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WO1997002354A1 - INTERACTION DE LA PROTEINE p53 AVEC UN FACTEUR DE TRANSCRIPTION DP-1 - Google Patents

INTERACTION DE LA PROTEINE p53 AVEC UN FACTEUR DE TRANSCRIPTION DP-1 Download PDF

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Publication number
WO1997002354A1
WO1997002354A1 PCT/GB1996/001560 GB9601560W WO9702354A1 WO 1997002354 A1 WO1997002354 A1 WO 1997002354A1 GB 9601560 W GB9601560 W GB 9601560W WO 9702354 A1 WO9702354 A1 WO 9702354A1
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WIPO (PCT)
Prior art keywords
protein
complex
assay
polynucleotide
transcription
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PCT/GB1996/001560
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English (en)
Inventor
Nicholas Barrie Lathangue
Original Assignee
Medical Research Council
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Filing date
Publication date
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to AU62373/96A priority Critical patent/AU6237396A/en
Priority to EP96921006A priority patent/EP0835319A1/fr
Priority to JP9504018A priority patent/JPH11509089A/ja
Publication of WO1997002354A1 publication Critical patent/WO1997002354A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to complexes of the protein p53, which is a transcription modulator, with DP proteins (e.g. DP-1) or E2F proteins (e.g E2F-1).
  • DP proteins e.g. DP-1
  • E2F proteins e.g E2F-1
  • the invention relates to use of these complexes in assays for potential therapeutic agents.
  • DRTF1/E2F and the tumour suppressor protein p53 play important roles in controlling early cell cycle events
  • DRTF1/E2F is believed to co-ordinate and integrate the transcription of cell cycle regulating genes, for example those involved in DNA synthesis, with the activity of regulatory proteins, such as the retinoblastoma tumour suppressor gene product (pRb), which modulate its transcriptional activity
  • pRb retinoblastoma tumour suppressor gene product
  • Gene ⁇ c DRTF1/E2F DNA binding activity and transcriptional activation arise when a member of two distinct families of proteins interact as DP/E2F heterodimers, such as DP-1 and E2F-1
  • DP-1 is a widespread component of DRTF1/E2F DNA binding activity which when expressed at high levels oncogenically transforms embryonic fibroblasts.
  • DRTF1/E2F a group of proteins which negatively regulate the cell cycle, including the retinoblastoma tumour suppressor protein (pRb) and its relatives pl07 and pl30 (collectively known as pocket proteins) bind to and inactivate the transcriptional activity of DRTF1/E2F (Bandara and La Thangue, 1991 , Chellappan et al., 1991 ; Schwarz et al , 1993. Cobrinik et al.
  • tumour cells for example, through the action of viral oncoproteins, such as adenovirus Ela (Nevins, 1992), and furthermore are known to be temporally influenced by cell cycle progression (Shirodkar et al , 1992, Cobrinik et al..
  • the proteins E2F-4 and E2F-5 are the subject of International Patent Application Nos. PCT/GB95/00868 and PCT/GB95/00869, respectively.
  • Three members of the DP family are known to exist namely DP-1 , DP-2 and DP-3 (Girling et al, 1993 and 1994; Ormondroyd et al, 1995).
  • DP-1 is the most widespread member of DRTF1/E2F yet defined (Bandara et al , 1993 and 1994) being for example, in all the various forms of DRTF1/E2F which occur during the cell cycle in 3T3 cells (Bandara et al, 1994).
  • E2F and DP proteins are endowed with growth-promoting activity since in a variety of assays they have been shown to possess proto-oncogenic activity (Singh et al , 1994; Ginsberg et al, 1994; Johnson et al, 1994, Xu et al, 1995; Jooss et al, 1995).
  • over-expression of DP-1 or DP-2 together with activated Ha-ras causes transformation of rat embryo fibroblasts which, interestingly, is apparent in the absence of a co-transfected E2F family member (Jooss et al, 1995).
  • DP-1 exists in at least two distinct forms which differ in DNA binding properties. Further, we have found that p53 interacts with DP-1. Functionally, p53 regulates transcription driven by the DP-1/E2F-1 heterodimer by repression. Thus DP-1 is a common cellular target in two distinct pathways of growth control mediated through the activities of the pRb and p53 tumour suppressor proteins. Moreover, the integration of p53 and MDM2 with DP-1 defines a potential pathway through which p53 and MDM2 can influence cell cycle progression.
  • DP-1 appears to be a common cellular target in two distinct pathways of growth control, and these pathways, regulated by the pRb and p53 tumour suppressor proteins, now appear to be functionally integrated.
  • the invention is thus based on the su ⁇ rising finding that p53 is able to interact with DPI, and thereby modulate the transactivation activity of the DP/E2F complexes
  • the invention in a first aspect provides a complex (preferably in a substantially isolated form) which comprises p53 and a DP or E2F protein
  • DP protein is intended to encompass not only DP-1 but additionally DP-2 and DP-3 and related proteins of similar activity
  • E2F protem it is intended to encompass the five known members of the E2F family, E2F-1 to E2F-5 inclusive, as well as related proteins of similar activity
  • DP-1 protein of mammalian, preferably human or murine origin is preferred.
  • a fusion protem comprising a protein or defined in any of (a) to (e) fused to another (e.g. heterologous) protein
  • E2F protein is to be construed likewise although of course in (a) above one would instead insert the E2F proteins, E2F-1 to E2F-5, mclusive Mammalian, preferably human or murine, E2F-1 is preferred.
  • Mutants will possess one or more mutations which are additions, deletions, or substitutions of ammo acids residues Preferably these mutations will not affect, or not substantially affect, the structure and/or function and or properties of the protein Mutants will generally still possess the ability to be able to complex with p53.
  • Mutants can either be naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by performing a site- directed mutagenesis on the encoding DNA) It will be apparent that the protems used in the complexes of the invention can either be naturally occurring or recombinant
  • p53 likewise includes fragments, mutants, allelic variants and species homologues etc in the same manner as described and defined for the DP and E2F proteins
  • Particular p53 mutants include those p53 mutants which are found in tumours, for example substitutions at R175, G245, R248, R249, R273, R282 and also mutants in the region 100-150
  • transcriptional modulators Proteins that fall within the terms "DP protein” and "E2F protein” are referred to as transcription factors
  • the transc ⁇ ption modulator is (e g re ⁇ ers ⁇ bl> ) bound to the transcription factor Proteins unable to bind, or to complex with, one of the transcription modulator or transc ⁇ ption factor will not be included m complexes of the invention
  • DP-1 exists in two forms defined using immunochemical reagents
  • the two forms may be different b ⁇ virtue of the degree of phosphorylation p53 binds preferentially to the form and the resulting complex appears to contribute to the growth inactivating effects of DP-1
  • a transcription modulator or transcription factor will form a complex with the other b> providing the two proteins and determining whether or not a complex has formed, for example by determining the molecular weight of the complex by methods such as SDS- PAGE or more preferabl ⁇ by using a tag or label to detect a fusion protem formmg part of the complex
  • a complex of the invention will be in substantially isolated form if it is in a form in which it is free of other polypeptides with which it may be associated in its natural environment (eg the body) It will be understood that the complex may be mixed with carriers or diluents which will not interfere with the intended purpose of the complex and yet still be regarded as substantially isolated
  • the complex of the invention may also be in a substantially purified form, in which case it will generally comprise the complex in a preparation in which more than 90% , eg 95 % , 98% or 99% of the proteins m the preparation is a constituted by a complex of the invention
  • allelic variant will be a variant which will occur naturally in the same animal and which will function in a substantially similar manner to the proteins described herein
  • a species homologue will be the equivalent protein which occurs naturally in another species, and which performs the equivalent function in that species to the protein described herein Withm any one species, a homologue may exist as several allelic variants, and these will all be considered homologues
  • Proteins at least 70% homologous to the naturally occurring protein are also contemplated for use in the complexes of the invention, as are proteins at least 80 or 90% and more preferably at least 95% homologous This will generally be over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous ammo acids
  • Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context Homology is usuallv calculated on the basis of amino acid identity (sometimes referred to as "hard homology")
  • mutants fragments, allelic variants or species homologues thereof capable of forming a complex will be at least 10, preferablv at least 15, for example at least 20, 25, 30, 40, 50 or 60 ammo acids m length
  • a complex of the invention may be labelled with a revealing or detectable label
  • the (revealing) label may be any suitable label which allows the complex to be detected Suitable labels include radioisotopes, e g 125 I, enzymes, antibodies and linkers such as biotin Labelled complexes of the invention may be used in diagnostic procedures such as immunoassays
  • a complex (or labelled complex) according to the invention may also be fixed to a solid phase, for example the wall of an immunoassay dish
  • a second aspect of the invention relates to a polynucleotide (suitably in a substantially isolated form) which comprises
  • the polynucleotide may also comprise RNA. It may also be a polynucleotide which includes within it synthetic or modified nucleotides.
  • a number of different types of modification to oligonucleotides are known in the art These include methylphosphonate and phosphorothionate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the nucleotides described herein may be modified by any method available in the art Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotides of the invention used in methods of therapy.
  • a polynucleotide of the invention will be in substantially isolated form if it is in a form in which it is free of other polynucleotides with which it may be associated in its natural environment (usually the body). It will be understood that the polynucleotide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polynucleotide and it may still be regarded as substantially isolated.
  • a polynucleotide according to the invention may be labelled, e g with a revealing or detectable label, by conventional means using radioactive or non-radioactive labels, or may be cloned into a vector.
  • Polynucleotides such as a DNA polynucleotides according to the invention, may be produced recombinantly, synthetically, or by any means available to those of skill in the art It may be also cloned by reference to the techniques disclosed herein
  • the invention mcludes a double stranded polynucleotide comprising a polynucleotide according to the invention and its complement
  • a third aspect of the invention relates to an (eg expression) vector suitable for the replication and expression of the polynucleotide, in particular a DNA or RNA polynucleotide, according to the second aspect of the invention
  • the vector may be, for example, a plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulator of the promoter
  • the vector may contain one or more selectable marker genes, for example an ampicillin resistance gene m the case of a bacte ⁇ al plasmid or a neomycin resistance gene for a mammalian vector.
  • the vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell
  • the vector may also be adapted to be used in vivo, for example in a method of gene therapy
  • Vectors of the third aspect are preferably recombinant replicable vectors
  • the vector may thus be used to replicate the DNA
  • the DNA in the vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by a host cell
  • the term "operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences
  • Such vectors may be transformed or transfected into a suitable host cell to provide for expression of component proteins in complexes of the invention
  • the vector of the invention may comprise separate promoters operably linked to the sequences encoding the transcription modulator and the transcription factor or the vector may comprise a single coding sequence comprising both the modulator and factor In this event, the factor and modulator sequences may be in either order
  • the sequences are optionally linked by a further sequence such as a linker sequence designed to allow the two encoded polypeptide sequences to fold in a manner found in nature or it may be a linker sequence which can be selectively cleaved allowing the two encoded polypeptide sequences to be separated
  • a linker sequence designed to allow the two encoded polypeptide sequences to fold in a manner found in nature or it may be a linker sequence which can be selectively cleaved allowing the two encoded polypeptide sequences to be separated
  • An example of the former type of linker includes an IgG hmge region whereas cleavable sequences of the latter type are well known per se in the art
  • a fourth aspect of the invention thus relates to host cells transformed or transfected with the vectors of the third aspect, or able to express DNA sequence(s) encoding both the transcription modulator and transcription factor, as defined for the first aspect
  • This may allow for the replication and expression of a polynucleotide according to the invention
  • the cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian Preferred hosts include 3T3 and SAOS-2 cells (the latter cells lack functional retinoblastoma (pRb) protein and p53)
  • the host cells of the fourth aspect express, and preferably secrete, both a transcription modulator and transcription factor, those terms already having been defined in the first aspect
  • a transcription modulator and transcription factor those terms already having been defined in the first aspect
  • the transcription modulator and factor will combine to form a complex of the first aspect, such interaction occurring either inside the cell or outside in the medium surrounding the host cells (if any)
  • Such expression can be achieved either by using a single vector, such as of the third aspect, which encodes both a transcription modulator and a transcription factor
  • a host cell can be transformed or transfected with two or more vectors A first vector will encode the transcription modulator, while a second vector will encode the transcription factor Both vectors will then be co ⁇ transfected into a host cell to produce the cells of the fourth aspect
  • a polynucleotide according to the invention may also be inserted into the vectors described above in an antisense orientation in order to provide for the production of antisense RNA.
  • Antisense RNA or other antisense polynucleotides may also be produced by synthetic means.
  • Such antisense polynucleotides may be used in a method of controlling the levels of the transcription modulator, a DP protein and/or E2F protein in a cell. Such a method may include introducing into the cell the antisense polynucleotide in an amount effective to inhibit or reduce the level of translation of the E2F mRNA into protein.
  • the cell may be a cell which is proliferating in an uncontrolled manner such as a tumour cell.
  • the invention provides a process for preparing a complex according to the invention which comprises cultivating a host cell of the fourth aspect, preferably transformed or transfected with an (expression) vector of the third aspect, under conditions providing for expression of coding sequence(s) encoding the component proteins of the complex, allowing the transcription modulator to bind to the transcription factor, and recovering the complex.
  • a sixth aspect of the invention relates to complexes of the first aspect, a polynucleotide of the second aspect, vectors of the third aspect and host cells of the fourth aspect for use in medicine.
  • all such substances find use in a method for treatment of the human or animal body by therapy.
  • the uses contemplated are the inhibition of cell growth, or the interference with transcription. End uses here would therefore include the treatment of proliferative diseases, such as cancer.
  • the transcription modulator need not be the naturally occurring p53 protein, as has been explained for the first aspect. Indeed, using the screening methods of the invention (which are discussed later) substances other than the naturally occurring proteins, which have similar properties, can be identified. These too may then be used in the same or similar manner to the p53 protein themselves.
  • a pharmaceutical or veterinary composition comprising a complex of the first aspect, a polynucleotide of the second aspect, a vector of the third aspect, or a host cell of the fourth aspect, together with a pharmaceutically, or veterinarily, acceptable carrier or diluent, respectively.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product
  • formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostatis and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the polypeptide to blood components or one or more organs
  • Complexes according to the invention or transcriptional modulators may be used for the treatment, regulation or diagnosis of conditions including proliferative diseases, in a mammal including man
  • conditions include those associated with abnormal (eg at an unusually high or low level) and/or aberrant (eg due to a mutation in the gene sequence) expression of one or more transcription factors such as the DP or E2F proteins or related family members
  • Treatment or regulation of conditions with the above-mentioned complexes or peptides will usually involve administering to a recipient in need of such treatment an effective amount of that complex or peptide as appropriate
  • Vectors carrying a polynucleotide according to the invention or a nucleic acid encoding a complex according to the invention may be used in a method of gene therapy Such gene therapy may aim to repress or inhibit cell growth and so treat uncontrolled proliferation of cells, for example a tumour cell Alternatively the complexes may be used to encourage or activate cell growth
  • Methods of gene therapy may also include delivering to a cell in a patient in need of treatment an effective amount of a vector capable of expressing in the cell either an antisense polynucleotide of the invention in order to inhibit or reduce the translation of E2F or DP mRNA into the corresponding protein which may interfere with the binding of an E2F protein to a DP protein or a related family member
  • Such a vector is suitably a viral vector
  • the viral vector may be any suitable vector available in the art for targeting tumour cells
  • Huber et al Proc Natl Acac Sci USA (1991) .88, 8039
  • Culver et al (Science (1992) 256, 1550-1552) also describe the use of retroviral vectors in virus-directed enzyme prodrug therapy, as do Ram et al (Cancer Research (1993) 53, 83-88)
  • Englehardt et al (Nature Genetics (1993) 4; 27-34 describe the use of adenovirus based vectors in the delivery of the cystic fibrosis transmembrane conductance product (CFTR) into cells.
  • CFTR cystic fibrosis transmembrane conductance product
  • a host cell of the fourth aspect of the invention in the manufacture of a medicament for treating uncontrolled proliferation of cells, for example in the treatment of cancer, viral disease, heart disease, self proliferation disorders as well as auto-immune disorders such as psoriasis.
  • the invention provides a novel assay for identifying putative chemotherapeutic agents for the treatment of proliferative or viral disease which comprises bringing into contact a DP protein, an E2F protein and a putative chemotherapeutic agent, and measuring the degree of inhibition of formation of the DP/E2F protein complex caused by the agent. It may not be necessary to use the complete DP and/or E2F protein in the assay, as long as sufficient of each protein is provided such that under the conditions of the assay in the absence of agent, they form a heterodimer.
  • a screening method or assay for identifying potential or putative chemotherapeutic agents comprising providing a transcription modulator and a potential or putative chemotherapeutic agent, and measuring the extent to which the agent is able to bind to the transcription modulator, or whether the agent forms a complex, of the invention with the modulator.
  • the ninth aspect also extends to a similar method where the transcription modulator is replaced by a transcription factor.
  • the transcription modulators and factors are as defmed for the first aspect of the invention.
  • the invention provides a screening method or assay for identifying potential or putative chemotherapeutic agents, the method or assay comprising :- (A) providing the following components: (i) a DP protein and/or an E2F protein; (ii) a p53 polypeptide; and
  • any one or more of the components may be labelled, eg with a radioactive or colorimetric label, to allow measurement of the result of the assay
  • Potential chemotherapeutic agents include transcription modulators and factors as described in the first aspect of the invention.
  • Mutants, homologues and fragments of the DP and E2F proteins have been defined earlier in a corresponding manner to the mutants, homologues and fragments of the MDM2 and p53 transcription modulator proteins, all of which find use in the assays of the invention
  • the assay can be performed without the need for a DP protein
  • the complex of (I) and (n) may be measured, for example, by its ability to bind an E2F DNA binding site in vitro
  • the assay may be an in vivo assay in which the ability of the complex to activate a promoter comprising an E2F binding site linked to a reporter gene is measured.
  • the in vivo assay may be performed in yeast, insect, amphibian or mammalian cells.
  • the assays of the invention extend to both to two component assays (in the ninth aspect) as well as three component assays (as in the tenth aspect)
  • one or both of the components may comprise a fusion protein
  • That fusion protein is encompassed by the terms p53, DP and E2F proteins
  • a first protein of interest may be fused to a protein comprising a foreign DNA binding domain, such as a GAL-4 domain That protein on its own would be inactive, and although it would bind DNA, it would not activate transcription.
  • a second protein of interest (for example a DP protein, if the first protein was a transcription modulator) may then be fused to be a protein comprising a foreign activation domain, such as a GAL-4 activator.
  • the first and second proteins of interest (which would usually be a transcription modulator and a transcription factor) will form a complex.
  • respective fusion proteins are present, such as a foreign DNA binding domain and foreign activation domain, they will cooperate to cause activation.
  • the affect of the chemotherapeutic agent on this process can then be monitored. The agent will usually be added to medium surrounding cells, while inside the cell the two fusion proteins are expressed.
  • proteins that find use in the complex of the invention may themselves be fusion proteins. They be fused with another (different) protein which can act as a label or tag for detection of that fusion protein.
  • Suitable tags include GST and a series of histidines residues (e.g. six), the latter being able to bind nickel.
  • fusion proteins may be used to purify the protein, or allow it to be used in assays.
  • the fusion protein or tag may have an affinity for another protein or substance (which may be present on a column) or an antibody.
  • an antibody specific for the tag on the fusion protein may be employed.
  • Candidate therapeutic agents which may be measured by the assay include not only the transcription modulators and transcription factors mentioned in the first aspect, but in particular fragments of 10 or more amino acids of:
  • the assays of the invention can be performed using standard techniques, the components (i) and (ii) being taken from known, publicly available sources.
  • One preferred method in values providing the proteins in recombinant form, expressed by a host cell (e.g. of the fourth aspect).
  • Host cells can be transfected or transformed in the assay performed in vitro.
  • the invention contemplates a number of assays. Broadly, these can be classified as follows.
  • Performing an assay to identify a substance that affects the interaction between the transcription modulator and the transcription factor Such substances may interfere with or alter the transactivation repressing properties of p53. Such substances may be more effective than the natural molecules for themselves. For example, they may be able to repress transcriptional activity more effectively than p53. On the other hand, they may have lower levels of transcription repressing activity than p53. Such substances may therefore act as either agonists or antagonists of p53, as appropriate. The substances may interfere with the interaction or binding between the transcription modulator and transcription factor, or alternatively competitively inhibit this interaction, or the formation of a complex between the transcription modulator and factor, by mimicking either of the transcriptional modulator or factor proteins.
  • Such an assay may be particularly useful in identifying compounds which are agonists of p53 or other substances which promote the presence in the cell of the form of DP-1 recognised by p53 which is likely to be growth inhibiting. For example such compounds may bind to DP-1 in a manner which mimics p53. Alternatively the compound may promote the binding of p53 to DP-1.
  • Such assays may also be used with p53 mutants such as those described above to assay for compounds which restore the ability of such p53 mutants to bind to DP- 1 where such p53 mutants have lost the ability to do so in tumour cells.
  • an assay to find an inhibitor of an E2F protein rrans-activation that is to say, inhibition of activation of transcription.
  • This inhibitor may therefore inhibit binding of an E2F protein to DNA (usually at the E2F binding site).
  • Potentially suitable inhibitors are proteins, and may have a similar or same effect as p53.
  • suitable inhibitory molecules may comprise fragments, mutants, allelic variants, or species homologues of p53 in the same manner as defined for complexes of the first aspect.
  • Assaying for inhibitors of (hetero)dimerisation which mimic the binding of p53 to DP-1.
  • the binding of DP-1 to an E2F protein promotes cell proliferation and thus p53 competes with E2F for binding to DP-1.
  • Such inhibitors may prevent dimerisation of an E2F protein (eg. E2F-1) with a DP protein, such as DP-1.
  • the assay can measure the degree of inhibition of binding by determining the ability of p53 and/or E2F to compete with each other and the potential inhibitor.
  • the inhibitor can be a fragment, mutant, allelic variant or species homologue of a DP or E2F protein as defmed for the complexes of the first aspect.
  • the invention thus contemplates the identification of substances for the treatment or prophylaxis of diseases that are based on the uncontrolled proliferation of cells, or where uncontrolled proliferation is an important or essential pathological aspect of the disease.
  • This includes cancer, viral disease, self proliferation itself as well as auto immune diseases such psoriasis.
  • Compounds contemplated here would include p53 agonists.
  • one is generally seeking to prevent, inhibit or interfere with the activity of an E2F protein.
  • some diseases and conditions can be treated by increasing E2F expression, for example by promoting or inducing overexpression. That could be achieved through the use of p53 antagonists.
  • This preferably results in apoptosis, sometimes known as programmed cell death.
  • Overexpression of the E2F protein can result in death of the cell (Qin et al J994) and therefore this aspect can also be used in the treatment of cancer.
  • E2F and p53 appear to modulate the activity of each other and disruption of the interaction by antagonists may promote the observed apoptotic effects of E2F.
  • Anti-DP-l(A) is a rabbit polyclonal anti-peptide serum raised against a peptide representing an N-terminal region in DP-1 , and has been previously described (Girling et al, 1993; Bandara et al, 1994).
  • Anti-DP- l(D) rabbit polyclonal anti-peptide serum (Bandara et al, 1994) and monoclonal antibody 32.3 were raised against a peptide representing a C-terminal region (residues 385 to 400) in DP-1.
  • Immunoblotting with anti-DP-l(A), anti-DP-1 (D) or 32.3 was performed as previously described. Either the homologous peptide. or a control unrelated peptide, peptide 1 , was added to assess specificity.
  • LSL buffer 50mM Tris-HCl pH 8.0, 150mM NaCl, 0.1 % NP40, 2 ⁇ g/ml aprotinin, 0.5mM PMSF
  • 32.3 was added in the presence of peptide and incubated for lhr on ice.
  • Immune complexes were collected with protein A-Sepharose and washed extensively (at least 3 times) in LSL buffer, released in SDS sample buffer, electrophoresed and immunoblotted with anti-DP-1 (A). The procedure for sequential immunoprecipitation and immunoblotting has been previously described (Bandara et al , 1993).
  • the effect of p53 on the E2F-site binding activity of the E2F-1/DP-1 heterodimer was assayed using in vitro transcribed and translated DP-1 (pG4DP-l ; 4) and E2F-1 (pSP72;27).
  • In vitro transcription and translation was carried out in a TNT T7/SP6 coupled reticulocyte lysate system (promega). pH6- mmp53 wt encodes a His-tagged complete mouse p53 protein (kindly supplied by Gunnar Weidt and Wolfgang Deppert). His-tagged murine p53 was purified from a 500ml pellet of IPTG-induced bacterial culture.
  • the bacterial pellet was resuspended in lOmls denaturing buffer (lOOmM sodium phosphate, lOmM tris base, 6.0M guanidine hydrochloride, 30mM imidazole) pH 8.0, and gently stirred for two hours at room temperature.
  • MgCl 2 was added to a final concentration of 5mM and cellular debris were cleared by repeated centrifugation at 4°C. 400 ⁇ l of nickel agarose (solid) QIAGEN) was added to the supernatant and rotated for one hour at room temperature.
  • the resin was washed stepwise with two 50ml volumes each of; denaturing buffer pH8.0, denaturing buffer pH 6.4 and renaturing buffer (25mM sodium phosphate pH 7.0, 300mM NaCl, lOmM /3-mercaptoethanol) containing IM then 0. IM and lastly no guanidine hydrochloride.
  • Protein was eluted off the resin by sequential washes with imidazole buffer ( 150 mM imidazole, lOOmM NaCl, 50mM Tris-HCl pH 8.0) and analysed by SDS-PAGE. An equal amount of heat-denatured or non-denatured His-tagged p53 was added to the binding reaction.
  • pH6-mmp53 wt encodes a His-tagged complete mouse p53 protein (kindly supplied by Gunnar Weidt and Wolfgang Deppert). pH6-mmp53, GST-pRb (Bandara et al, 1991) and GST alone were induced and purified by conventional procedures.
  • 15 ⁇ l of fusion protein bound to the appropriate agarose glutathione-agarose or nickel-agarose was incubated with F9 EC whole cell extract with constant rotation for 2hrs at 4°C. The suspension was centrifuged and repeatedly washed with LSL buffer, resuspended in SDS loading buffer and immunoblotted with anti-DP-l(A).
  • GST-DP- 1 encodes a complete DP-1 protein fused to GST in pGEX-3X and was induced and purified by conventional procedures.
  • E2F-1 , E2F-4 and wild-type p53 were transcribed and translated in the presence 35 S methionine in a TNT coupled lysate (Promega) as recommended by the manufacturer.
  • TNT coupled lysate Promega
  • fusion protein was added to the translate in PBSA containing ImM EDTA, ImM DTT, 0.5% Tween 20 and incubated for 30 min at 30°C. Glutathione beads were subsequently added and incubated for a further 30 min.
  • Binding assay for MDM2 pGST-MDM2 encodes a complete MDM2 protein fused to GST in pGEX-3X and was induced and purified by conventional procedures. DP-1 and wild-type p53 were transcribed and translated in the presence 35 S methionine in a TNT coupled lysate (Promega) as recommended by the manufacturer. For the in vitro binding assay, fusion protein was added to the translate in PBSA containing ImM EDTA, ImM DTT, 0.5 % Tween 20 and incubated for 30 min at 30°C. Glutathione beads were subsequently added and incubated for a further 30 min. Beads were collected and washed four times in the same buffer before being solubilized in SDS sample buffer.
  • the reporter construct p3xWT-GL, p3xMT-GL, pCMV- ⁇ gal, pCMV-E2F-l and pCMV-DP-1 have all been described previously (Girling et al. , 1994; Jooss et al, 1995).
  • pC53-SN3 encodes wild-type p53 driven by the CMV enhancer/promoter region (Baker et al, 1990).
  • pJ4 ⁇ -MDM2 encodes the complete MDM2 protein (Oliner et al., 1992).
  • the total amount of DNA in each transfection was made up with empty vector which, in the case of the p53 titration was with pCMV-neoBam, and for the MDM2 titration waswith pJ4 ⁇ Cells were transfected by the conventional calcium phosphate procedure. Luciferase and ⁇ -galactosidase assays were peformed as described previously (Girling et al, 1994) Each treatment was performed in duplicate
  • DP-1 polypeptides of 55kD can be resolved during cell cycle progression in 3T3 cells, referred to as p55L (lower) and p55U (upper), p55L appearing towards the end of Gl as cells begin to enter S phase (Bandara et al , 1994)
  • p55L lower
  • p55U upper
  • p55L appearing towards the end of Gl as cells begin to enter S phase
  • an anti-peptide was prepared monoclonal antibody, 32 3. which recognises p55L.
  • anti-DP-1 By immuno-blotting extracts prepared from a synchronous cultures of F9 EC cells a polyclonal antiserum, anti-DP-1 (A), revealed both forms of p55 in contrast to monoclonal antibody 32.3 which defined p55L.
  • anti-DP-l(A) specifically immunoprecipitated a group of polypeptides with a range of molecular weights.
  • anti-DP-l(A) immunoprecipitate was released and subsequently re-immunoprecipitated with anti-DP-l(D), which possesses similar specificity to 32.3 for p55L, a subset of the anti-DP-l(A) associated polypeptides were apparent.
  • the efficiency of interaction of DP-1 and MDM2 was compared with the binding of p53 and MDM2.
  • An assay was used in which the ability of MDM2, expressed as a GST fusion protein, to bind to in vitro translated DP-1 and p53 was assessed It was found that p53 and MDM2 were able to associate m the conditions of this assay and likewise, DP-1 could interact with MDM2, similar results being obtained with the other members of the DP family, DP-2 and DP-3
  • the efficiency of interaction between DP-1 and MDM2 appeared to be somewhat lower than the p53/MDM2 interaction although both interactions were specific since neither DP-1 or p53 could bind to a control GST protein.
  • this result reflects the data derived by immunoprecipitation from mammalian cell extracts where p53 co-immunoprecipitated with p55U, a DP-1 protein recognised by anti-DP-l(A) but not anti- DP-l(D).
  • the specificity of p53 for DP-1 in the in vitro binding assay therefore possesses some similarity with the interaction in mammalian cells, and supports the conclusion that p53 interacts with an immunochemcially distinct form of DP-1.
  • N-terminal region in p53 is required for binding to DP-1.
  • DP-1 is a frequent component of DRTF1/E2F (Bandara et al, 1993 and 1994)
  • the functional consequence of an interaction of either p53 with DP-1 was assessed by studying the effects on E2F site-dependent transcription driven by DP-1 and E2F-1 a situation in which it is known that both proteins co-operate in transcriptional activation as a DNA binding heterodimer (Bandara et al, 1993. In these assay conditions DP-1 alone possesses insignificant transcriptional activity (Bandara et al., 1993).
  • DP-1 Distinct forms of DP-1. It is known that DP-1 is the most widespread DNA binding component of physiological DRTF1/E2F DNA binding activity (Bandara et al, 1993, 1994) where it exists in a heterodimer with an E2F family member (Bandara et al, 1993; Helin et al, 1993; Krek et al, 1993). Previous studies have shown that the polypeptide encoded by DP-1, p55, is subject to cell cycle control since it is regulated during cell cycle progression, the level of p55L increasing as cells progress through the cycle (Bandara et al., 1994).
  • p53 associates with DP-1.
  • DP-1 sequesters p53, titrating out its activity, and thus over-riding the growth-regulating effects of p53.
  • DP-1 may act in an analogous fashion to certain viral oncoproteins, such as the adenovirus Elb and papilloma virus E6 proteins, since their ability to inactivate p53 correlates with oncogenic activity (Moran, 1993).
  • Adenovirus Ela prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature 351:494-497.
  • DP-1 a cell cycle-regulated and phosphorylated component of transcription factor DRTF1/E2F which is functionally important for recognition by pRb and the adenovirus E4 orf 6/7 protein.
  • E2F-4 a new member of the E2F gene family, has oncogenic activity and associates with pl07 in vivo. Genes Dev. 8:2680-2690. Buck, V. et al, 1995. Molecular and functional characterisation of E2F-5, a new member of the E2F family. Oncogene 11:31-38
  • the E2F transcription factor is a cellular target for the Rb protein.
  • a cyclin A-specific protein kinase complex possesses sequence-specific DNA binding activity: p33 cdk2 is a component of the E2F-cyclin A complex.
  • E2F-4 a new member of the E2F transcription factor family, interacts with pl07. Genes Dev. 8:2665-2679. Girling, R. et al, 1993. A new component of the transcription factor DRTF1/E2F. Nature. 362:83- 87.
  • E2F-5 a new E2F family member that interacts with pl30 in vivo. Mol Cell. Biol. 15:3082-3089.
  • E2F-2 a novel protein with the biochemical properties of transcription factor E2F. Mol. Cell. Biol. 13:7802-7812.
  • DRTF1/E2F an expanding family of heterodimeric transcription factors implicated in cell cycle control.
  • the retinoblastoma protein binds a family of E2F transcription factors. Mol. Cell. Biol 13:7813-7825.
  • Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 362:857-860.
  • the transcription factor E2F interacts with the retinoblastoma product and a pl07-cyclin A complex in a cell cycle-regulated manner.

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Abstract

L'invention porte sur des complexes comprenant la protéine p53 avec les facteurs de transcription DP-1 (et d'autres membres de la famille DP) ou E2F-1 (ou d'autres protéines E2F, jusqu'à la protéine E2F-5 incluse). Il est possible d'utiliser ces nouveaux complexes dans le cadre d'épreuves concernant des agents chimiothérapeutiques potentiels qui sont des agonistes ou antagonistes de la protéine p53, en particulier des agonistes ou antagonistes modulant une progression d'un bout à l'autre du cycle cellulaire par l'interruption de la fixation normale de la protéine p53 à DP-1 et/ou à E2F-1.
PCT/GB1996/001560 1995-06-30 1996-06-28 INTERACTION DE LA PROTEINE p53 AVEC UN FACTEUR DE TRANSCRIPTION DP-1 WO1997002354A1 (fr)

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AU62373/96A AU6237396A (en) 1995-06-30 1996-06-28 Interaction of p53 with transcription factor dp-1
EP96921006A EP0835319A1 (fr) 1995-06-30 1996-06-28 INTERACTION DE LA PROTEINE p53 AVEC UN FACTEUR DE TRANSCRIPTION DP-1
JP9504018A JPH11509089A (ja) 1995-06-30 1996-06-28 ｐ５３と転写因子ＤＰ−１との相互作用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043647A1 (fr) * 1996-05-15 1997-11-20 Medical Research Council Signaux de localisation nucleaire des proteines dp et e2f et leur utilisation
WO1999027091A1 (fr) * 1997-11-21 1999-06-03 The University Court Of The University Of Glasgow Proteine interagissant avec le facteur de transcription dp et utilisation de cette derniere
US6159691A (en) * 1996-05-15 2000-12-12 Prolifix Limited Assay for a putative regulator of cell cycle progression
WO2000056905A3 (fr) * 1999-03-19 2000-12-28 Cropdesign Nv Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture
JP2002504325A (ja) * 1998-02-19 2002-02-12 ノヴィミュンヌ・エス・アー 転写因子ｓｔａｔ１と転写因子ｕｓｆ１の間の結合を阻害する能力をもつ化合物のスクリーニング方法
US6387649B1 (en) 1996-09-30 2002-05-14 Prolifix Limited Assay for a regulator of cell cycle progression
US8123977B2 (en) 2005-11-07 2012-02-28 Lg Chem, Ltd. Copolymer for liquid crystal alignment, liquid crystal aligning layer including copolymer for liquid crystal alignment, and liquid crystal display including liquid crystal aligning layer

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WO1994000603A1 (fr) * 1992-06-26 1994-01-06 The Trustees Of Princeton University Procede de detection de cellules precancereuses ou cancereuses au moyen d'anticorps ou de sondes de p90

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WO1994000603A1 (fr) * 1992-06-26 1994-01-06 The Trustees Of Princeton University Procede de detection de cellules precancereuses ou cancereuses au moyen d'anticorps ou de sondes de p90

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ALMASAN ET AL.: "Deficiency of retinoblastoma protein leads to inappropriate S-phase entry, activation of E2F-responsive genes, and apoptosis", PROC. NATL ACAD. SCI., vol. 92, no. 12, 6 June 1995 (1995-06-06), pages 5436 - 5440, XP002012354 *
MARTIN ET AL.: "Regulation of transcription by E2F1/PD1", J. CELL. SCI., vol. 19, 1995, pages 91 - 94, XP000601457 *
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QIN ET AL.: "Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis", PROC. NATL ACAD. SCI., vol. 91, no. 23, 8 November 1994 (1994-11-08), pages 10918 - 10922, XP002012350 *
VON KNEBEL DOEBERITZ ET AL.: "Reversible repression of Papillomavirus oncogene expression in cervical carcinoma cells : consequences for the phenotype and E6-p53 and E7-pRB interactions", J. VIROLOGY, vol. 68, no. 5, May 1994 (1994-05-01), pages 2811 - 2821, XP002012352 *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997043647A1 (fr) * 1996-05-15 1997-11-20 Medical Research Council Signaux de localisation nucleaire des proteines dp et e2f et leur utilisation
US5859199A (en) * 1996-05-15 1999-01-12 Prolifix Limited Transcription factor DP-3 and isoforms thereof
US6159691A (en) * 1996-05-15 2000-12-12 Prolifix Limited Assay for a putative regulator of cell cycle progression
US6387649B1 (en) 1996-09-30 2002-05-14 Prolifix Limited Assay for a regulator of cell cycle progression
WO1999027091A1 (fr) * 1997-11-21 1999-06-03 The University Court Of The University Of Glasgow Proteine interagissant avec le facteur de transcription dp et utilisation de cette derniere
JP2002504325A (ja) * 1998-02-19 2002-02-12 ノヴィミュンヌ・エス・アー 転写因子ｓｔａｔ１と転写因子ｕｓｆ１の間の結合を阻害する能力をもつ化合物のスクリーニング方法
WO2000056905A3 (fr) * 1999-03-19 2000-12-28 Cropdesign Nv Procede pour accelerer et/ou ameliorer la croissance et/ou le rendement de vegetaux ou pour modifier leur architecture
US8123977B2 (en) 2005-11-07 2012-02-28 Lg Chem, Ltd. Copolymer for liquid crystal alignment, liquid crystal aligning layer including copolymer for liquid crystal alignment, and liquid crystal display including liquid crystal aligning layer

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