutation is referred to as K91R Mak V, and is predicted to act as a trans-dominant inhibitor of endogenous Mak V activity.
  • sequence of the mutated protein is shown as seq.ID No 1.
  • An expression vector constructed to direct the synthesis of a fusion protein between K91R and GFP was transfected into cultured tumour cells. Wild type Mak V fused to the C terminus of GFP was also expressed in cells. The construct pEGFP:Mak V directs expression of wild type Mak V fused to GFP, whilst pEGFP:KDMak V directs expression of K91R Mak V fused to GFP.
  • B16F10 melanoma cells were synchronised in late G2 phase by treatment with 500nM nocodazole (Sigma). For release of the nocodazole block, cells were washed three times in PBS and incubated in fresh medium.
  • centrosomes single centrosome (Gl phase), duplicated centrosomes (S phase) and
  • transfected cells were analysed by fluoresence activated cell sorting.
  • B16F10 melanoma cells were again transfected with either wild type Mak V:GFP fusion protein, or with the K91R:GFP fusion protein.
  • the cells were incubated for 24 hours, and then stained with the DNA-binding fluorescent dye propidium iodide.
  • the DNA content of GFP expressing cells was examined by Fluorescent Activated Cell Sorter (FACS) analysis.
  • FACS Fluorescent Activated Cell Sorter
  • the first requirement is a substrate, either a protein or a
  • the usual approach to performing the assay is to incubate the kinase enzyme and the substrate together with ATP.
  • the ATP acts as a donor for the phosphate group which is transferred by the kinase onto the substrate.
  • To analyse the outcome it is necessary to be able to measure the proportion of the substrate which has become phosphorylated during the incubation. This is done by a variety of methods.
  • the phosphate which is transferred from ATP known as the terminal or gamma phosphate of ATP, is radiolabelled, i.e. Is either 32 P or 33 P.
  • the substrate needs to be separated from the unused ATP and the amount of radiolabel incorporated into the substrate measured.
  • Approaches to the separation include the use of gel electrophoresis or filter binding to separate the protein or peptide substrate from the ATP.
  • the amount of radioactivity and thus phosphate incorporated by the action of the kinase is measured by scintillation counting or using an instrument such as a Phosphorlmager to quantify radioactivity.
  • An alternative approach is to immobilise the substrate onto a surface before conducting the assay and then detecting phosphorylated substrate using an antibody which recognises specifically phosphotyrosine or phosphoserine (i.e. The phosphorylated amino acid resulting from kinase action).
  • This approach has the advantage that it is easily utilised in a proximity detection assay, which means that automation is possible.
  • Such assay systems are marketed by PerkinElmer. Essentially, the substrate is bound to the bottom of the wells
  • the substrate which may now be phosphorylated, is incubated with a radiolabelled antibody which binds to the phosphorylated amino acid.
  • the radiolabel on the antibody is in proximity of the scintillant and the signal can be detected by a

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des kinases associées au centrosome, en particulier des kinases associées au centrosome présentant une activité de kinase réduite ou ne présentant aucune activité de kinase. L'invention concerne également des molécules d'acides nucléiques codant de telles kinases, ainsi que des méthodes servant à identifier des inhibiteurs ou des activateurs de la progression du cycle cellulaire, lesdites méthodes consistant à (a) mettre à disposition une cellule comprenant une kinase associée au centrosome ; (b) ajouter un composé à la cellule ; et (c) déterminer si le composé stimule ou inhibe la progression du cycle cellulaire. Cela permet d'étudier la régulation du cycle cellulaire et d'identifier de nouveaux composés pouvant réguler la progression du cycle cellulaire.
PCT/GB2002/004940 2001-11-02 2002-10-31 Enzymes et inhibition d'enzymes Ceased WO2003038078A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002339077A AU2002339077A1 (en) 2001-11-02 2002-10-31 Controsome-associated kinases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0126415A GB0126415D0 (en) 2001-11-02 2001-11-02 Enzymes and enzyme inhibition
GB0126415.9 2001-11-02

Publications (2)

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WO2003038078A2 true WO2003038078A2 (fr) 2003-05-08
WO2003038078A3 WO2003038078A3 (fr) 2003-06-19

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* Cited by examiner, † Cited by third party
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US6365372B1 (en) * 1999-05-27 2002-04-02 Saint Louis University SNF2 related CBP activator protein (SRCAP)
CA2392685C (fr) * 1999-11-29 2011-02-22 Avi Biopharma, Inc. Methodes et compositions antibacteriennes antisens

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AU2002339077A1 (en) 2003-05-12
WO2003038078A3 (fr) 2003-06-19

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