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WO2001090404A1 - Systemes et methodes de criblage de medicaments - Google Patents

Systemes et methodes de criblage de medicaments Download PDF

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Publication number
WO2001090404A1
WO2001090404A1 PCT/GB2001/002180 GB0102180W WO0190404A1 WO 2001090404 A1 WO2001090404 A1 WO 2001090404A1 GB 0102180 W GB0102180 W GB 0102180W WO 0190404 A1 WO0190404 A1 WO 0190404A1
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WIPO (PCT)
Prior art keywords
dna
stimulatory
nhej
protein kinase
inositol
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Ceased
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PCT/GB2001/002180
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English (en)
Inventor
Steve Craig West
Leslyn Ann Akemi Hanakahi
Michael Bartlet-Jones
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Cancer Research Technology Ltd
Cancer Research Horizons Ltd
Original Assignee
Imperial Cancer Research Technology Ltd
Cancer Research Technology Ltd
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Priority claimed from GBGB0012179.8A external-priority patent/GB0012179D0/en
Application filed by Imperial Cancer Research Technology Ltd, Cancer Research Technology Ltd filed Critical Imperial Cancer Research Technology Ltd
Priority to JP2001586599A priority Critical patent/JP2004500849A/ja
Priority to EP01929850A priority patent/EP1283903A1/fr
Priority to US10/296,014 priority patent/US20040029130A1/en
Priority to CA002408749A priority patent/CA2408749A1/fr
Priority to AU2001256530A priority patent/AU2001256530A1/en
Publication of WO2001090404A1 publication Critical patent/WO2001090404A1/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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to assays and drug screening systems involving components of the non-homologous end joining (NHEJ) pathway, and to screening systems which make use of the protein kinase known as DNA-PK (DNA-dependent protein kinase) and related protein kinases such as ATR, ATM and FRAP.
  • the invention also relates to inositol hexakisphosphate (IP ⁇ ), inositol pentakisphosphate (IP 5 ), inositol tetrakisphosphate (IP 4 ), diphosphoinositol pentakisphosphate (IP 7 ) and bis- diphosphoinositol tetrakisphosphate (IPs).
  • DSBs double strand breaks
  • DSBs double strand breaks
  • Two mechanisms for the repair of DSBs have been described, involving either homologous recombination or non-homologous end-joining (NHEJ).
  • Homologous recombination is particularly effective in S-phase when the break can be repaired using genetic information from a sister chromatid, whereas NHEJ is thought to be effective at all times in the cell cycle (Essers et al, 2000; Takata et al, 1998).
  • NHEJ also plays an important role in DSB repair during V(D)J recombination (Blunt et al, 1995; Taccioli et al, 1993).
  • XRCC4 encodes a protein (XRCC4) that forms a heterodimer with DNA ligase IV
  • XRCC5 and XRCC6 encode the 70 and 80 kDa subunits of the DNA end- binding protein Ku
  • XRCC7 encodes the catalytic subunit of the DNA- dependent protein kinase DNA-PK cS .
  • DNA-PK cs is a large protein (-3500 amino acids, M w ⁇ 465 kDa) (Smith and Jackson, 1999), the carboxyl terminus of which contains a catalytic domain that is related to that found in the phosphatidylinositol 3 (PI 3)-kinase family (Hartley et al, 1995). This similarity initially suggested that DNA- PK cs might be capable of phosphorylating inositol phospholipids, but no such activity has been detected. Instead, DNA-PK cS was shown to be a serine/threonine protein kinase.
  • PI 3-kinase related family include ATM, a protein deficient in Ataxia telangiectasia, and ATR, defects in which lead to an AT-related disorder (Keith and Schreiber, 1995; Smith and Jackson, 1999). Why these proteins should have retained the protein motifs characteristic of a phosphatidylinositol kinase remains a mystery.
  • WO 90/00057 relates to a method for moderating the rate of cellular mitosis in a living mammalian tissue having a pathologically elevated rate of cellular mitosis, which comprises perfusing the tissue with inositol hexaphosphate (or salt) and a source of inositol (or salt) to moderate the elevated rate of cellular mitosis.
  • the method allegedly is useful in human and mammalian diseases wherein NK cell activity is altered, eg tumours, other cancers including leukaemia, immunosuppressed individuals, and in viral, fungal or protozoal infections.
  • WO 95/05380 relates to a method of modulating selectin by administering an effective amount of inositol polyanion (including inositol hexakisphosphate) which binds to the selectin.
  • inositol polyanion including inositol hexakisphosphate
  • Selectin binding is apparently associated with infection with a microorganism, malignancy or other disorders including inflammation and autoimmunity.
  • WO 98/30902 relates to modulation of the NHEJ system via regulation (using protein and/or natural or synthetic compounds) of the interactions of XRCC4 and DNA ligase IV, and XRCC4 and DNA-PK to effect cellular DNA repair activity. It also relates to screens for individuals predisposed to conditions in which XRCC4 and/or DNA ligase IV are deficient.
  • WO 99/04266 relates to the interaction of p53 with, and its phosphorylation by, ATM and related protein kinases such as ATR and DNA-PK.
  • the activity of the proteins is shown to increase in the presence of DNA.
  • Assays for modulators of phosphorylation by the interaction between the proteins and p53 or other proteins having similar phosphorylation sites are provided. Methods of purifying ATM or ATR are also claimed.
  • WO 00/00644 relates to a method for increasing the susceptibility of a cell to DNA-damaging agents by using an antisense oligonucleotide so as to prevent expression of a DNA dependent protein kinase subunit.
  • This invention also relates to a method of treating a tumour in a subject, comprising administering to the subject an antisense DNA-PK oligonucleotide.
  • Gao et al (2000) Nature 404, 897-900 describes the interplay of p53 and XRCC4 in tumourigenesis, genome stability and development.
  • IP 6 inositol hexakisphosphate
  • IP 6 and IP 7 bind the Ku70/80 heterodimer which, in combination with the catalytic subunit of DNA-PK (DNA-PK cS ), makes up DNA-PK.
  • IP 6 has no effect on T4 DNA ligase activity. It is possible that IP 6 binds the Ku70 subunit or the Ku80 subunit.
  • the present invention makes use of these observations in order to develop further methods of performing NHEJ and assays of NHEJ; screening assays for compounds which may modulate NHEJ and which may be therapeutically useful; screening assays for compound which may modulate DNA-PK and related protein kinases and which may be therapeutically useful; compositions and kits of part which may be useful in performing the assays and methods; and methods of modulating NHEJ.
  • the present invention also relates to methods of modulating NHEJ of DNA and therapeutic methods wherein NHEJ of DNA is enhanced or reduced.
  • the present invention also relates to methods of measuring IP 6 or other stimulatory inositol phosphates in an individual in order to determine whether the individual may have, or be susceptible to, a defect in DNA repair or cell cycle checkpoint control.
  • a first aspect of the invention provides a method of stimulating non- homologous end-joining (NHEJ) of DNA the method comprising performing NHEJ of DNA in the presence of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate.
  • IP 6 inositol hexakisphosphate
  • IP 6 or other stimulatory inositol phosphate is added to a NHEJ reaction in order to stimulate j oining of DNA.
  • Non-homologous end-joining is the ligation of DNA termini, typically intermolecular ligation. It includes the joining of DNA ends which exhibit little or no complementarity to each other (and so, typically, each end does not hybridise to the other) and, in any event, is a term well known in the art as is evidenced by its use in many of the papers and patent applications referred to herein, all of which are incorporated herein by reference.
  • a NHEJ reaction requires a suitable DNA substrate, and suitable components for the reaction of joining the DNA ends to proceed.
  • Suitable DNA substrates are those that, typically, are linear DNA molecules the length of which need only be large enough to accommodate the factors which participate in NHEJ.
  • each DNA fragment to be joined is, independently, at least 50 bp, preferably at least 70 bp, more preferably at least 100 bp but may be bigger.
  • one or both of the DNA molecules (or DNA ends) to be joined are detectably labelled such as with radiolabelled phosphorus or with fluorescent labels.
  • two separate DNA molecules to be joined in the NHEJ two ends of the same molecule can be joined such as the ends of a linearised plasmid.
  • NHEJ typically takes place in a eukaryotic cell, such as a vertebrate cell including mammalian cells (although it can also occur in some circumstances in prokaryotes) but, as is described in detail in Baumann & West (1998) Proc. Natl. Acad.
  • DNA-PK DNA-dependent protein kinase
  • stimulating NHEJ we include the meaning that the rate of NHEJ of DNA is increased by the presence of IP 6 in a NHEJ reaction mixture compared to the rate when IP 6 is not present and the reaction mixture is otherwise the same. It will be appreciated that the stimulation will reach a threshold level and that, typically, stimulation according to the method is achieved when IP 6 or other stimulatory inositol phosphate is included in a NHEJ reaction to which no IP 6 or other stimulatory inositol phosphate has been added previously.
  • IP 6 or other stimulatory inositol phosphate is essential for NHEJ and so the presence of IP 6 or other stimulatory inositol phosphate may stimulate a NHEJ reaction from there being no joining to there being some joining of substrate DNA.
  • human cell-free extracts can perform NHEJ in the presence of a suitable substrate, as described in Baumann & West (1998) Proc. Natl. Acad. Sci. USA 95, 14066-14070, without the addition of IP 6 ; however, in this instance it is possible that the cell-free extract already contains a small amount of IP 6 or other stimulatory inositol phosphate.
  • the NHEJ reaction mixture contains a semi-purified cell extract which contains the necessary protein components but from which any natural stimulatory inositol phosphates have been removed.
  • This semi-purified cell extract is then supplemented with a suitable amount of a stimulatory inositol phosphate, such as IP 6 , in order to stimulate NHEJ.
  • a stimulatory inositol phosphate such as IP 6
  • other stimulatory inositol phosphates we include any other inositol phosphates or derivatives of inositol phosphate (such as derivatives with one or more pyrophosphates) which have an effect on the stimulation of NHEJ which is qualitatively the same as the effect of IP 6 on the stimulation of NHEJ as defined using the reaction conditions in Example 1.
  • the stimulatory inositol phosphate will have at least 2% of the stimulatory activity of IP 6 and preferably at least 5% or at least 10% of the stimulatory activity of IP 6 on a molar basis under the same conditions as described in Example 1.
  • Certain stimulatory inositol phosphates may have greater stimulatory activity than IP 6 ; thus, the stimulatory inositol phosphate may have about the same stimulatory activity as LP 6 or it may be greater, such as 150% or 300% or 500% or even 1000% of the stimulatory activity of IP 6 .
  • IP 7 that is to say inositol wherein five positions are occupied by phosphate residues, and one by a pyrophosphate residue
  • IP 6 is better at stimulatory NHEJ than IP 6 .
  • IP 8 (ie inositol wherein four positions are occupied by phosphate residues and two by pyrophosphate residues) are also included as "stimulatory inositol phosphates".
  • the stimulatory inositol phosphate is typically an inositol polyphosphate (ie it has several phosphate groups).
  • the inositol phosphate may be an oligomer or polymer of inositol phosphate moieties wherein the inositol phosphate moieties are joined by a suitable linker.
  • the oligomer or polymer may be a homo-oligomer/polymer in which case each inositol phosphate moiety is the same, or it may be a hetero-oligomer in which case at least some of the inositol phosphate moieties may be different.
  • the "stimulatory inositol phosphate” may be an inositol phosphate derivative wherein one or more of the phosphate groups have been replaced by phosphonate groups.
  • IP 6 when interacting with DNA-PK is modified or further phosphorylated (eg to a pyrophosphate form). Any such modification, if it leads to a stimulatory inositol phosphate as defined is included within the scope of the invention.
  • inositol phosphate include derivatives in which an inositol phosphate moiety is attached to another moiety, for example by linkage through a free hydroxyl position (if present) or through phosphate.
  • the stimulatory inositol phosphate is a naturally-occurring inositol phosphate or derivative thereof.
  • the stimulatory inositol phosphate is any one of IP 6 , IP5 or IP 4 .
  • IP 6 is o-inositol 1,2,3,4,5,6-hexakisphosphate.
  • IP 5 we include any stereoisomer of inositol pentakisphosphate. It is preferred that the IP 5 is my ⁇ -inositol 1,3,4,5,6 pentakisphosphate.
  • IP 4 we include any stereoisomers of inositol tetrakisphosphate. It is preferred that the IP 4 is D- voinositol 1,3,4,5-tetrakisphosphate.
  • the stimulatory inositol phosphate may be IP 7 or IPg or an inositol phosphate with further pyrophosphate residues.
  • IP 7 we include any stereoisomer of diphosphoinositol pentakisphosphate. It is preferred that the IP 7 is mv ⁇ -5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (IP 7 -pp5) or yo-6-diphosphoinositol 1,2,3,4,5- pentakisphosphate (IP 7 -pp6).
  • IP 8 we include any stereoisomer of bis-diphosphoinositol tetrakisphosphate.
  • inositol phosphates particularly those which have four, five or six phosphate groups may have substantially the same stimulatory effect in NHEJ as yo-inositol 1,2,3,4,5,6-hexakisphosphate and the skilled person will be able to determine this using the methods described in the Examples.
  • the methods and assays of the invention therefore include the use of such inositol phosphates with substantially the same stimulatory effect.
  • wyo-inositol 1,3,4,5,6 pentakisphosphate and D-my ⁇ -inositol 1,3,4,5 tetrakisphosphate are considered to have substantially the same stimulatory effect as myo- inositol 1,2,3,4,5,6 hexakisphosphate, although they are not as effective in stimulating NHEJ as the particular IP 6 .
  • IP 7 is more effective than the particular IP 6 in stimulating NHEJ.
  • Inositol hexasulphate (IS 6) is unable to stimulate end-joining; my ⁇ -inositol 1,4,5-trisphosphate was shown to inhibit end-joining.
  • a second aspect of the invention provides an assay of non-homologous end- joining (NHEJ) of DNA wherein the assay comprises inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate.
  • NHEJ non-homologous end- joining
  • the assay contains sufficient components in order to carry out NHEJ of DNA.
  • typical assays of NHEJ are those which can be performed in vitro such as described in Baumann & West supra.
  • NHEJ in cell-free systems is also described in Labhart (1999) Eur. J. Biochem. 265, 849-861, incorporated herein by reference.
  • Reconstitution of NHEJ may be achieved by using recombinantly expressed protein components (such as expressed using a baculovirus system); typically, such a reconstituted system includes DNA-PK, XRCC4, DNA ligase IV, a suitable DNA substrate and a stimulatory inositol phosphate such as IP 6 .
  • the assay may also be carried out in vivo using DNA substrates which, for example, are designed to observe V(D)J joining (see Smith et al (1998) J. Mol. Biol. 281, 815-825 for an example).
  • the method of the first aspect of the invention or the assay of the second aspect of the invention (and indeed for all aspects of the invention which rely on IP 6 or other stimulatory inositol phosphate in an assay or method) that the IP 6 or other stimulatory inositol phosphate is added exogenously, although it is possible that the IP stimulatory inositol phosphate is released from a source present in a cell or cell extract. Typically, the IP 6 or other stimulatory inositol phosphate is added at the start of a NHEJ reaction.
  • the concentration of stimulatory inositol phosphate in the reaction is between 10 nM and 50 ⁇ M; more preferably between 50 nM and 10 ⁇ M; still more preferably between 100 nM and 1 ⁇ M. These ranges are particularly preferred for IP 6 .
  • Preferred ranges for IP 7 are between 0.1 and 1 ⁇ M.
  • the method of the first aspect of the invention or the assay of the second aspect of the invention may be carried out in vivo, it is preferred if it is carried out in vitro; in vitro methods are particularly suitable for the drug screening methods described in more detail below.
  • the NHEJ of DNA in the method of the first aspect of the invention and in the assay of the second aspect of the invention typically make use of a NHEJ reaction mixture which includes DNA-dependent protein kinase (and preferably all components thereof, namely DNA-PKcs, Ku70 and Ku80), XRCC4, DNA ligase IV and a suitable DNA substrate.
  • the NHEJ reaction contains all of these components. It may also include other components which are required for, or enhance, the reaction or make detection of the joined DNA products more readily detected. Such components include ATP and Mg as is described in Example 1.
  • dNTPs deoxynucleotides
  • Amino acid and nucleic acid sequences of polypeptides useful in various aspects of the invention are available from GenBank under the followmg Accession Nos: human Ku70 - J04611; human Ku80 - M30938; human DNA ligase TV - X83441; human XRCC4 - U40622; human DNA - PKcs - U47077; S. cerevisiae Ku70 - X70379; S. cerevisiae Ku80 - Z49702; S. cerevisiae DNA ligase IV - Z74913.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral eg 'phage, or phagemid, as appropriate.
  • plasmids viral eg 'phage, or phagemid
  • Many known techniques and protocols for manipulation of nucleic acid for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al, eds. John Wiley & Sons, 1992.
  • Polypeptides can be purified and isolated from the host cells using techniques well known in the art.
  • polypeptide components of the NHEJ reaction mixture are human (for example extracted from human cells or produced by recombinant techniques from human coding sequences, whether or not expressed in human cells), it will be appreciated that they may be from any suitable source, for example from other mammals or other vertebrates. Because of the conserved nature of the NHEJ reaction in eukaryotic cells, the components may come from lower eukaryotes such as Saccharomyces cerevisiae or Schizosaccharomyces pombe.
  • polypeptide components in a given NHEJ reaction mixture are from the same source (eg all are human), it may be possible to "mix and match" the components, for example by using a DNA ligase IV from one source and a DNA-PKcs from another source, provided that the components are able to perform NHEJ of DNA.
  • the polypeptide components of the NHEJ reaction mixture may have the same amino acid sequence as the polypeptide as found in nature or it may be a variant thereof which retains the necessary activity for use in a NHEJ reaction.
  • protein kinases lower
  • variants are ones which retain their protein kinase activity (ie catalytic activity and/or ability to interact with another component).
  • Variants include variants in which one or more amino acids have been inserted, deleted or replaced.
  • a particularly useful variant is a fusion of the polypeptide with another peptide or polypeptide which facilitates purification.
  • Such a polypeptide is the well known glutathione S- transferase.
  • a reference to a polypeptide includes a reference to a variant as defined.
  • the term variant included a fragment which retains a defined activity.
  • suitable fragments include those which retain the domain, such as a C-terminal domain, homologous to PI 3-kinase. The position of the PI 3-kinase-related domain in this family of proteins is detailed in Featherstone & Jackson (1999) Br. J. Cancer 80 (Suppl 1), 14- 19.
  • the invention also includes aqueous compositions that contain at least one of DNA-PKcs, Ku70, Ku80, XRCC4, DNA ligase IV and a suitable DNA substrate (and preferably all of these) in addition to IP 6 or other stimulatory inositol phosphate wherein the IP 6 or other stimulatory inositol phosphate is at a concentration of at least 10 nM, preferably at least 50 nM, more preferably at least 100 nM, still more preferably at least 1 ⁇ M.
  • a crude estimate of the K d for IP 6 is 1 ⁇ M and so, conveniently, to saturate the system 10 ⁇ M IP 6 may be used. At this level, increases in IP 6 do not appear to result in further increase in NHEJ.
  • the estimate of the K ⁇ j for IP 7 is 100 nM and saturation occurs at 1 ⁇ M.
  • polypeptide components of the aqueous composition consist only of the specific components for the NHEJ and does not include other polypeptide components, such as those derived from a cell extract, which are not required for or enhance NHEJ.
  • each polypeptide component of the aqueous composition is recombinantly produced and purified.
  • a third aspect of the invention provides the use of IP 6 or other stimulatory inositol phosphate for stimulating non-homologous end-joining of DNA. It will be appreciated that before the present invention, and despite the extensive study of NHEJ, it was not realised that IP 6 or other stimulatory inositol phosphate could stimulate (or may even be essential for) NHEJ.
  • the invention also provides the use of IP 6 or other stimulatory inositol phosphate in assays for compounds which modulate NHEJ by whatever means, and in methods which modulate NHEJ by whatever means.
  • the assays may involve changes in NHEJ activity, changes in the recognition of substrates by the NHEJ components and/or changes in subcellular localisation of components of the NHEJ reaction such as DNA-PK, XRCC4 or DNA ligase IV.
  • a fourth aspect of the invention provides a kit of parts comprising IP 6 or other stimulatory inositol phosphate and one or more of a DNA-dependent protein kinase (or its constituent parts DNA-PKcs, Ku70 and Ku80), XRCC4, DNA ligase IV and a suitable DNA substrate.
  • the kit of parts comprises IP 6 or other stimulatory inositol phosphate and each of a DNA-dependent protein kinase, XRCC4, DNA ligase IV and a suitable DNA substrate.
  • the kit of parts may also include other components that are required to carry out an NHEJ assay such as ATP, Mg and, in some circumstances, dNTPs.
  • DNA-dependent protein kinase, XRCC4 and DNA ligase IV are provided as a fraction from a cell free extract, such as the fraction as is described in Example 1. They may also be provided by expression of the individual components by recombinant means. This method is particularly preferred when the NHEJ is to be reconstituted from purified components.
  • a fifth aspect of the invention provides a kit of parts comprising inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphates and a host cell expressing one or more of a DNA-dependent protein kinase, XRCC4 and DNA ligase.
  • IP 6 inositol hexakisphosphate
  • XRCC4 DNA-dependent protein kinase
  • DNA ligase DNA-dependent protein kinase
  • the host cells expressing one or more of the polypeptide components of the NHEJ reaction mixture as said may be produced using standard recombinant methods and the appropriate genes/cDNAs encoding the components as is well known in the art.
  • IP 6 and IP 7 interact with, and may modulate the activity of, protein kinases.
  • IP 6 and IP 7 or indeed any other stimulatory inositol phosphates as defined
  • combinations of IP 6 and other stimulatory inositol phosphates such as IP 7 or IPg with a protein kinase are useful in drug screening assays and the like.
  • a sixth aspect of the invention provides an assay of a protein kinase wherein the assay comprises IP 6 or other stimulatory inositol phosphate.
  • An assay of a protein kinase contains the said protein kinase and, when the catalytic activity of the protein kinase is being measured, a suitable phosphorylatable substrate (such as the target of the said protein kinase or a phosphorylatable peptide therefrom) and a suitable phosphate donor such as ATP or an analogue thereof which has a transferable ⁇ -phosphate group.
  • the assay contains the protein kinase and the other component.
  • Protein kinases are known to interact with their protein substrate and with other components. These other components may be allosteric effectors and they may be macromolecular components such as other proteins or DNA.
  • a seventh aspect of the invention provides a kit of parts comprising a protein kinase and IP 6 or other stimulatory inositol phosphates.
  • the protein kinase is expressed from a recombinant DNA molecule.
  • the protein kinase is substantially free of other components with which it is naturally associated.
  • the invention also includes aqueous compositions that contain at least one protein kinase (preferably substantially free of other components with which it is naturally associated, for example as produced by recombinant expression) and a stimulatory inositol phosphate such as IP 6 or IP 7 .
  • the protein kinase included in an assay, kit of parts or aqueous composition is substantially pure (eg at least 90% pure).
  • An eighth aspect of the invention provides a kit of parts comprising IP 6 or other stimulatory inositol phosphate and a host cell expressing a protein kinase.
  • Protein kinases can be expressed recombinantly as is well known in the art. For example, the cloning, expression and isolation of protein kinases can be carried out using the methodology described above.
  • DNA-PK binds IP 6 and IP 7 but not IP 3 . More particularly, we have shown that IP 6 and IP 7 binds the Ku70/80 heterodimer portion of DNA-PK. IP 6 or IP 7 may bind to the Ku70 subunit or to the Ku80 subunit. The presence of the catalytic subunit (DNA-PK cS ) is not required for binding of IP 6 or IP 7 to Ku70/80.
  • DNA-PK along with ATM, ATR and FRAP belong to a family of related protein kinases wherein the protein kinase is a protein kinase which has a domain, preferably a C-terminal domain, with similarity to the catalytic domain of phosphatidylinositol 3-kinase.
  • DNA-PK, ATM, ATR and FRAP have an inositol head group binding domain.
  • Other members of the family include the Saccharomyces cerevisiae gene products Tellp, Meclp, Torlp and Tor2p, and the Schizosaccharomyces pombe gene product Rad3. It is preferred that the protein kinase in the sixth, seventh or eighth aspects of the invention is a member of this family, most preferably one of DNA-PK, ATR, ATM or FRAP.
  • ATM is the protein encoded by the gene mutated in human ataxia- telangiecstasia (or equivalent genes in other species).
  • DNA-PK DNA dependent protein kinase
  • DNA-PKcs DNA dependent protein kinase catalytic subunit
  • WO 99/04266 incorporated herein by reference, describes methods of purifying ATM and ATR.
  • PI3 kinase related kinases share, in their C-terminal domain at least 25% amino acid sequence identity with the C-terminal domains of DNA-PKcs, more preferably at least 30% or 35% or 40% or 50% or 70% or 90% sequence identity.
  • DNA-PK shows 28% homology
  • RAD3 shows 39% (see Hunter (1995) Cell 83, 1-4).
  • the carboxy termini of the relevant proteins are compared in Keith & Schreiber (1995) Science 270, 50-51.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (Thompson et al., 1994). The parameters used may be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
  • NHEJ is involved in many important biological processes, and modulation of NHEJ has been proposed in relation to a number of medical practices. Compounds that modulate these processes may be useful as potential drugs or for further studies on NHEJ. Thus, these compounds may be useful for any of a variety of purposes.
  • anti-tumour or anti-cancer therapy particularly augmentation of radiotherapy or chemotherapy.
  • Ionising radiation and radiomimetic drugs are commonly used to treat cancer by inflicting DNA damage.
  • Cells deficient in DNA repair are hypersensitive to ionising radiation and radiomimetics.
  • Chemotherapy includes the use of topoisomerase II poisons and other compounds involved in DNA control.
  • Another is the potentiation of gene targeting and gene therapy. Modulation of NHEJ may be used to increase efficiencies of gene targeting, of interest and ultimate use in gene therapy.
  • a further, related, purpose is in anti-retroviral therapy, since DNA repair pathways such as involving the components XRCC4 and DNA ligase IV are involved in effecting retroviral and retrotransposon integration into the genome of a host cell. Retroviruses are of considerable risk to the health of humans and animals, causing, inter alia, AIDS, various cancers and human adult T-cell leukaemia/lymphoma. Integration of retroviral DNA into the genome is essential for efficient viral propagation and may be targeted by inhibition of DNA repair pathway components.
  • modulators of NHEJ may be used in modulation of immune system function, since such factors are required for generation of mature immunoglobulin and T-cell receptor genes by site-specific V(D)J recombination.
  • a ninth aspect of the invention provides a method of identifying a compound which modulates or mimics the effect of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate in stimulating non-homologous end-joining (NHEJ) of DNA the method comprising performing NHEJ of DNA in the presence of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate and determining the effect of a test compound on the NHEJ of DNA.
  • IP 6 inositol hexakisphosphate
  • NHEJ non-homologous end-joining
  • the method is carried out in vitro, for example, using a suitable fraction from a cell-free extracts as described in Example 1, or by using reconstituted systems for NHEJ as described above.
  • the method may also be carried out in vivo as described above.
  • the NHEJ of DNA is carried out in a NHEJ reaction mixture which includes DNA-PK, XRCC4, DNA ligase JV and a suitable DNA substrate.
  • DNA-PK, XRCC4 and DNA ligase IV are the only polypeptide components of the reaction mixture.
  • the DNA-PK, XRCC4 and DNA ligase IV are each substantially pure (eg at least 90% pure) before combination.
  • NHEJ is measured using the methods described in Example 1.
  • the DNA substrates which are joined by the NHEJ reaction are preferably radioactively labelled and the joining is measured by size-separating the DNA, and for example by agarose gel electrophoresis, and the DNA detected by phosphorimaging.
  • High throughput screening assays may be developed, and an example is given in Example 2.
  • the NHEJ is based on the ligation of two linear DNA molecules (although ligation/recircularisation of a linearised plasmid may be measured).
  • High throughput screens may be based on the detection of the retention of a labelled (eg radiolabelled or fluorescently labelled) DNA molecule on a solid support (eg microtitre well) following ligation to a second DNA molecule immobilised on the solid support.
  • PCR amplification across the ligation junction may also be used.
  • test compound typically be added to the NHEJ reaction either before or after the addition of IP 6 or other stimulatory inositol phosphate such as IP 7 to determine whether the order of addition has an effect on the NHEJ reaction.
  • comparisons may be made between a reaction which contains the test compound and contains IP 6 or other stimulatory inositol phosphate and reactions which do not contain IP 6 or other stimulatory inositol phosphate or do not contain the test compound.
  • Compounds which specifically enhance the effect of the stimulatory inositol phosphate in the NHEJ typically lead to increased NHEJ activity, whereas compounds which specifically reduce the effect of the stimulatory inositol phosphate in the NHEJ typically lead to decreased NHEJ activity.
  • Compounds which mimic the stimulatory inositol phosphate in a structural sense may increase the NHEJ activity if the mimic is a functional analogue but may decrease NHEJ activity if the mimic is not a functional analogue.
  • the methods of the ninth aspect of the invention are suitable for identifying compounds which modulate or mimic the effect of IP 6 or other stimulatory inositol phosphate on the catalytic (ie DNA joining activity) of the NHEJ reaction.
  • the invention also includes the identification of compounds that modulate the interactions between components of a NHEJ reaction whether or not such modulation leads directly to a change in DNA joining activity.
  • a tenth aspect of the invention provides a method of identifying a compound which modulates or mimics the effect of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate in stimulating non-homologous end-joining (NHEJ) of DNA the method comprising determining, in the presence of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate, the effect of a test compound on the interactions between the components NHEJ reaction.
  • IP 6 inositol hexakisphosphate
  • NHEJ non-homologous end-joining
  • the components of the NHEJ reaction between which an interaction is measured is any one or more of a DNA-dependent protein kinase (or its components ie DNA-PK cS , Ku70 and Ku80), XRCC4, DNA ligase IV and a suitable DNA substrate.
  • Other interactions include interaction of any one of XRCC4, DNA ligase IV and the DNA substrate with any one of Mrel l, NBS and Rad50 which themselves form a complex (Mrel l/NBS/Rad50).
  • the Mrel l/NBS/Rad50 complex is believed to net act upstream of DNA-PK in the processing of DNA ends. Further details of the Mrel 1/NBS/Rad50 complex are found in Labhart (1999) Eur. J. Biochem. 265, 849-861.
  • IP 6 and other stimulatory inositol phosphates such as IP 7 bind to the Ku70/80 heterodimer of DNA- PK, it is particularly preferred that this heterodimer is present and that interactions between it and other NHEJ components are measured.
  • the interaction between Ku 70/80 and DNA-PK cS may be measured, as may the interaction between Ku 70/80 and DNA. Some of these interactions also require the presence of DNA.
  • Ku70/80-IP 6 and DNA-PK cS may require DNA to form a supercomplex (see Figure 10).
  • the interaction between the Ku70 and Ku80 subunits in the heterodimer may also be measured.
  • the test compound may be added to the sample containing components of the NHEJ reaction whose interaction is to be measured either before or after the addition of IP 6 or other stimulatory inositol phosphate to determine whether the order of addition has an effect on the interaction between components.
  • comparisons may be made between the interactions in a sample containing components of the NHEJ reaction whose interaction is to be measured which sample contains the test compound and IP 6 or other stimulatory inositol phosphate with equivalent samples (in terms of the NHEJ components) which do not contain IP 6 or other stimulatory inositol phosphate or do not contain the test compound.
  • Compounds which modulate or mimic the effect of the stimulatory inositol phosphate can be selected by their activity to modulate or mimic the interactions of the components when in the presence of the stimulatory inositol phosphate.
  • Preferred embodiments of this aspect of the invention measure the interaction (in the presence of IP 6 or other stimulatory inositol phosphate) between DNA-PK and its DNA effector; DNA-PK and a cosubstrate (such as ATP); DNA-PK and XRCC4; DNA-PK and DNA ligase IV; XRCC4 and DNA ligase JV; and DNA ligase IV and its DNA substrate and/or a co- substrate such as ATP.
  • Further preferred embodiments of this aspect of the invention measure the interaction (in the presence of IP 6 or other stimulatory inositol phosphate) of Ku70/80 with DNA-PK cS , and the interaction between Ku70 and Ku80 (whether or not in the presence of DNA-PK cs ).
  • the interaction between Ku70/80 and DNA-PK cS may require DNA.
  • the method is used to identify compounds which modulate or mimic the effect of IP 6 or other stimulatory inositol phosphate in stimulating NHEJ by altering the interaction of DNA-PK with another component (such as its effector DNA or DNA ligase IV or XRCC4 or its cognate DNA or a substrate or cosubstrate) or by altering the interactions between the components of DNA-PK (ie DNA-PKcs and Ku70 and/or Ku80).
  • another component such as its effector DNA or DNA ligase IV or XRCC4 or its cognate DNA or a substrate or cosubstrate
  • An eleventh aspect of the invention provides a method of identifying a compound which modulates or mimics the effect of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate on a protein kinase the method comprising determining, in the presence of IP 6 or other stimulatory inositol phosphate, the effect of a test compound on the catalytic activity of the protein kinase or on the ability of the protein kinase to interact with another component.
  • IP 6 inositol hexakisphosphate
  • Protein kinases phosphorylate, generally in a specific manner, proteins in hydroxyl-containing amino acid residues (serine, threonine or tyrosine), by transferring the ⁇ -phosphate group from ATP to the protein.
  • the substrate for the protein kinase is typically the cognate protein but, conveniently, it may be a synthetic peptide derived from the protein and which contains the phosphorylatable amino acid residue. Protein kinases are also able to autophosphorylate.
  • the protein kinase is a protein kinase which has a domain, preferably a C-terminal domain, with similarity to the catalytic domain of phosphoinositide 3-kinase.
  • the protein kinase is a protein kinase involved in the maintenance of genome stability, such as those which are involved in a DNA repair response.
  • the protein kinase is any one of a DNA-dependent protein kinase, ATR, ATM, FRAP, or the Saccharomyces cerevisiae gene products Tellp, Meclp, Torlp or Tor2p, or the Schizosaccharomyces pombe gene product Rad3. It is particularly preferred if the protein kinase is any one of DNA-PK, ATR, ATM or FRAP, and most preferred if the protein kinase is DNA-PK.
  • DNA-PK is known to phosphorylate XRCC4 which is, therefore, a suitable substrate. Peptide portions of XRCC4 may also be suitable as substrates.
  • Each of DNA-PK, ATR, ATM and FRAP can phosphorylate p53 in vitro. This phosphorylation has been mapped to Ser 15 in p53 for ATM, ATR and DNA-PK so an N-terminal peptide of p53 may be used as a substrate (Hall- Jackson et al (1999) and references cited therein). Yarosh et al (2000) J. Invert. Dermatol. 114, 1005-1010 shows that FRAP is a DNA-dependent protein kinase which is associated with UV-induced damage. FRAP also phosphorylates PHAS-1 (Brunn et al (1997) Science 111, 99-101).
  • Catalytic activity of a protein kinase can readily be determined using methods well known in the art, such as by measuring the incorporation of a radiolabelled phosphate group in the substrate following transfer from ATP. Typically, a series of reactions are carried out which assess the effect of the test compound in order to confirm (or deny) that it is a compound which modulates or mimics the effect of IP 6 or other stimulatory inositol phosphate on a protein kinase rather than a compound which has a non-specific effect.
  • test compound may be added to the protein kinase reaction either before or after the addition of IP 6 or other stimulatory inositol phosphate to determine whether the order of addition has an effect on the catalytic activity.
  • comparisons may be made between a reaction which contains the test compound and IP 6 or other stimulatory inositol phosphate and reactions which do not contain IP 6 or other stimulatory inositol phosphate or do not contain the test compound.
  • the catalytic activity of the protein kinase can be assessed by the transfer of the ⁇ -phosphate from ATP to a substrate (which may include itself ie autophosphorylation).
  • a substrate which may include itself ie autophosphorylation.
  • the ⁇ -phosphate is radiolabelled and so phosphorylation of the substrate can be detected by detecting the radioactivity (eg by scintillation counting, autoradiography or phosphorimaging).
  • it can be detected by separating the phosphorylated substrate from non-phosphorylated substrate, or by using phosphoprotein specific antibodies, for example by fluorescence.
  • the protein kinase is the catalytic subunit of DNA-PK (ie DNA-PKcs) and the other component is any one of Ku70, Ku80, DNA ligase IV, XRCC4 or a suitable DNA effector thereof.
  • DNA-PKcs DNA-PK
  • the other component is XRCC4.
  • the protein kinases ATM and ATR are activated by DNA.
  • FRAP can be stimulated by a DNA damage response.
  • the protein kinase is DNA-PK, ATM, ATR or FRAP and the interaction with its effector DNA is determined.
  • test compound may be added to a sample containing the protein kinase and a compound with which it interacts (such as its substrate but in the absence of ATP or such as an effector DNA as is the case with DNA-PK or ATR or ATM or FRAP) before or after the addition of IP 6 or other stimulatory inositol phosphate to determine whether the order of addition has an effect on the interaction between components.
  • a compound with which it interacts such as its substrate but in the absence of ATP or such as an effector DNA as is the case with DNA-PK or ATR or ATM or FRAP
  • comparisons may be made between the interactions in a sample containing the protein kinase and a component with which it interacts which sample contains the test compound and IP 6 or other stimulatory inositol phosphate with equivalent samples (in terms of protein kinase and interacting component) which do not contain IP 6 or other stimulatory inositol phosphate or do not contain the test compound.
  • a component with which a protein kinase interacts includes ATP or an analogue thereof, such as a non-hydrolysable analogue as is well known in the art.
  • a further aspect of the invention provides a method of identifying a compound which modulates the binding of IP 6 or other stimulatory inositol phosphate to a protein kinase, the method comprising determining whether a test compound reduces or increases the binding of IP 6 or other stimulatory inositol phosphate to the said protein kinase or a subunit thereof.
  • the subunit of the protein kinase is the Ku70/Ku80 heterodimer of DNA-PK.
  • the interaction (association) of the stimulatory inositol phosphate (such as IP 6 ) with, and dissociation from the protein kinase or subunit thereof may be measured using methods well known in the art.
  • the protein kinase is immobilised and the binding of detectably-labelled stimulatory inositol phosphate is measured.
  • the stimulatory inositol phosphate, such as IP 6 is radiolabelled or fluorescently labelled. Scintillation proximity assays, as described below, are particularly useful in binding assays. We have shown that IP 6 and IP 7 binds to DNA-PK.
  • IP 6 and IP 7 have been shown to bind to the Ku70/80 heterodimer which forms part of DNA-PK.
  • the protein kinase is a protein kinase which has a domain, preferably a C-terminal domain, with similarity to the catalytic domain of phosphoinositide 3-kinase.
  • the protein kinase is preferably any one of the members of this family of protein kinases as discussed above. It is particularly preferred if it is DNA-PK. As noted DNA-PK is made up of three subunits.
  • the binding assay may use any one of DNA-PK cS , Ku70 or Ku80 or, as noted above, any functional variants (eg fragments) thereof which retain the binding site for the stimulatory inositol phosphate such as IP 6 .
  • the binding assay uses the Ku70/Ku80 heterodimer.
  • a still further aspect of the invention provides a method of identifying a compound which modulates the binding of IP 6 or other stimulatory inositol phosphate to the Ku70/Ku80 heterodimer of DNA-PK or Ku70 subunit thereof or Ku80 subunit thereof, the method comprising determining whether a test compound reduces or increases the binding of IP 6 or other stimulatory inositol phosphate to the said Ku70/Ku80 heterodimer or the Ku70 subunit thereof or the Ku80 subunit thereof.
  • the Ku70/Ku80 heterodimer is used.
  • the inositol phosphate (for example, IP 5 or IP 6 or IP 7 ) is immobilised onto a solid substrate (such as the floor and/or wall of a microtitre plate) and the Ku 70/80 heterodimer is bound thereto.
  • a competition binding assay is carried out with test compounds.
  • the Ku 70/80 used may be suitably detectably labelled so that the presence or absence (or depletion) of Ku 70/80 bound to the inositol phosphate can readily be detected.
  • a detectably labelled antibody which selectively binds to Ku 70/80 may be used to detect the presence or absence (or depletion) of Ku 70/80 bound to the inositol phosphate.
  • Suitable antibodies are commercially available from Neo Markers, LabVission Corporation, 47790 Westinghouse Drive, Fremont, CA 94539, USA.
  • the Ku70/80 heterodimer or Ku70 subunit thereof or Ku80 subunit thereof do not require the presence of DNA-PK cS .
  • the Ku protein (independent of DNA-PK cS ) is involved in telomere biology and, in particular, Peterson et al (2001) Nature Genet. 27, 64-67 indicate that the function of a stem-loop in telomerase RNA is linked to the DNA repair protein Ku.
  • compounds which modulate the binding of IP 6 or other stimulatory inositol phosphate to Ku70/80 heterodimer or Ku70 subunit thereof or Ku80 subunit thereof may modulate teleomeres or telomerase function.
  • the compound is one which modulates the binding of IP 6 or other stimulatory inositol phosphate to the Ku70/80 heterodimer.
  • a still further aspect of the invention provides a method of identifying a compound which modulates the binding of IP 6 or other stimulatory inositol phosphate to XRCC4 or DNA ligase IV, the method comprising determining whether a test compound reduces or increases the binding of IP 6 or other stimulatory inositol phosphate to the said XRCC4 or DNA ligase IV. Binding of the stimulatory inositol phosphate to XRCC4 or DNA ligase IV can be done using analogous methods as for binding to protein kinases.
  • test compound or substance which may be added to a screening method or assay of the invention will normally be determined by trial and error depending on the type of compound or method used.
  • the test compound may be used at a concentration of around 0.01 nm to 100 ⁇ M.
  • Compounds which may be used as test compounds may be natural or synthetic compounds. Extracts of plants which may contain several characterised or uncharacterised compounds may also be used and are considered "test compounds".
  • screening assays which are capable of high throughput operation will be particularly preferred.
  • Examples may include cell based assays and protein-protein binding assays.
  • An SPA-based (Scintillation Proximity Assay; Amersham International) system may be used.
  • an assay for identifying a compound capable of modulating the activity of a protein kinase may be performed as follows. Beads comprising scintillant and a polypeptide that may be phosphorylated may be prepared. The beads may be mixed with a sample comprising the protein kinase and 32 P-ATP or 33 P-ATP and with the test compound. Conveniently this is done in a 96-well format.
  • the plate is then counted using a suitable scintillation counter, using known parameters for 32 P or 33 P SPA assays. Only 32 P or 33 P that is in proximity to the scintillant, i.e. only that bound to the polypeptide, is detected. Variants of such an assay, for example in which the polypeptide is immobilised on the scintillant beads via binding to an antibody, may also be used. Methods of detecting polypeptide/polypeptide interactions include ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Fluorescence Energy Resonance Transfer (FRET) methods, for example, well known to those skilled in the art, may be used, in which binding of two fluorescent labelled entities may be measured by measuring the interaction of the fluorescent labels when in close proximity to each other.
  • FRET Fluorescence Energy Resonance Transfer
  • a polypeptide to macromolecules for example DNA, RNA, proteins and phospholipids
  • a surface plasmon resonance assay for example as described in
  • the yeast two-hybrid system may be used to detect interactions between polypeptides.
  • yeast two-hybrid system An example of the use of the yeast two-hybrid system is the use of two compounds, such as two components of the NHEJ reaction mixture, and, which interact to form a complex involved in NHEJ, to facilitate the identification of compounds that modulate NHEJ. These compounds are detected by adapting yeast two-hybrid expression systems known in the art for use as described herein. These systems which allow detection of protein interactions via a transcriptional activation assay, are generally described by Gyuris et al, Cell 75:791-803 (1993) and Fields & Song, Nature 340:245- 246 (1989), and are commercially available from Clontech (Palo Alto, CA).
  • the yeast two-hybrid assay is carried out in the presence of IP 6 in order to identify compounds which modulate or mimic the effect of IP 6 on NHEJ.
  • a region of, for example, DNA-PKcs, which interacts with, for example, Ku70 is fused to the GAL4-DNA-binding domain by subcloning a DNA fragment encoding this into the expression vector, pGBT9, provided in the MATCHMAKER Two-Hybrid System kit commercially available from Clontech (catalogue number K1605-1).
  • a fusion of the GAL4 activation domain with the region of Ku70 is generated by subcloning the Ku70 domain-encoding DNA fragment into the expression vector, PGAD424, also provided in the Clontech kit.
  • Analagous expression vectors may also be used.
  • Yeast transformations and colony lift filter assays are carried out according to the methods of MATCHMAKER Two-Hybrid System and various methods known in the art. Prior to the colony filter assay, transformed yeast may be treated with candidate compounds and, as appropriate, with IP 6 or other stimulatory inositol phosphate being screened for the ability to modulate NHEJ.
  • the interaction results obtained using the candidate compound in combination with the yeast system may then be compared to those results observed with the yeast system not treated with the candidate compound (or not treated with IP 6 ), all other factors (eg cell type and culture conditions) being equal.
  • a compound capable of modulating NHEJ is able to alter the interaction between DNA-PKcs and Ku70.
  • a compound capable of decreasing NHEJ by disrupting the binding of DNA-PKcs to the Ku70 may be isolated using the modified yeast two-hybrid system described above, in which the reporter gene encodes a protein, such as ricin, that is toxic to yeast. Yeast cells containing such a ricin reporter die unless the binding of DNA-PKcs to Ku70 is disrupted. Yeast cells treated with a compound that disrupts the DNA-PKcs/Ku70 interaction form viable colonies, and from this result it may be inferred that the compound is capable of decreasing, and possibly inhibiting, NHEJ.
  • the assay is carried out, as appropriate, with IP 6 or other stimulatory inositol phosphate to determine that the compound is one which modulates or mimics the effect of IP 6 or other stimulatory inositol phosphate on NHEJ.
  • the present invention relates to screening methods for drugs or lead compounds.
  • the compound selected following the screen may be a drug-like compound or lead compound for the development of a drug-like compound.
  • a drug-like compound is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble.
  • a drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.
  • the drug-like compound may have increased stability and lower toxicity than, for example, IP 6 . It may also have an improved stimulatory profile compared to IP 6 .
  • lead compound is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • test compounds are inositol derivatives, more preferably inositol phosphates.
  • the test compounds may be phosphoinositides or analogues of IP 6 .
  • the test compounds may be any test compounds, provided that they can be introduced into a suitable assay. Libraries of test compounds may be designed by reference to the general structure and charge distribution of IP 6 , and libraries of compounds may be synthesised using combinatorial chemistry techniques as are well known in the art. It will be appreciated that the test compound is not the stimulatory inositol phosphate present in the assay. It may be possible to select test compounds for screening in the assays and methods of the invention, for example by using computer aided design to select compounds which eg have a similar spatial structure and/or charge distribution to IP 6 .
  • the compounds identified in the screening assays of the invention may themselves be useful in medical treatment or may be useful in developing agents for medical treatment.
  • the compounds identified by the screening methods of the invention maybe useful in developing agents for the treatment of conditions where there is abnormal or inappropriate non- homologous end-joining of DNA. It is envisaged that the compounds may be useful in the development of agents for treating cancer, augmenting cancer radiotherapy and/or chemotherapy regimes, improving gene therapy regimes, enhancing homologous recombination, treating retroviral infections, and modulating the immune system.
  • Compounds which, following the screening method of the ninth and tenth aspects of the invention, are ones which mimic or modulate the effect of IP 6 or other stimulatory inositol phosphate in a NHEJ reaction are selected for further study.
  • Compounds which modulate the binding of the stimulatory inositol phosphate (such as IP 6 ) to DNA-PK (or a subunit thereof) or XRCC4 or DNA ligase IV may also be selected for further study.
  • these compounds are then tested further in another screen which is designed for the selection of compounds which are suitable for treating cancer, augmenting cancer radiotherapy and/or chemotherapy regimes, improving gene therapy regimes, enhancing homologous recombination, treating retroviral infections, or modulating the immune system.
  • the screens are ones which involve cell-based assays which look at end-points relevant to the condition in question.
  • the screens may also involve animal models of the relevant condition.
  • Cell-based screens and animal models are available for at least some of cancer, augmentation of cancer radiotherapy and/or chemotherapy, gene therapy, homologous recombination, retroviral infections and immune system modulation.
  • Compounds which, following the screening method of the eleventh aspect of the invention are ones which mimic or modulate the effect of IP 6 or other stimulatory inositol phosphate on protein kinases. Conveniently, these compounds are then tested in another screen which is designed for the selection of agents which modulate protein kinase or its interactions.
  • the protein kinase is any one of DNA-PK, ATM, ATR or FRAP and the method of the invention is used to identify compounds which may be useful in developing agents which modulate cell cycle checkpoint control.
  • further screens for compounds selected in the method of the eleventh aspect of the invention include screens specifically designed to identify agents which modulate cell cycle checkpoint control.
  • a twelfth aspect of the invention provides a compound identifiable by the screening methods of the invention.
  • a thirteenth aspect of the invention provides a compound identified by the screening methods of the invention.
  • Such compounds are useful in medicine, particularly in the conditions mentioned above.
  • the compounds may be packaged and presented for use in medicine, or may be used in the manufacture of a medicament for treating conditions in which the patient may benefit from modulation of non-homologous end-joining of DNA or from modulation of protein kinase function.
  • compounds obtained in the screens may disrupt DNA-PK-IP 6 interaction and thereby sensitise cells to therapy (eg cancer chemo- or radiotherapy) by disrupting NHEJ.
  • a fourteenth aspect of the invention provides a method of reducing non- homologous end-joining (NHEJ) of DNA the method comprising reducing the amount of, or inhibiting the stimulatory effect of, inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate in a NHEJ reaction.
  • NHEJ non- homologous end-joining
  • myo-inositol trisphosphate (IP 3 ) is inhibitory in a NHEJ and may be used to reduce NHEJ.
  • a fifteenth aspect of the invention provides a method of enhancing non- homologous end-joining (NHEJ) of DNA the method comprising increasing the amount of, or enhancing or mimicking the stimulatory effect of, inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate in a NHEJ reaction.
  • NHEJ non- homologous end-joining
  • the methods may be used in vitro, but it is particularly preferred if they are used in vivo, for example in a cell where it is desirable to reduce or enhance NHEJ of DNA. It is preferred ifthe cell where reduction or enhancement of NHEJ of DNA takes place according to the method of the invention is a cell in a human or animal in need of reduction or enhancement of NHEJ of DNA.
  • conditions where reduction of NHEJ of DNA in an animal or human may be desired include cancer, augmentation of cancer radiotherapy and/or chemotherapy, gene therapy, homologous recombination, retroviral infections and immune system modulation.
  • the invention includes a method of treating these conditions or carrying out these procedures, the method comprising administering to the patient an effective amount of a compound which reduces the amount of, or enhances the stimulatory effect of, IP 6 or other stimulatory inositol phosphate in NHEJ reaction.
  • the invention also includes the use of a compound which reduces the amount of, or enhances the stimulatory effect of, IP 6 or other stimulatory inositol phosphate in NHEJ reaction in the manufacture of a medicament for treating a condition where reduction of NHEJ of DNA is desired.
  • Anti-inositol phosphate antibodies may be used to reduce the amount of, or inhibit the stimulatory effect, of the stimulatory inositol phosphate, such as IP 6 .
  • Anti-inositol phosphate antibodies may be made using methods well known in the art using the inositol phosphate as an immunogen (or hapten) or by using it to screen synthetic antibody-display libraries (eg phage display libraries).
  • antibody we include antibody-like molecules such as antigen binding fragments of antibodies or synthetic antibodies. Thus, the term antibody includes Fab, Fv, ScFv, dAb and the like as are well known in the art.
  • Antibodies to IP 3 are described in Shieh & Chen (1995) Biochem. J. 311, 1009-1014 and Goa et al (1994) Biorg. Medicinal Chem. 2, 7-13 and antibodies to IP 6 may be made by analogous methods.
  • the screening methods of the invention may be used to identify high affinity IP 6 analogues which may specifically compete with IP 6 for the DNA-PK binding site.
  • IP 6 levels in the cell may be up- or downregulated by targeting the synthetic enzyme (eg IPK1 in Saccharomyces cerevisiae) or enzymes involved in IP 6 turnover (eg phosphatases).
  • the synthetic enzyme eg IPK1 in Saccharomyces cerevisiae
  • enzymes involved in IP 6 turnover eg phosphatases.
  • Antisense compounds which are well known in the art and may be designed by reference to a particular nucleotide sequence (eg to the mRNA or gene encoding the IP 6 synthetic enzyme or to an IP 6 phosphatase), may be used.
  • Enhancement of NHEJ may be desirable in patients who are cancer prone or who are immuno-compromised. It may also be desirable in A-T patients.
  • the invention also includes a method of treating these conditions, the method comprising administering to the patient an effective amount of a compound which increases the amount of, or enhances or mimics the stimulatory effect of, IP 6 or other stimulatory inositol phosphate in a NHEJ reaction.
  • the invention also includes the use of a compound which increases the amount of, or enhances or mimics the stimulatory effect of, IP 6 or other stimulatory inositol phosphate in a NHEJ reaction in the manufacture of a medicament for treating a condition where enhancement of NHEJ of DNA is desired.
  • histamine has been shown to alter IP 6 levels in a cell, and antibodies may be used to reduce the amount of, or inhibit the stimulatory effect of the stimulatory inositol phosphate.
  • a sixteenth aspect of the invention provides a method of modulating the activity or interaction of a protein kinase the method comprising changing the amount of inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate present with the protein kinase, or inhibiting or enhancing the effect of IP 6 or other stimulatory inositol phosphate on the protein kinase.
  • IP 6 inositol hexakisphosphate
  • the protein kinase is a protein kinase which has a domain, preferably a C-terminal domain, with similarity to the catalytic domain of phosphatidylinositol 3-kinase.
  • the method may be used in vitro, but it is particularly preferred if it is used in vivo, for example in a cell where it is desirable to modulate the activity or interaction of the protein kinase.
  • the modulation of the protein kinase takes place in a cell in a human or animal body, in need of such modulation.
  • the protein kinase is any one of DNA-PK, ATR, ATM or FRAP and the method is for modulating cell cycle checkpoint control.
  • the protein kinase is ATM or ATR.
  • Modulators of ATM or ATR, as identified herein, may be useful for treating any of a variety of purposes such as in therapy of ataxia-telangiectasia (A- T); modulation of the immune system (ATM appears to be required for the generation of a fully functional immune system); modulating teleomere length (cells of A-T patients lose their telomeres more quickly than normal individuals) - this may be useful ageing, AIDS and other conditions; tumour/cancer therapy; and in augmenting of cancer radiotherapy or chemotherapy.
  • the aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a compound of the invention Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • a seventeenth aspect of the invention provides a method of determining whether an individual has or is predisposed to a defect in DNA repair or cell cycle checkpoint control, the method comprising the steps of (1) obtaining a sample from the patient, (2) determining the concentration of, or subcellular localisation of, inositol hexakisphosphate (IP 6 ) or other stimulatory inositol phosphate in the sample, and (3) comparing the result with a standard.
  • IP 6 inositol hexakisphosphate
  • the sample obtained from the patient may be any suitable sample.
  • the sample is a sample of a tissue in which DNA repair or cell cycle checkpoint control defects are known to occur, such as skin and in tumour samples.
  • concentration, or subcellular localisation, of IPg or other stimulatory inositol phosphate may be determined by any suitable method.
  • anti-IPg antibodies may be made using methods well known in the art which may be used to quantify IPg, and also to detect its sub cellular location, for example by using suitable staining and microscopic techniques such as confocal microscopy, immunofluorescence microscopy and by immunohistochemistry.
  • IP 6 and other stimulatory inositol phosphates as defined can be measure by suitable analytical techniques such as ELISA.
  • Subcellular localalisation can be compared, for example, by measuring IPg in a nuclear extract compared to a cytoplasmic extract.
  • the invention may also be considered to relate to a method of modulating DNA repair activity comprising performing DNA repair in the presence of an effective modulating amount of an inositol phosphate, or isomer thereof.
  • an inositol phosphate is IPg.
  • any inositol phosphate it includes reference to a salt thereof, particularly a physiologically acceptable salt.
  • FIG. 1 Repair of double-strand breaks by non-homologous end-joining.
  • DSBs caused by either irradiation or chemical assault, are bound by the Ku heterodimer (Ku70/80) and the catalytic subunit of the DNA-dependent protein kinase, DNA-PK cS . Binding protects the free ends from nuclease attack while simultaneously initiating the assembly of the NHEJ apparatus. Through an as yet undefined process, DNA ends are bridged, and the XRCC4/DNA ligase TV complex is recruited to the DSB where it effects repair.
  • FIG. 1 Purification of SFA.
  • A Schematic representation of the chromatographic steps taken to purify SFA from HeLa cytoplasmic extracts.
  • B Complementation of DNA end-joining by the addition of undiluted DEAE fractions to PC-C. Samples were analyzed by gel electrophoresis and 32 P-labeled DNA visualized by autoradiography.
  • C Fractions eluting from mono Q were diluted 1:50 in L buffer and assayed for the ability to complement end-joining by PC-C. End-joining by PC-C alone (-), and a selected region of each column elution profile is shown. Mobilities of linear, dimer and trimer DNA species are indicated.
  • Figure 3. Physical characteristics of SFA. A.
  • Proton decoupled phosphorus spectra revealed four peaks (ratio 1:2:2:1) close to the phosphoric acid reference at 1.4909 ppm (intensity 2.582), 1.0003 ppm (intensity 5.465), 0.5554 ppm (intensity 6.002) and -0.0850 ppm (intensity 2.927), suggesting phosphate groups and showing no evidence of phosphorus-phosphorus coupling.
  • Ion-trap spectrum revealed a mass of 660.9 Da, which represents the [mass +l] +1 -ion or [659.9 + l] +1 -ion followed by a series of related sodium salts at 682.9 Da ( ⁇ lNa*), 704.9 Da (+2Na + ), 726.9 Da (+3Na + ).
  • FIG. 4 Stimulation of DNA-PK dependent end-joining by inositol phosphates.
  • A Schematic representation of IP 6 .
  • B Complementation of PC-C by IPg. End-joining assays were carried out using PC-C complemented with increasing amounts of IPg.
  • C Effect of inositol phosphates on DNA-PK dependent NHEJ.
  • Inositol hexakisphosphate IPg
  • inositol pentakisphosphate IP 5
  • inositol tetrakisphosphate IP 4
  • inositol trisphosphate IP 3
  • inositol hexasulphate ISg
  • FIG. 5 SFA and IPg co-fractionate by strong anion exchange chromatography. A trace amount (4 nM) of 3 H-IP 6 was added to a 1 ml aliquot of SFA. The resulting sample was applied to AG 1-X8 resin and eluted as described in Materials and Methods. Top, elution profile of 3 H-IP 6 as measured by scintillation counting. Bottom, elution profile of SFA, determined by the complementation of PC-C mediated end-joining. Figure 6. Specificity of IPg for DNA-PK mediated end-to-end ligation. A. DNA end-joining reactions catalyzed by PC-C were analyzed in the presence or absence of 2 ⁇ M IPg. B. Similar reactions carried out using T4 DNA ligase (0.1 u/ ⁇ l) in place of PC-C.
  • FIG. 7 Binding of IPg by DNA-PK.
  • A Schematic representation of DNA-PKcs. The grey bar represents the C-terminal 380aa which share sequence similarity to the catalytic domain of the PI 3-kinases (Hartley et al, 1995) and the black box indicates the location of the putative inositol phosphate headgroup binding domain of the PI 3-kinases (Wymann and Pirola, 1998).
  • the lysine (K) residue believed to be the target of wortmannin interaction is shown as are the two aspartate (D) residues believed to be located in the ATP binding active site of the protein kinase.
  • Figure 8 shows that both IP 7 - ⁇ 5 and IP 7 -pp6 stimulate NHEJ by PC-C and that this stimulation is approximately 10- fold better than that achieved by IPg.
  • Figure 9 shows that ISg does not compete with IP 6 in binding to DNA-PK.
  • Figure 10 shows the distribution of 3 H-IPg along a gel filtration column under various conditions.
  • Superdex 200 gel filtration, 50 ⁇ l fractions, scint. counting 20 ⁇ l/fraction 100 nM 3 H-IP 6 , 100 nM Ku, 100 nM DNA (200 nM DNA ends), 100 nM PK cS .
  • Running buffer 50 mM HEPES, pH 8.0, 40 mM KOAc, 10% Glycerol, 0.1 M KCl, 1 mM DTT.
  • Figure 11 is an overlay of the Ku + 3 H-IP 6 and Ku + DNA-PK cS + 3 H-IP 6 curves from Figure 10 which emphasises that the presence of DNA-PK cS does not alter the mobility of the Ku- 3 H-IP 6 curves from Figure 10 which emphasises that the presence of DNA-PK cs does not alter the mobility of the Ku- 3 H-IP 6 complex along the gel filtration column.
  • Figure 12 shows the result of gel filtration carried out on DNA-PK + 3 H-IP 6 in the presence or absence of DNA.
  • Figure 13 shows the result of competition analysis of IPg binding by Ku using IP 3 , IPg and IP 7 .
  • Figure 14 shows the results of specificity trials using the spin column method.
  • Example 1 Stimulation of DNA-PK dependent non-homologous end- joining by inositol phosphate
  • PC-C phosphocellulose chromatography
  • RNA or DNA SFA was treated with either RNAse A, NaOH (0.1-1.0 M at 60°C), DNAse I or micrococcal nuclease. These treatments had no effect on the ability of SFA to stimulate end-joining (data not shown). UV absorbance spectroscopy demonstrated that a sample of concentrated SFA did not absorb at 260 nm, indicating a lack of purine or pyrimidine moieties in the sample (data not shown). These data demonstrate that SFA is not a nucleic acid.
  • the molecular mass of SFA was determined by mass spectroscopy (Figure 3C). While no significant signal was observed in the range commonly associated with macromolecules, polypeptides or polymeric nucleic acids, the SFA sample was found to contain a number of species of low molecular mass. Although the SFA sample was found to be heterogeneous, a clear peak was detected at a mass of 660.9 Da. Additionally, an array of peaks which differed from the original 660.9 Da peak by 22 Da (the mass of sodium Na*) were observed downstream of the 660.9 Da peak. These masses appear to correspond to the +1 (Na ), +2 (Na*) and +3 (Na ) salts of the 660.9 Da species.
  • Inositol is a fully hydroxylated six-carbon ring which is found in a number of phorphorylation states ranging from mono- through hexakisphosphate.
  • Inositol hexakisphosphate (IP 6 ) ( Figure 4A) shares the same molecular weight as SFA (659.9 Da) and has the same phosphorus content.
  • IP 6 was assayed for its ability to stimulated end-joining by PC-C. As shown in Figure 4B, IP 6 stimulated end-joining at concentrations in the region of 100 nM and stimulation was maximal at 1 ⁇ M.
  • IP 5 inositol phosphates
  • inositol hexasulphate (ISg) - an inositol compound which would provide a charge distribution similar to that of IPg, while presenting sulfate rather than phosphate groups - was also assayed. It was found that ISg was unable to stimulate end-joining, demonstrating a clear requirement for phosphate groups ( Figure 4C). Indeed, we found that IPg proved to be the most effective inositol phosphate compound of those tested. IP 5 and IP 4 were also able to stimulate end-joining, but the efficiency of this stimulation was reduced relative to IPg. These data show that end-joining requires a phosphorylated inositol species, and that the stimulation of NHEJ is directly related to the extent of phosphorylation.
  • IP 6 is the active component in SFA was obtained by strong anion exchange (SAX) chromatography, using a resin that is commonly utilized to separate highly charged molecules such as the inositol phosphates.
  • SAX strong anion exchange
  • IPg is of small molecular size (660 Da)
  • its high charge to mass ratio and the hydrogen bonding observed between phosphate groups results in a larger apparent molecular size in aqueous solutions at low ionic strength. This has been observed by the retention of LP 6 by dialysis membranes at low ionic strength, and the passage of IP 6 through the same membrane at high ionic strength (data not shown).
  • Equilibrium dialysis trials were performed to compare the movement of SFA and H-IPg across a dialysis membrane (12-14 kDa cutoff).
  • IPg for DNA-PK dependent end-joining
  • IPg inositol hexakisphosphate
  • the catalytic domain of DNA-PK cS is related to that found in the PI 3- kinases which phosphorylate inositol phospholipids.
  • Previous studies have shown that in the PI 3-kinases recognition of the inositol phosphate headgroup is mediated by defined sequences within this conserved domain (Figure 7A) (Bondeva et al, 1998). Given that NHEJ is dependent upon DNA-PK and is stimulated by the addition of IPg it was plausible that this stimulatory effect is due to a physical interaction between LP 6 and DNA-PK.
  • IPg inositol hexakisphosphate
  • IPg transition upon ligand
  • IPg binding by DNA-PK might be structural in nature, possibly due to an allosteric shift upon association with IPg.
  • binding of IPg could simply alter the surface charge distribution of DNA- PK.
  • Such an alteration of local electrostatic potential has been observed in the binding of IPg to phosphoglycerate mutase (Rigden et al, 1999).
  • ligand binding was mediated by both hydrogen bonding interactions and by the strong positive electrostatic potential of the active site cleft. Occupancy of this highly charged cleft by IPg exposes several phosphates to the solvent, which has a pronounced effect on the local electrostatic potential relative to the unbound state.
  • DNA-PK is the only protein known to participate in NHEJ that has been demonstrated to bind IPg.
  • DNA-PK is a member of the phosphatidylinositol 3-kinase (PI3K)-related kinase family, as are ATM and ATR. All three proteins exhibit a strong sequence homology to the PI 3- kinases, especially in the catalytic core domain that binds and phosphorylates the phosphoinositol headgroup of phosphatidylinositol.
  • PI3K phosphatidylinositol 3-kinase
  • IP 3 D- yo-inositol 1,3,4,5- tetrakisphosphate
  • IP 4 D- yo-inositol 1,3,4,5- tetrakisphosphate
  • IP 5 yo-inositol 1,3,4,5,6-pentakisphosphate
  • ISg inositol hexasulphate
  • IPg yo-inositol 1,2,3,4,5,6-hexakisphosphate
  • H-IPg (10-30 Ci/mmol) was purchased from NEN.
  • HeLa whole cell extracts were prepared and fractionated step-wise over phosphocellulose as described (Baumann and West, 1998). Fraction PC-C was dialyzed for 2 hours against L buffer (20 mM Tris-HCl pH 8.0, 25 mM KOAc, 0.5 mM EDTA, 10% glycerol, 1 mM DTT) and stored at -80°C.
  • End joining reactions (10 ⁇ l) were carried out in 50 mM HEPES pH 8.0, 40 mM KOAc, 0.5 mM Mg(OAc) 2 , 1 mM ATP, 1 mM DTT, 0.1 mg/ml BSA, contained 2-3 ⁇ l (3-5 ⁇ g) of PC-C and Hindlll-linearized 5'- 32 P-labeled pDEA7Z DNA (10 ng). Incubation was for 1 hour at 37°C. 32 P-labeled DNA products were deproteinized and analyzed by electrophoresis through 0.6% agarose gels followed by autoradiography.
  • HeLa cells 300 L were cultured in suspension to a density of 5 x 10 5 cells/ml, harvested by centrifugation, washed twice with PBS, flash-frozen on liquid nitrogen and stored at -80°C.
  • hypotonic lysis buffer 10 mM Tris-HCl pH 8.0, 1 mM EDTA,
  • DEAE-SFA fractions were heat denatured by boiling for 15-20 minutes, then centrifuged for 45 min at 45,000 rpm at 4°C in a Beckman Ti45 rotor to remove insoluble aggregates. The supernatant was dialyzed for 6 hours against L buffer, applied to a 2.6 x 37 cm affi-gel heparin column (Bio-Rad) equilibrated in the same buffer, then eluted with a 15 column volume 0-1.0 M KCl linear gradient in L buffer. Active fractions eluting between 0.28- 0.33 M KCl were pooled, dialyzed for 6 hours against L buffer, then loaded onto a Mono P Hr 5/20 column (Pharmacia).
  • Mass spectrometry SFA sodium salt was acidified with IM HCl to approximately pH 2.0, diluted with 1 equivalent of 70% methanol/30% formic acid and a lO ⁇ l sample was loaded into a carbon coated nano spray capillary needle (Protana) and the molecular weight determined by mass spectrometry using an ion-trap (LCQ Thermoquest).
  • Reactions (10 ⁇ l) containing T4 DNA ligase (NEB) were carried out in lx T4 DNA ligase buffer (NEB) at 25°C in the presence and absence of inositol phosphates. Reactions were stopped, deproteinized and the products analyzed by agarose gel electrophoresis.
  • Binding reactions (55 ⁇ l) were carried out in 25 mM HEPES pH 7.5, 50 mM KCl, 10 mM MgCl 2 , 1 mM DTT, 10% glycerol, 0.1% NP-40 with 5000 units of DNA-PK (Promega) or 1.8 mg/ml of protein size standards for gel filtration (BioRad) and 100 nM 3 H-IP 6 or 3 H-IP 3 at 4°C for 30 min. Complexes were resolved on a Superose 12 PC3.2/30 column run in 50 mM HEPES pH 8.0, 40 mM KOAc, 0.1M KCl, 10% glycerol, 1 mM DTT at 40 ⁇ l/min.
  • DNA-dependent protein kinase catalytic subunit a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 82, 849-856.
  • PIK-related kinases DNA repair, recombination, and cell-cycle checkpoints. Science 270, 50-51.
  • the XRCC4 gene product is a target for and interacts with the DNA-dependent protein kinase. J. Biol. Chem. 273, 1794-1801. Modesti, M., Hesse, J. E., and Gellert, M. (1999). DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase TV activity. EMBO J. 18, 2008-2018.
  • Taccioli G. E., Rathbun, G., Oltz, E., Stamato, T., Jeggo, P. A., and Alt, F.
  • DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497-5508.
  • Example 2 High throughput screening assay for NHEJ A microtiter-plate (96- wells) assay which would work like an ELISA assay is used.
  • a "target” duplex DNA is adhered to the plate (call the sequence oligos 1 and 2 which are complementary and have an exposed, unmodified terminus).
  • Cell extracts are mixed with a "detection" duplex DNA (linear duplex - oligos 3 and 4 which are complementary - which have one exposed, unmodified terminus (which will be ligated to the target DNA) and the other terminus is both 5' and 3' blocked by biotin; oligo 3 is 5' biotinylated, oligo 4 is 3' biotinylated.
  • the biotin moieties serve two functions: 1) block the end of the DNA and prevent additional ligation which would result in multimerization and non-linear report of activity and 2) for detection.
  • NHEJ reaction components including IP 6 , are incubated in the plate (which presents the target duplex) in the presence of the detection DNA (and buffer and Mg ⁇ and ATP). The plate is washed to remove proteins and excess detection DNA. The only way for the detection DNA to remain on the plate is by ligation with the target DNA. The presence of the detection duplex indicated NHEJ - this retention should be quantitative.
  • Detection quantification of the biotin may be carried out as with an ELISA assay - using avidin coupled enzymes and a chromogenic enzyme substrate.
  • IPg can be additionally phosphorylated to IP 7 . This results in a species that is pyrophosphorylated at one carbon of the inositol ring.
  • IP pp5 and IP 7 -pp6 were tested to see if these polyphosphorylated inositols would stimulate end-joining by PC-C. As shown in Fig 8 both IP 7 species do stimulate NHEJ by PC-C and this stimulation is approximately 10-fold better than that achieved with IPg.
  • DNA-PK (composed of the Ku 70/80 heterodimer and PI-3-like kinase DNA-PK cS ) binds IP 6 with specificity.
  • the Ku 70/80 heterodimer is responsible for this binding activity.
  • the Ku-IPg complex is capable of binding DNA forming a higher-order complex.
  • a super- complex can be formed that contains Ku, IPg, DNA-PK cS and DNA.
  • IP 6 recognition by DNA-PK was assessed by competition trials using inositol hexasulphate (IS 6 ). As shown in Fig 9, tritiated IPg ( 3 H-IP 6 ) is not bound by any of the molecular weight standards used to calibrate the gel filtration column (diamonds, STDs) and is detected in the far-included volume which elutes late in the column profile. Addition of DNA-PK results in an increase in the mobility of the 3 H-IPg indicating complex formation. ISg, presented in 10- and 100-fold molar excess have no effect on the amount of H-IP 6 bound by DNA-PK. These data, when taken together with binding trials using 3 H-IP 3 , indicate specific binding of IPg by DNA-PK.
  • IPg binding by Ku was examined by competition analysis (Fig 13) using IP 3 , IPg and IP 7 . As shown in Fig 13, one molar equivalent of IPg or IP 7 resulted in an approximate 50% loss m H-IPg binding, while a 3- fold molar excess of IP 3 had no effect.
  • IPg and IP 7 are recognized equally by Ku indicates that the binding of an inositol polyphosphate (IP) by Ku is not the source of the 10-fold increase in NHEJ stimulation observed with IP 7 .
  • IP inositol polyphosphate
  • Example 5 Spin columns used to examine inositide phosphate binding species

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Abstract

L'invention porte sur un procédé de stimulation de mécanismes d'assemblage non homologue des terminaison d'ADN, ce procédé consistant à réaliser l'assemblage non homologue des terminaisons de l'ADN en présence d'hexakisphosphate inositol (IP6) ou autre phosphate inositol stimulateur. L'invention porte également sur le dosage d'une protéine kinase, ce dosage comprenant hexakisphosphate inositol (IP6) ou autre phosphate inositol stimulateur. L'invention porte encore sur des méthodes de criblage de composés qui peuvent moduler l'assemblage non homologue des terminaisons et être utiles d'un point de vue thérapeutique, ainsi que sur des méthodes de criblage de composés qui peuvent moduler ADN-PK et des protéines kinases apparentées et qui peuvent être utiles d'un point de vue thérapeutique. Des procédés de modulation des mécanismes d'assemblage non homologue des terminaisons, ainsi que des protéines kinases sont également décrits.
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FR2839726A1 (fr) * 2002-05-17 2003-11-21 Commissariat Energie Atomique Methode de mesure du processus de recombinaison non-homolgue de l'adn dans des cellules de mammifere et ses applications
WO2016073990A3 (fr) * 2014-11-07 2016-08-11 Editas Medicine, Inc. Procédés pour améliorer l'édition génomique médiée par crispr/cas
EP2986709A4 (fr) * 2013-04-16 2017-03-15 University Of Washington Through Its Center For Commercialization Activation d'une autre voie pour la réparation dirigée par homologie afin de stimuler la correction génétique et le génie génomique
WO2017147056A1 (fr) * 2016-02-22 2017-08-31 Caribou Biosciences, Inc. Méthodes de modulation de résultats de réparation d'adn

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
FR2839726A1 (fr) * 2002-05-17 2003-11-21 Commissariat Energie Atomique Methode de mesure du processus de recombinaison non-homolgue de l'adn dans des cellules de mammifere et ses applications
WO2003097842A1 (fr) * 2002-05-17 2003-11-27 Commissariat A L'energie Atomique Mesure du processus de recombinaison non-homologue de l'adn dans des cellules de mammifere
EP2986709A4 (fr) * 2013-04-16 2017-03-15 University Of Washington Through Its Center For Commercialization Activation d'une autre voie pour la réparation dirigée par homologie afin de stimuler la correction génétique et le génie génomique
WO2016073990A3 (fr) * 2014-11-07 2016-08-11 Editas Medicine, Inc. Procédés pour améliorer l'édition génomique médiée par crispr/cas
US11680268B2 (en) 2014-11-07 2023-06-20 Editas Medicine, Inc. Methods for improving CRISPR/Cas-mediated genome-editing
EP4464338A3 (fr) * 2014-11-07 2025-02-12 Editas Medicine, Inc. Systèmes pour améliorer l'édition génomique médiée par crispr/cas
WO2017147056A1 (fr) * 2016-02-22 2017-08-31 Caribou Biosciences, Inc. Méthodes de modulation de résultats de réparation d'adn
US11155814B2 (en) 2016-02-22 2021-10-26 Caribou Biosciences, Inc. Methods for using DNA repair for cell engineering

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