EP0859839A1 - ACTIVATION DE LA PROTEINE p53 - Google Patents

ACTIVATION DE LA PROTEINE p53

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Publication number
EP0859839A1
EP0859839A1 EP96935079A EP96935079A EP0859839A1 EP 0859839 A1 EP0859839 A1 EP 0859839A1 EP 96935079 A EP96935079 A EP 96935079A EP 96935079 A EP96935079 A EP 96935079A EP 0859839 A1 EP0859839 A1 EP 0859839A1
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European Patent Office
Prior art keywords
dnak
protein
activation
substance
peptide
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EP96935079A
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German (de)
English (en)
Inventor
David Philip Magicwell House Balmullo LANE
Theodore Robert Hupp
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University of Dundee
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University of Dundee
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • 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

  • the present invention relates to the p53 tumour suppressor protein, and more particularly to the activation of latent p53 protein using substances comprising or based on the C- terminal negative regulatory domain of p53 or the epitope of antibody Pab241.
  • the present invention also relates to the identification of substances based on motifs in the C- terminal domain of p53 that interact with DnaK.
  • the present invention also includes applications of these substances, both therapeutic applications and the use of the substances in screening for mimetics.
  • p53 appears to play a central role in the cellular response to irradiation damage by activating an apoptotic or growth arrest pathway in proliferating cells (Malzman and Czyzyk, 1984; Kastan et al 1992; Kuerbitz et al 1992; Hall et al 1993; Lu and Lane 1993; Zhan et al 1993; Clarke et al 1993; Lowe et al 1993; Merritt et al 1994; Yonish et al 1991) .
  • p53 is activated by cellular stress
  • cellular stress may involve both increases in p53 protein level and in p53's specific activity by covalent modification.
  • direct activation of the latent sequence-specific DNA binding activity of human p53 can occur through UV and serum- responsive signalling pathways in insect cell systems (Hupp and Lane, 1995)
  • upstream intracellular signalling pathways involved in the direct activation of the biochemical function of p53 in mammalian cells following irradiation have not been clearly delineated nor have the actual enzymes activating p53 function in vivo been identified.
  • Biochemical analysis of p53 has shown that it interacts with many proteins implicated in regulation of p53 protein function, including; protein kinases and phosphatases, heat shock protein, and DNA binding proteins.
  • the biochemical activity of p53 may also be regulated by interaction of the C-terminus with single stranded RNA or DNA (Oberosler et al 1993; Bakalkin et al 1994; Jayaraman and Prives 1995) .
  • the activity of p53 most tightly linked to its tumour suppressor activity is the ability of the protein to bind to DNA sequence-specifically (Kern et al 1992; El-Deiry et al 1992) .
  • Inactivating point mutations usually map within the active site for sequence-specific DNA binding or within the central core DNA binding domain (Cho et al 1994; Halazonetis and Kandil 1993; Bargonetti et al 1993) .
  • sequence-specific DNA binding is a biologically relevant function of p53 and understanding its regulation may reveal mechanisms whereby the cell regulates a key damage- responsive pathway.
  • a monoclonal antibody or bacterial Hsp70 whose binding sites reside in the C-terminal negative regulatory domain, mimics the effects of protein kinases and activate latent p53 through a concerted transition of sub-units in the tetramer (Hupp and Lane 1994a) .
  • the present invention provides a substance which has the property of activating the sequence specific DNA binding activity of latent p53, said substance consisting of: (i) a fragment of the C-terminal regulatory domain of p53 protein, or an active portion or derivative thereof;
  • an active portion means a portion of the p53 peptide which is less than the full amino acid sequence of the fragment above, but which retains the property of activating the DNA binding activity of latent p53.
  • a “derivative” is a protein modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion or substitution of one or more amino acids, without fundamentally altering the essential activity of the protein.
  • “functional mimetic” means a substance which may not contain a fragment or active portion of the p53 amino acid sequence, and probably is not a peptide at all, but which has some or all of the properties of the p53 fragment, in particular the property of activating the DNA binding activity of latent p53.
  • “functional moiety” means a non- p53 derived molecule, for example a label, a drug, or a carrier molecule.
  • the functional moiety is a carrier molecule is a 16 aa peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name "Penetratin”) , which can be coupled to one of the above substances via a terminal Cys residue.
  • the "Penetratin” molecule and its properties are described in W091/18981.
  • the functional moiety is a p53 activating molecule.
  • the substances could be coupled to a molecule such as PEG to affect function and prolong the in vivo half-life of the substances (see Kuan et al, J. Biol. Chem., 269:7610-7616, (1996)) .
  • the substance comprises a fragment of the C-terminal regulatory domain of p53 protein between amino acid residues 369 and 383. More preferably, the fragment of the C-terminal regulatory domain of p53 includes residues corresponding to the amino acids K370, K372, K373, R379, K381, K382. As demonstrated below, replacement of these residues in the 369-383 p53 fragment reduces the ability of the p53 fragment to activate latent p53.
  • the substance preferably consists of fragments of p53 localised between amino acid residues residues 287 to 310 of murine p53, corresponding to residues 293 to 316 of human p53.
  • the present invention provides a substance which has the property of binding to DnaK, said substance consisting of a fragment of the C-terminal regulatory domain of p53 protein from amino acid residues 381 to 388, or an active portion or derivative thereof.
  • the present invention includes composition comprising one or more of the above substances in combination with known p53 activators, e.g. heat shock proteins such as DnaK, antibodies such as Pab421 or Pab241, or kinases such as casein kinase II.
  • heat shock proteins such as DnaK
  • antibodies such as Pab421 or Pab241
  • kinases such as casein kinase II.
  • modification of p53 by cdc2/cyclin kinase may also interact synergistically with the substances described herein.
  • the present invention provides pharmaceutical compositions comprising any of the above substances and the use of these compositions in methods of medical treatment.
  • the present invention relates to the use of these substances in the preparation of medicaments for the treatment of disorders in which the activation of latent p53 is required, such as cancer or other hyperproliferative disorders.
  • compositions according to the present invention may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such.materials should be non ⁇ toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • a parenterally acceptable aqueous solution may be employed which is pyrogen-free and has suitable pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions.
  • Dosage levels can be determined by the those skilled in the art, taking into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.
  • the present invention also provides nucleic acid encoding these proteins.
  • nucleic acid encoding the proteins can readily construct from the amino acid sequences disclosed herein, taking account of factors such as codon preference in the host used to express the nucleic acid sequences.
  • nucleic acid encoding the carrier protein can be linked to the sequence encoding the peptides and the sequences expressed as a fusion.
  • the present invention provides vectors incorporating the above nucleic acid sequences operably linked to control sequences to direct their expression, and host cells transformed with the vectors .
  • the present invention provides the use of any one of the above substances in screening for (i) compounds having one or more of the biological activities of the substances described above or (ii) compounds which are binding partners of one of the substances, e.g. antibodies or complementary peptides specific for the p53 fragments or p53 mimetics, or substances having the same binding properties as DnaK.
  • the candidate compounds can be selected from a synthetic combinatorial library. Examples of screening procedures for mimetics or binding partners include:
  • yeast two hybrid screens to detect candidate peptides which bind to the substances or to oligonucleotides derived from the p53 fragments (for a description of yeast two hybrid screens see our earlier application W096/14334) ;
  • the present invention provides the use of a fragment of p53 including (i) amino acid residues corresponding to K370, K372, K373, R379, K381, and K383 or (ii) amino acid residues comprising the epitope of murine p53 bound by antibody Pab241, or a fragment from the corresponding region of human p53 , in the design of an organic compound which is modelled to resemble the three dimensional structure of said amino acid residues, the organic compound having the property of activating latent P 53.
  • the present invention provides the use of a fragment of p53 including amino acid residues corresponding to 381-388 (KKLMFKT) of p53, in the design of an organic compound which is modelled to resemble the three dimensional structure of said amino acid residues, the organic compound having the property of binding to DnaK.
  • the designing of mimetics to a known pharmaceutically active compound is a known -approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, eg peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties, eg stereochemistry, bonding, size and/or charge, using data from a range of sources, eg spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between ' atoms) and other techniques can be used in this modelling process.
  • a range of sources eg spectroscopic techniques, X-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between ' atoms
  • other techniques can be used in this modelling process.
  • the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining ' the biological activity of the lead compound.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • (IB) The level of ⁇ - galactosidase produced by p53 transcriptional activity was assessed at the single cell level using an immunohistochemical method and the jS-gal substrate: (IBi) OJ/m 2 , (IBii) 10J/m 2 , (IBiii) 20J/m 2 , (IBiv) 40J/m 2 .
  • FIG. 1 Activation of the sequence-specific transcriptional response of p53 in vivo by microinjection of Pab421.
  • Cells were microinjected with control buffer (2i) , the activating antibody Pab421 (2ii) , or the inhibiting antibody Pab246 (2iii) , incubated for 16 -hours, and then stained for 0-galactosidase production as in Figure 1.
  • control buffer (2i) the activating antibody Pab421
  • FIG. 1 The complete results of two similar experiments are detailed in Table 1.
  • FIG. 3 (Top panel) . Binding sites of activating proteins within the C-terminal regulatory domain of p53. The high affinity amino acid contacts required for Pab421 (Stephen et al 1995) and DnaK binding (data not shown) , and the sites of phosphorylation of protein kinase C and casein kinase II are as indicated. The overlap and sequence of synthetic peptides C ⁇ C ⁇ , relative to the carboxy-terminus of p53, are also as indicated. Peptides C-i-C,, are not biotinylated, while peptides C 3 -C 12 contain an amino terminal biotin group linked to the amino acid sequence SGSG.
  • FIG. 4 (Top panel) Synergistic activation of phospho- p53 with a synthetic peptide derived from the C-terminal regulatory domain.
  • p53 50 nM was left unphosphorylated (lanes 1 to 6) or phosphorylated at 30°C with recombinant human casein kinase II ((Hupp et al 1993) ; 2ng) for 1 minute. After phosphorylation, the reactions were incubated further for 1, 2, 5, and 10 minutes at 30°C. Subsequently, peptide C x (20 ⁇ M) was added (lanes 6-11) during DNA binding at 0°C and products were analysed as indicated in the Experimental Procedures.
  • p53 50 nM was phosphorylated with casein kinase II (2 ng) for one minute at 30°C (lanes 1-12) or was left unphosphorylated (lanes 13-15) . Subsequently at 0°C either peptide C_ (lanes 2-5; 5, 10, 20 or 40 ⁇ M) ; potassium chloride (lanes 6-9; 50, 100, 150, and 200 mM) ; DnaK (lanes 10-12; 1.4, 2.8, or 5.6 ⁇ M protein) ; and peptide C_ (lanes 13-15; 10, 20, or 40 ⁇ M) were added and DNA binding was assayed as indicated in Materials and Methods.
  • Figure 8 Model for peptide activation of latent p53 tetramers.
  • p53 exists stably in the latent state due to interactions between the basic negative regulatory domain amino acid side-chains (shaded cylinders) and a peptide binding pocket within the tetramer.
  • Deletion of the regulatory domain or its phosphorylation and subsequent incubation at high temperature (30°C) disrupts regulatory domain interactions and converts the latent tetramer to an activated tetramer through a concerted transition of subunits.
  • Activation by post-translational modification at the regulatory site can now be separated into two stages.
  • the first step is a rapid event in which the regulatory domain is modified (ie by covalent modification) , but the energy barrier required to disrupt the regulatory domain- tetramer interaction is not overcome. As a result, a stable and latent phospho-intermediate can be isolated.
  • Figure 9 Peptides derived from the C-terminal regulatory domain of human p53 compete with DnaK activation.
  • A Competitive binding experiments with peptide 379-393 and all three activating proteins: lane 1 (p53, no activator) , lane 2 (p53 + PAb421) , lane 3 (p53 + DnaK), lane 4 (p52 + casein kinase II), lanes 5-7 (p53 + PAb421 with l ⁇ g, O.l ⁇ g, and O.Ol ⁇ g of peptide) , lanes 8-10 (p53 + DnaK with l ⁇ g, O.l ⁇ g, and O.Ol ⁇ g of peptide) , and lanes 11-13 (p53 + casein kinase II with l ⁇ g, O.l ⁇ g, and O.Ol ⁇ g of peptide) .
  • (B) Competitive binding experiments using peptide 369-383 which contains the 421 epitope : lanes 1-3 (p53 + PAb421 with l ⁇ g, O.l ⁇ g, and O.Ol ⁇ g of peptide), lanes 4-6 (p53 + DnaK l ⁇ g, O.l ⁇ g, and O.Ol ⁇ g of peptide) , and lanes 7-9 (p53 + casein kinase II l ⁇ g, O.l ⁇ g, and O.Ol ⁇ g of peptide) .
  • Figure 10 Binding of DnaK to human p53 peptides.
  • Figure 11 Binding of human Hsc70 to human p53 synthetic peptides.
  • 1.5 ⁇ M human Hsc70 was incubated with 0.05, 0.5 and 5 ⁇ M of .the respective C-terminal biotinylated peptides of human p53. Protein bound to the peptides was detected with Streptavidin-peroxidase and ECL following a native gel electrophoresis and blot onto nitrocellulose.
  • DnaK 1.5 ⁇ M incubated with equivalent amounts of peptide 376-390 were run on the same gel as an internal standard for the strength of the interaction.
  • Figure 12 Binding of DnaK to murine p53 peptides .
  • the binding of 1.5 ⁇ M DnaK to biotinylated peptides of the C- terminus of murine p53 was established using titrations of the relevant peptides (0.05, 0.5, 5 ⁇ M) .
  • Protein-peptide complexes were resolved by native gel electrophoresis and detected with Streptavidin-peroxidase and ECL.
  • the human peptide 376-390 was run as a control for the strength of the signal.
  • Figure 13 Activation of murine p53 by DnaK.
  • Latent murine p53 (150ng) produced in bacteria was activated with the monoclonal antibody PAb421 or co-incubated for 30 minutes at 30°C with increasing concentrations (0.05, 0.1, 0.5, 3, 6 ⁇ M) of the bacterial heat shock protein DnaK prior to analysis for DNA binding by gel electrophoresis at 0°C.
  • Figure 14 A. Activation of murine p53 by DnaK is dependent on the phosphorylation state of p53. 150ng of wild-type murine p53 produced in insect cells was incubated for 30 minutes at 30°C with increasing concentrations of
  • the DnaK binding site was defined to aa381-388 and human Hsc70 binds to the same site on p53.
  • the sequence of murine p53 was obtained from the Swiss protein database and it is assumed that the second ATG is used as the initiation codon, which predicts a 387-amino acid protein) .
  • Within the DnaK binding site on human p53 there are four amino acid changes in murine p53 which reduce the binding affinity of DnaK and lower the activation extent of murine p53 by DnaK.
  • murine p53 is efficiently activated by DnaK if it is assembled and phosphorylated in recombinant insect cell expression systems.
  • cdc2 phosphorylation site (aa309) . Consistent with the regulatory role played by this second domain, the monoclonal antibody PAb241 also activates p52 protein for DNA binding.
  • Figure 16 shows the sequences of the regulatory domains of p53 identified in this application, in addition showing the DnaK binding site.
  • Hsp70 isoforms with C-terminal peptides of p53. Protein sequences were obtained from the Swiss Protein data base and analysed for sequence homology with DnaK using the GCG software. Hsc70, sharing the least overall homology with DnaK, interacts strongly with p53 at the same site as DnaK. -Hsp70 binds very weakly to peptide 376-390. No interaction could be demonstrated for p53 peptides and the Grp78 (BiP) heat shock protein which was expressed in E. coli as recombinant hamster Grp78. Materials and Methods
  • Recombinant latent forms of human p53, casein kinase II 5 from rabbit muscle, DnaK, and monoclonal antibodies DO-1, Pab421-Fab fragments, Pab421 were purified as described (Hupp and Lane 1994a; Hupp et al 1992) . Assembly of activation reactions, sequence-specific DNA binding reactions, ELISA, DnaK and p53 peptide-binding reactions,
  • the cells of a DnaK over-expressing strain were grown shaking in LB media at 30°C to an O.D. (600nm) of 0.5, when they were quickly heat-shocked by two fold dilution in prewarmed (55°C) LB media. Subsequently, the incubation was
  • Sepharose column at a protein(mg) :resin(ml) ratio of 10:1.
  • Bound protein was eluted using a linear gradient from 0.05 to 1M KCl (in buffer B, containing 10% glycerol, 20mM HEPES
  • 35 DnaK were dialysed against a buffer containing 10% sucrose, 20mM Imidazole (pH 7.0), 20mM MgCl 2 , 20mM KCl, 5mM DTT, ImM Benzamidine and leupeptin to l ⁇ g/ml (buffer A) .
  • Dialysed protein was applied to an ATP-agarose column (Sigma A-2767) in buffer A at a protein(mg) :resin(ml) ratio of 20:1. After washing in buffer A containing IM KCl and equilibration in buffer A, DnaK was eluted in buffer A containing lOmM ATP.
  • Recombinant human p53 was expressed in BL21 E. coli cells at room temperature using a T7 expression system (Midgley et al, 1992) , in which latent p53 tetramers were purified from soluble lysates (Hupp et al, 1992) .
  • p53 was purified by a modification of a published protocol (Hupp et al 1994a) . Briefly, human p53 was purified using Heparin- Sepharose (Hi-Trap, Pharmacia Biotech Inc.) , Phosphocellulose (P-ll, Whatmann) , and Superose-12 (Pharmacia) column chromatography.
  • Murine wild-type p53 was expressed in E. coli BL21 cells as described for human p53 (Hupp et al 1994a) .
  • Murine wild- type p53 and the point mutant Ala309 were also expressed in Spodoptera frugiperda cells (Sf9 cells) as described elsewhere (Hansen et al) .
  • the protein was purified using Heparin-Sepharose (Hi-Trap) , Pharmacia Biotech Inc.) with a linear KCL gradient from 0.1.to IM KCl in buffer B.
  • Activation of p53 was added to lO ⁇ l of Activation buffer (10% glycerol, 1.0 mg/ml BSA, 0.05 M KCl, 0.1 mM EDTA, 5 mM DTT, 0.05% Triton X-100, 10 mM MgCl 2 , 0.5 mM ATP (or 50 ⁇ M ATP when activated using protein kinases) and 25 mM HEPES (pH 7.6)), followed by incubation at 30°C for 30 minutes with indicated activating factor or peptide.
  • Activation buffer (10% glycerol, 1.0 mg/ml BSA, 0.05 M KCl, 0.1 mM EDTA, 5 mM DTT, 0.05% Triton X-100, 10 mM MgCl 2 , 0.5 mM ATP (or 50 ⁇ M ATP when activated using protein kinases) and 25 mM HEPES (pH 7.6)
  • Reactions were placed at 0°C at lO ⁇ l of a DNA binding buffer (20% glycerol, 1.0 mg/ml BSA, 0.05 M KCl, 0.1 mM EDTA, 5 -mM DTT, 0.05% Triton X-100, 10 mM MgCl 2 , 0.5 mM ATP, 5 ng of radiolabeled consensus site oligonucleotide (Hupp et al 1992) , and 100 ng of supercoiled pBluescript competitor DNA. Reaction products were processed using native gel electrophoresis as indicated previously (Hupp et al 1992) .
  • the S-galactosidase activity was measured 12 hours after the cells were exposed to 10 J/m 2 of UV to induce the endogenous wild type p53 and the mot responsive clone was used to carry out further study.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • l ⁇ g/ml G4108 culture medium was removed and the cells were exposed to UV in a XL-1500UV cross-linker (Spectronics Corporation) .
  • the energy of UV light delivered was precisely controlled by the cross-linker. After exposure the cells were grown in fresh culture medium for 16 hours before LacZ expression was determined.
  • Murine p53 ELISA assay Levels of murine p53 were determined in a two site -immunoassay as follows. Falcon microtitre dish wells were coated with 50 ml of purified mouse monoclonal antibody Pab 2487 at 30 mg/ml in carbonate coating buffer for 8 h at 4°C. The capture antibody was then removed and the plates blocked with 200 ml 3% BSA in PBS overnight at 4°C. The plates were washed (1 X PBDS, 2 X 0.1% NP-40 in PBS, l X PBS) and 50 ml of serially two fold diluted cell extract added for 3 hours at 4°C.
  • Cell extracts were prepared by lysis in ice-cold NET buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40) containing 2 mM phenylmethylsulphonyl fluoride, for 30 minutes at 4°C. Debris was removed from the lysate by centrifugation at 14000 rpm in a refrigerated Eppendorf centrifuge. Cell lysates were removed avoiding cross contamination and the plates washed as before. Captured murine p53 was detected using 50 ml of the rabbit anti-p53 serum CM5 diluted 1:1000 in 1% BSA in PBS for 2 h at 4°C.
  • the plates were washed and 50 ml horse radish peroxidase conjugated swine anti- rabbit IgG diluted 1:1000 in 1% BSA in PBS was added for 2 h at 4°C. Following washing, bound p53 was visualized with 50 ml TMB substrated per well. The colour reaction was stopped by the addition of 50 ml 1 M H 2 S0 4 per well and the optical density at 450 nm measured.
  • the murine p53 ELISA assay was standardized by including known quantities of recombinant murine p53 expressed in E. coli and resolubilised from inclusion bodies .
  • a microinjection buffer 100 mM glutamic acid, 140 mM KOH, 1 mM MgS4, pH7.2
  • Peptides used in this assay were purified and obtained from Chiron Mimotopes and contain a biotin group at the N- terminus linked to the amino acid sequence SGSG. Peptides are 15-mers representing the linear amino acid sequence of human or murine p53. DnaK (1.5 ⁇ M) was incubated with 0.05, 0.5 and 5 ⁇ M peptide in reaction buffer (15% glycerol, 25mM HEPES, pH 7.5, 50 mM KCl, 5mM DTT, 0.02% Triton X-100, 10 mM MgCl 2 , ImM ATP) for 30 minutes at 30°C.
  • reaction buffer (15% glycerol, 25mM HEPES, pH 7.5, 50 mM KCl, 5mM DTT, 0.02% Triton X-100, 10 mM MgCl 2 , ImM ATP
  • p53 protein (12ng) was incubated in lO ⁇ l of a buffer (10% glycerol, lOmM MgCl 2 , 20mM HEPES, pH 7.6, 0.ImM ATP, 0.ImM EDTA, 5mM DTT, 0.1% Triton X-100) with casein kinase II from rabbit muscle (Hupp et al, 1993) or recombinant human casein kinase II obtained from Boehringer Mannheim (O.lmU) . Incubations were performed for the indicated times at 30°C and reactions were added to lO ⁇ l of DNA binding " buffer containing radioactive target DNA as described below.
  • DNA binding assay Binding conditions are described in detail elsewhere (Hupp et al, 1992) . Briefly, the DNA binding buffer contained 20% (v/v) glycerol, 50mM KCl, 40mM HEPES (pH 7.5), 0.05mM EDTA, 5mM DTT, 0.1% Triton X-100, lOmM MgCl 2 , and 1. Omg/ml bovine serum albumin.
  • a double-stranded oligonucleotide representing the specific p53 consensus site (El Deiry et al, 1992, Hupp et al,_ 1992) was end-labelled with [ ⁇ 32 - P]ATP and used with a 20-fold excess of supercoiled non- specific, non-labelled competitor DNA (pBluescript II SK+, Stratagene) . Incubations of p53 with DnaK or peptide were performed for 30 minutes at 30°C in lO ⁇ l reaction buffer.
  • UV induced activation of the transcriptional function of p53 does not require an increase in p53 protein levels: support for a model of p53 activation by postranslational modification.
  • a synthetic peptide derived from the C-terminal regulatory domain activates latent p53
  • Peptide C_ (C369-383), which harbors both the protein kinase C site and Pab421 epitope, activates p53 at peptide concentrations of lOO ⁇ M
  • DnaK targets the C-terminal negative regulatory domain at a site within peptide C 3 (data not shown) and (iii) activation of p53 by DnaK can now be divided into two stages; a latent-DnaK-p53 intermediate complex can be stably isolated at 0°C (see below) .
  • Alanine scan of the activating peptide defines specific amino acids required to activate latent forms of p53.
  • Truncation of the activating peptide defines endpoints required for activation of p53.
  • a panel of biotinylated peptides which contain N-terminal or C-terminal deletions of the activating peptide C 7 (a biotinylated version of the activating peptide C_ , Figure 3) , were used to define the end limits required for peptide activation with DnaK at 0°C.
  • a titration of peptide C 7 activates p53 using DnaK with an apparent K- of activation being 9 ⁇ M ( Figure 6) , which is approximately four-fold lower than that obtained with the non-biotinylated peptide C_ in the presence of DnaK.
  • DnaK targets the C-terminal regulatory site of human p53.
  • DnaK can activate latent, wild-type and some mutant forms of p53 protein for sequence-specific DNA binding (Hupp et al, 1993) . Although the mechanism is undefined, it is clear that the tetramerization status of p53 is preserved after activation, indicating that DnaK does not dissociate tetramers into smaller units. DnaK activation of p53 also requires energy in the form of heat, as the reaction cannot proceed at 0°C (see below) . In addition, DnaK partner proteins DnaK and GrpE are not required for this interaction, -reducing the likelihood that substrate targeting and turnover, putatively associated with GrpE and DnaK function, are required for DnaK function in this system.
  • studying the mechanism of p53 interaction with DnaK may not only provide insight into how p53 protein activity is altered by interacting proteins, but it may give insight into the mechanism whereby DnaK interacts with a native protein substrate.
  • a series of synthetic peptides was screened for the ability to interfere with the activation of human p53 DNA binding by DnaK. Reactions were divided into two stages by first incubating p53 and DnaK in the absence or presence of synthetic peptides at 30°C and then assaying for sequence- specific DNA binding at 0°C. Synthetic peptide 379-393 derived from the C-terminus of p53 competed effectively with DnaK (Fig. 9A, lanes 8-10 versus lane 3) . The concentration of DnaK in the reaction was 1.4 ⁇ M and the amount of peptide required for 50% inhibition of DnaK activity in this assay was approximately 0.5 to 1 ⁇ M.
  • DnaK protein targets the C- terminal domain of p53.
  • the requirement for stoichiometric amounts of inhibitory peptide suggest that, although DnaK protein is required in a 50-fold molar excess over p53 protein, inhibition of most of the DnaK is required to block p53 activation.
  • the requirement for high levels of DnaK protein may depend on an altered conformation of DnaK at higher protein concentrations, as in one biochemical system, it is the absolute DnaK protein concentration and not the molar ratio of DnaK to its substrate that dictates chaperone activity. Indeed, the specific activity of HSP70 isoforms may be sensitive to changes in their conformation or oligomeric structure (Blond-Elguindi, 1993) .
  • peptide 379-393 The inhibition by peptide 379-393 is specific for DnaK since PAb421 (lanes 5-7 versus lane 2) and casein kinase II (lanes 11-13 versus lane 4) activation of p53 were not blocked by this peptide.
  • Peptide 379-393 was unable to compete with casein kinase II activation of p53 despite harboring the phosphorylation site for this kinase.
  • Short synthetic peptides containing the casein kinase II phosphorylation site of p53 have only a relatively weak homology to the defined consensus motif of the casein kinase II site and have previously been shown to be ineffective as substrates for this enzyme. Additional structural determinants within the p53 homotetramer may contribute to casein kinase II specificity.
  • Peptide 376- 390 formed a stoichiometric complex with DnaK and exhibited the strongest binding to DnaK.
  • the peptide 379-393 bound weaker to DnaK, as the presence of the last three acidic amino acids in the peptide may reduce the stability of complex formation with DnaK. Therefore, the last few amino acids do not contribute to the DnaK binding site, but rather seem to reduce binding. This is consistent with the fact that DnaK is a more potent activator of latent p53 protein when the final four amino acids are removed (Hupp and Lane, 1995) and that peptide 379-393 has a slightly reduced inhibitory activity towards DnaK in comparison with peptide 374-388 (Fig.
  • Bacterial DnaK belongs to the heat shock protein 70 family and is very homologous to its human members (Table 2) .
  • HSP70 homologues can exhibit quite distinct substrate specificities, peptides that bind to bacterial DnaK with a high affinity contain some similar hydrophobic properties that match the consensus binding sequence for some mammalian heat shock proteins, suggesting the possibility that some conservation of substrate specificity may exist.
  • HSP70 family members studied include Grp79, Hsc70, and Hsp70 (see below) .
  • the constitutively expressed Grp78/BiP is a resident protein of the endoplasmic reticulum (ER) and a member of the glucose-regulated protein family.
  • ER endoplasmic reticulum
  • An affinity panning approach of peptide libraries in bacteriophages has identified an optimal heptameric motif for the molecular chaperone BiP with a high content of aromatic and hydrophobic amino acids.
  • Recombinant hamster Grp78 (BiP) did not show any binding to our C-terminal p53 peptides (Table 2) .
  • Hsc70 is a constitutive member of the Hsp70 family found in the nucleus and cytosol, just like stress-induced Hsp70
  • Human Hsp70 and Hsc70 proteins have been well characterized biochemically with respect to their ability to interact with denatured protein substrates (Freeman et al, 1995) . Although human Hsp70 protein did not bind to the C-terminal p53 peptides (Table 2) , human Hsc70 protein exhibited strong binding to the same peptides as observed with DnaK (figure 11) .
  • Hsc70 protein binding to the C-terminal p53 peptides was less avid than that observed for DnaK, which has been inferred previously, based on the relative ability of a C-terminal p53 peptide to compete with bovine Hsc70 and DnaK protein binding to unfolded lactalbumin.
  • the functional significance of this conserved interaction of DnaK and Hsc70 with the C-terminal p53 peptide is not yet clear.
  • Our data indicate that one interaction site for human Hsc70 protein may reside in the p53 protein C-terminus and that human Hsp70 does not interact similarly.
  • Hsc70 was not able to substitute for DnaK in activating latent human or murine p53 for DNA binding (data not shown) .
  • DnaK binds with a lower affinity to C-terminal peptides derived from murine p53.
  • Murine p53 is 85.8% homologous to human p53 and both proteins include well described functional features like the DNA binding domain in the central region and a C- terminal tetramerization domain (Wang et al, 1994) . Both proteins can activate transcription of target genes containing p53 binding sites (Farmer et al, 1992) and they can both function as transcription factors in yeast
  • Human and murine p53 also form hetero-tetramers when co-translated.
  • the cellular human proteins TBP (TATA-binding protein) and hdm2 (human double minute 2) also bind murine p53.
  • TBP TATA-binding protein
  • hdm2 human double minute 2
  • murine and human p53 have conserved their C-terminal cdc2/cyclin, protein kinase C and casein kinase II phosphorylation sites, there is a striking degree of divergence in the region of human p53 implicated in binding to DnaK protein (figure 15) .
  • the murine p53Ala309 mutant protein was significantly (50-65%) less active than wild-type p53 after incubation with DnaK protein (figure 14) . This indicates a concerted synergistic action between binding of a factor at the C-terminal regulatory domain and phosphorylation at the cdc2 site. In fact, PAb421 was still efficient in activating insect cell-derived murine p53 protein mutated at this site (Ala309) . Identification of a second regulatory domain on p53 whose modification contributes to activation.
  • That activation of the transcriptional activity of p53 can occur without an increase in p53 protein levels is consistent with the existence of distinct cellular signalling pathways capable of modulating the conversion between latent and activated forms of p53 (Hupp and Lane 1995) .
  • the results also explain how microinjection of antibodies to the carboxy-terminus of p53 can block cell cycle progression (Mercer et al 1982; Mercer et al 1984; Deppert et al 1990) , by virtue of activating the biochemical function of endogenous p53 protein.
  • determining the mechanism of p53 activation may have future therapeutic relevance.
  • the stability of the negative regulatory domain interactions are also manifested in the ability to purify to homogeneity latent p53 tetramers using three sequential chromatographic matrices (Hupp and Lane 1994a) ; ie a spontaneous shift in equilibrium from the latent to activated state during purification would have reduced latent tetramer yield if p53 negative regulatory domain- tetramer interactions were of low affinity and subject to destabilisation.
  • Immunoaffinity purification of p53 using a Pab421 antibody-linked matrix utilizes a peptide similar to the activating peptide C x ( Figure 3) to elute p53 from the antibody column (Bargonetti et al 1991) . That specific peptides can synergise with p53-activating factors ( Figures 4-6) , indicates that conclusions based on studies of regulation of p53 by enzymes may- be misleading due to contamination of protein preparations with the peptides.
  • the allosteric model of negative regulation model can also be used to predict that reduction in net basic charge in the vicinity of the carboxy-terminal regulatory site would be destabilising and could displace the negative regulatory domain from its binding site.
  • three other independent experiments support this prediction.
  • site directed mutagenesis producing the substitution of four basic amino acid residues with hydrophobic amino acids from the C-terminal negative regulatory domain has already been shown to strikingly increase the specific activity of p53 as a sequence-specific transcriptional activator in vivo (Tarunina and Jenkins 1993) , presumably by allowing spontaneous activation of p53 during tetramer assembly in cells.
  • peptide-dependent activation provides the opportunity to formulate biochemical models of p53 latency which can be tested experimentally and thus shed new light on the role phosphorylation plays in allosterically altering polypeptide conformation.
  • These studies also hold promise for the design of low molecular weight agents which can also activate mutant forms of p53 defective in allosteric activation by protein kinases, as activation of wild type p53 can now be shown in vivo using a monoclonal antibody. Definition of an Hsc70 binding site on p53.
  • Hsp/c70 proteins immunoprecipitates with a fraction of mutant, but not wild-type p53 protein.
  • overexpression of Hsc70 can suppress focus formation of cell lines induced by mutant p53 protein.
  • the bacterial heat shock protein DnaK is a potent activator of latent human p53 and allosteric-class of mutant p53 proteins in vi tro . Since other activators (like PAb421, protein kinase C, and casein kinase II) also target the extreme C-terminal regulatory domain of p53 and another activator (cdc2/cyclin kinases) targets a site flanking the tetramerization domain, we tried to determine if the site of interaction with DnaK was also in this region.
  • activators like PAb421, protein kinase C, and casein kinase II
  • cdc2/cyclin kinases targets a site flanking the tetramerization domain
  • the peptide KKLMFKT which corresponds to the DnaK binding side on p53, has been shown to compete efficiently with the binding of reduced and carboxymethylated lactalbumin (RCMLA) to DnaK. Indeed, by changing the second amino acid to a tyrosine (KYLMFKT) the binding of bovine Hsc70, which only bound moderately to the original peptide, changes dramatically to reach the same degree as binding to DnaK. In murine p53 the sequence is changed in that particular region to KKTMVKKV, which reduces the affinity of DnaK to the target sequence. Thus, the exchange of three hydrophobic residues and the introduction of a positive C- terminal charge reduces the affinity, but do not abolish critical amino acids for DnaK binding.
  • human and murine p53 are very similar on a structural level, they may respond differently to post- translational modifications that target the C-terminus.
  • the carboxy-terminal serine 392 site of human p53 which is phosphorylated by casein kinase II in vivo and in vi tro, does not contribute to wild-type human p53's biochemical and biological growth suppressor activity.
  • a mutation of the casein kinase II site of murine p53 inactivates its ability to suppress growth in rodent cell lines.
  • the activation of murine p53 from insect cells by DnaK represents a new model system, which helps to describe the mechanism of activation for DNA binding of murine p53.
  • the activation of murine p53 by DnaK appears to be similar to the model proposed previously for activation of human p53 by phosphorylation, in which phosphorylation of latent human p53 can "prime" the protein and convert it to a form that can be activated by weak activators, like peptides.
  • DnaK is a weak activator of unphosphorylated, murine p53, due to differences in amino acids of the binding region reflected in its reduced affinity for murine peptides.
  • Phosphorylation of murine p53 in insect cells may prime for a more efficient activation by DnaK.
  • Wang and Prives (1995) have shown that phosphorylation by cyclin B/cdc2 and cyclin A/cdk2 complexes can lead to increased DNA binding by human and murine p53.
  • Mutation of serine 309 to Alanine precludes cdc2 phosphorylation of murine p53 in vivo and reduces the activation by DnaK by approximately 65%.
  • PAb421 a strong activator in this system, to activate for DNA binding of Ala309 is only reduced by 15-30%.
  • Hupp T R Meek D W, Midgley C A and Lane D P (1992) . Regulation of the Specific DNA Binding Function of p53. Cell 71, 875-886. Hupp T R, Meek DM, Midgley C A, and Lane D P (1993) . Activation of the cryptic DNA binding Function of Mutant Forms of p53. Nucl . Acids Res. 21, 3167-3174.
  • Wild type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA 89, 7491-7495.
  • UV irradiation stimulates levels of p53 cellular tumour antigen in nontransformed mouse cells. Mol. Cell. Biol. 4, 1689-94.
  • Himan wild-type p53 adopts a unique conformational and phosphorylation state invivo during growth arrest of glioblastoma cells. Oncogene 7, 1635-1643.

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Abstract

L'invention concerne des substances ayant la propriété d'activer la liaison de l'ADN spécifique de séquence de la protéine p53 latente, lesdites substances étant basées sur des fragments du domaine régulateur C-terminal de la p53 ou sur des fragments de la p53 murine comportant l'épitope lié par l'anticorps Pab241 (ou la région correspondante de la p53 humaine). Il a été démontré que ces substances activent la p53 latente et agissent en synergie avec les activateurs connus de la p53 tels que la DnaK, l'anticorps monoclonal Pab421 et les kinases. L'invention concerne également l'utilisation de ces substances à des fins thérapeutiques, ainsi que pour la sélection de mimétiques et de partenaires de liaison. Elle concerne enfin un motif de la p53 qui entre en interaction avec la protéine de choc thermique DnaK.
EP96935079A 1995-10-20 1996-10-21 ACTIVATION DE LA PROTEINE p53 Withdrawn EP0859839A1 (fr)

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EP0989136A4 (fr) * 1997-05-15 2002-10-09 Kyowa Hakko Kogyo Kk Peptides a structures cycliques et a effet restaurateur d'activite de la proteine p53 sur les mutants de ladite proteine
JP2001509396A (ja) * 1997-07-10 2001-07-24 メディカル リサーチ カウンシル シャペロン断片
DE19735221C1 (de) 1997-08-14 1999-05-06 Wolfgang Willi Prof Dr Deppert Beeinflussung der Bindung von p53 an Zielgene
DE19802792A1 (de) * 1998-01-26 1999-07-29 Wolfgang Willi Prof Dr Deppert Hemmung der durch mutiertes p53 verursachten Modulation von DNA
GB9810040D0 (en) 1998-05-11 1998-07-08 Univ Nottingham Blood borne tumour markers
WO2000022115A2 (fr) * 1998-10-13 2000-04-20 Board Of Regents, The University Of Texas System Dosages permettant d'identifier des alterations fonctionnelles dans le gene suppresseur de tumeur p53
GB9827228D0 (en) 1998-12-10 1999-02-03 Univ Nottingham Cancer detection method and reagents
AU2003202689A1 (en) * 2002-01-23 2003-09-09 El-Gewely, Mohamed, Raafat Methods of screening molecular libraries for active molecules that restore the function of target proteins and peptides that restore the function of mutant p53
GB2395270B (en) 2002-11-14 2006-08-16 Univ Nottingham Tumour marker proteins and uses thereof
GB2426581A (en) 2005-05-27 2006-11-29 Univ Nottingham Immunoassay methods
SI1889059T1 (sl) 2005-05-27 2009-12-31 Oncimmune Ltd Izboljšani imunotestni postopek
CN101632020B (zh) 2006-09-13 2013-11-27 昂西免疫有限公司 改进的免疫测定方法
DK2118123T3 (en) 2007-01-31 2016-01-25 Dana Farber Cancer Inst Inc Stabilized p53 peptides and uses thereof
US8592377B2 (en) 2007-03-28 2013-11-26 President And Fellows Of Harvard College Stitched polypeptides
ES2711526T3 (es) 2010-08-13 2019-05-06 Aileron Therapeutics Inc Macrociclos peptidomiméticos
TW201806968A (zh) 2011-10-18 2018-03-01 艾利倫治療公司 擬肽巨環化合物
EP2819688A4 (fr) 2012-02-15 2015-10-28 Aileron Therapeutics Inc Macrocycles peptidomimétiques réticulés par triazole et par thioéther
BR112014020103A2 (pt) 2012-02-15 2018-10-09 Aileron Therapeutics, Inc. macrociclos peptidomiméticos
WO2014071241A1 (fr) 2012-11-01 2014-05-08 Aileron Therapeutics, Inc. Acides aminés disubstitués et procédés de préparation et d'utilisation de ceux-ci
AU2015320545C1 (en) 2014-09-24 2020-05-14 Aileron Therapeutics, Inc. Peptidomimetic macrocycles and formulations thereof
EP3197478A4 (fr) 2014-09-24 2018-05-30 Aileron Therapeutics, Inc. Macrocycles peptidomimétiques et leurs utilisations
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