EP0942986A1 - Systems for determining substances active against hpv-associated diseases - Google Patents
Systems for determining substances active against hpv-associated diseasesInfo
- Publication number
- EP0942986A1 EP0942986A1 EP97952025A EP97952025A EP0942986A1 EP 0942986 A1 EP0942986 A1 EP 0942986A1 EP 97952025 A EP97952025 A EP 97952025A EP 97952025 A EP97952025 A EP 97952025A EP 0942986 A1 EP0942986 A1 EP 0942986A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- cell
- cell regulator
- dnas
- substances
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This invention relates to systems which are suitable for the determination of substances active against HPV-associated diseases, particularly genital wards or carcinomas, and to the use of such systems.
- HPVs human papillo- ma viruses
- the underlying object of the present invention is thus to provide a means with which substances active against HPV-associated diseases, particularly genital wards or carcinomas, can be found.
- the present invention thus relates to a system comprising an E7 protein of HPV and a cellular regulator protein bound by the E7 protein.
- the present invention is based on the recognition by the Applicant that an E7 protein of HPV is capable of binding cellular regulator proteins (hereinafter called cell regulator proteins) and can thus intervene in cell regulation.
- cell regulator proteins cellular regulator proteins
- the cell cycle regulator p27KIP1 and the glycolysis control enzyme M2PK are examples of cell regulator proteins such as these.
- E7 protein of HPV comprises an E7 protein of any HPV type, particularly of HPV1 , 5, 6, 1 1 , 1 6 or 1 8.
- An E7 protein may comprise a wild type sequence or a sequence which differs therefrom by one or more amino acids.
- a wild type sequence of an E7 protein from HPV1 6 is given by Seedorf, K., et al., in Virology 145, ( 1 985), 1 81 -5, for example.
- E7 proteins from HPV 1 , 5, 6, 1 1 or 18 are given by the following GeneBank Accession numbers: PAPAPI (HPV1 ) No: V01 1 1 6, PPH5CG (HPV5) No: M 1 7463, PAPA6B (HPV6) No: X00203, PPH 1 1 (HPV1 1 ) No: M 141 1 9, or PAPHPV1 8 (HPV1 8) No: X0501 5.
- an E7 protein may exist in a shortened form, i.e. it may be present only in the form of that fragment which is necessary for binding to a cell regulator protein.
- Such a fragment may be a C- terminal fragment of E7 protein, particularly a fragment comprising amino acids 39-98 of E7 protein.
- the protuberant fragment can also exist in multiple copies within a polypeptide molecule.
- an E7 protein or a protuberant fragment thereof may exist in the form of a fusion protein. In a fusion protein such as this, for example, the E7 protein or a protuberant fragment thereof may be combined with a polypeptide which allows binding to a solid support.
- Such a polypeptide is a His-tag sequence, a glutathione S-transferase (GST) fraction, a sequence tag derived from SV40 T-antigen, a DNA binding domain (DBD) of the bacterial LexA repressor or a trans activation domain (TAD) of the bacterial protein B42, for example. It may be advantageous if the E7 protein or the protuberant fragment thereof is combined with a DNA binding domain (DBD) of the bacterial LexA repressor.
- cell regulator protein comprises any regulator protein of a cell, particularly a cell derived from mammals, e.g. human, mouse or rat, which can be bound by an E7 protein of HPV.
- the cell cycle regulator p27K1 P1 see Polyak, K. et al., Cell 78, ( 1 994), 59-66
- the glycolysis control enzyme M2PK see Tani, K. et al., Gene 73 ( 1988), 509-1 6) are examples of cell regulator proteins such as these.
- a cell regulator protein may comprise a wild type sequence or a sequence which differs therefrom by one or more amino acids.
- a wild type sequence of p27KIP1 is given, for example, by Polyak, K., et al. (see above).
- a cell regulator protein may exist in shortened form, i.e. it may be present only in the form of that fragment which is necessary for binding to an E7 protein of HPV. This fragment can also exist in multiple copies within a polypeptide molecule.
- a cell regulator protein or the above fragment thereof may exist in the form of a fusion protein. In a fusion protein such as this, for example, the cell regulator protein or the above fragment thereof may be combined with a polypeptide which allows binding to a solid support.
- Such a polypeptide is a His-tag sequence, a glutathione S-transferase (GST) fraction, a sequence tag derived from SV40 T-antigen, a DNA binding domain (DBD) of the bacterial LexA repressor or a trans activation domain (TAD) of the bacterial protein B42, for example. It may be advantageous if the cell regulator protein or the above fragment thereof is combined with a trans activation domain (TAD) of the bacterial protein B42.
- the above system is suitable for the determination of substances which intervene in binding of an E7 protein to a cell regulator protein.
- the above system is usable for the determination of substances active against HPV- associated diseases, especially genital wards or carcinomas.
- the preparation of the above (fusion) proteins can be effected by customary methods.
- a recombinant preparation is selected, for example, it is advantageous to insert the DNA which codes for the (fusion) protein in an expression vector and then to use the latter for the transfection or transformation of host cells. It may be advantageous if the expression vector contains DNA which codes for the E7 protein and the cell regulator protein as mentioned above. According to the invention, a DNA is preferred which codes for the fusion protein B42 (TAD)- p27KIP1 .
- TAD fusion protein B42
- E. coii these are pGEMEX, pUC derivatives, pGEM-T, pGEX-2T, pet3b and pQE-8, for example, pY100, Ycpad l and pREP-L20 can be cited for expression in yeast, for example, pKCR, pEFBOS, cDM8 and pCEV4 can be cited for expression in animal cells, for example, whilst the baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in insect cells.
- host cells such as these comprise the E. coli strains HB1 01 , DH 1 , x 1 776, JM 1 01 , JM 1 09, BL21 and SG 1 3009, the yeast strains Saccharomyces cerevisiae and Saccharomyces pombe and the animal cells L, NIH-3T3, FM3A, CHO, COS, Vero, Hela, U2-0S and C33A, and also the insect cells sf9.
- E. coli strains HB1 01 , DH 1 , x 1 776, JM 1 01 , JM 1 09, BL21 and SG 1 3009 the yeast strains Saccharomyces cerevisiae and Saccharomyces pombe and the animal cells L, NIH-3T3, FM3A, CHO, COS, Vero, Hela, U2-0S and C33A, and also the insect cells sf9.
- the above system can be used in the usual manner for the in vitro determination of substances which intervene in binding of an E7 protein to a cell regulator protein.
- the above system is usable for the invitro determination of substances active against HPV-associated diseases, especially genital wards or carcinomas.
- an E7 protein in the form of a fusion protein e.g. as a (GST) E7 protein
- GST a cell regulator protein
- p27KIP1 can be added in the form of a fusion protein, e.g. as His-p27KIP1 . After a washing procedure, it can be determined using a labelled antibody directed against p27KIP1 whether binding has occurred between the E7 protein and the p27KIP1 cell regulator protein or whether this has been prevented by one or more substance. In this manner substances are determined to intervene in binding of an E7 protein to a cell regulator protein. These substances can be active against HPV-associated diseases, especially genital wards or carcinomas.
- a system is also provided which is suitable for the in vivo determination of substances which intervene in binding of an E7 protein to a cell regulator protein.
- the above system is usable for the in vivo determination of substances active against HPV-associated diseases, especially genital wards or carcinomas.
- a system such as this preferably comprises an E7 protein in the form of a lexA (DBD) E7 protein fusion protein and a cell regulator protein in the form of a B42 (TAD) cell regulator protein fusion protein, wherein the fusion proteins are present in the form of DNAs which are expressed together with a DNA coding for a reporter protein, and wherein the expression of the reporter protein-DNA is under the control of a LexA (DBD)-dependent promotor.
- DBD lexA
- TAD B42
- DNAs are expressed indicates that the DNAs which code for the said fusion proteins and the reporter protein can be expressed jointly in one cell.
- One skilled in the art is aware of expression vectors, host cells and methods for an expression such as this. Reference is made to the above statements in this respect.
- reporter protein comprises any reporter protein, the expression of which may be under the control of a lexA (DBD)-dependent promotor.
- LacZ, "green fluorescent protein” or luciferase are examples of a reporter protein such as this.
- the fusion proteins Due to the expression of the DNAs which code for the said fusion proteins, i.e. due to the provision of the fusion proteins comprising the lexA (DBD) E7 protein and B42 (TAD) cell regulator protein, these fusion proteins can bind to each other, and can bind via lexA (DBD) to the lexA (DBD)-dependent promoter of the DNA which codes for the reporter protein and can activate this promoter.
- B42 (TAD) of the B42 (TAD)-cell regulator protein-fusion protein is particularly important for the latter.
- one to all of the said DNAs are present in one cell in the above system for the in vivo determination of substances as mentioned above. It is advantageous if this presence is a stable one.
- the present invention also relates to cells which contain one to all of these DNAs. One of these cells which contains all the said DNAs in stable form was deposited as 1 6 E7/p27 at the DSMZ on November 26th 1 996, under DSM 1 1 308.
- DSM 1 1 308 expresses, in stable form, the fusion proteins lexA (DBD) E7 protein and B42 (TAD)-p27KIP1 , as well as the reporter protein lac Z.
- DBD fusion proteins lexA
- B42 B42
- p27KIP1 the reporter protein lac Z.
- the latter expression is indicated by the detection of ⁇ -galactosidase activity.
- a reduced ⁇ -galactosidase activity is measured, or ⁇ -galactosidase activity can even no longer be measured at all. In this manner, substances as mentioned above are determined.
- the above system can be present in the form of a kit.
- This kit comprises the E7 protein and the cell regulator protein as mentioned above, and optionally other components, e.g. a support as mentioned above.
- the present invention it is possible to determine substances which intervene in binding of an E7 protein to a cell regulator protein.
- the present invention is usable for the determination of substances active against HPV-associated diseases, especially genital wards or carcinomas. This can be effected in a rapid and simple manner.
- the determination can be made both in vitro and in vivo.
- Mutual confirmation or verification of the data obtained is thus possible.
- the present invention thus constitutes a considerable step in the search for possible therapies for HPV-associated diseases, particularly genital wards or carcinomas.
- the present invention is illustrated by the following examples.
- Example 1 In vitro identification of substances intervening the binding of E7 protein to a cell regulator protein
- a GST-E7 fusion protein is expressed in E. coli.
- an expression vector is used coding for glutathione-S-transferase (GST)tagged full length HPV-1 6 E7 protein.
- GST glutathione-S-transferase
- Such an expression vector is described by Ciccolini, F. et al., Oncogene 9, ( 1 994), 2342-2348.
- the expression vector is used for the transformation of E.coli strain ⁇ DH5 and the expressed fusion protein is purified as follows: After an induction of 4 hours with 1 mM IPTG at 30°C, bacteria are pelleted, washed once in cold PBS, and sonicated in lysis buffer ( 1 x PBS, 0, 1 % Triton-X-100, 1 mM DTT, 0,2 mM PMSF, 1 mM NaF) in the presence of glass beads. Lysates are centrifuged at 5000 rpm in a SS34 rotor and clarified supernatants are then incubated for 30 min at room temperature with glutathione Sepharose 4B beads (GSH-beads) preequilibrated with lysis buffer.
- lysis buffer 1 x PBS, 0, 1 % Triton-X-100, 1 mM DTT, 0,2 mM PMSF, 1 mM NaF
- GSH-beads-coupled GST-E7 fusion protein is obtained. After washing with lysis buffer (five times 10 bed volumes) GST-E7 fusion protein is eluted from the GSH-beads using 50 mM Tris-HCI (pH 8.0), 1 0 mM red. glutathione. The GST-E7 fusion protein is adsorbed to 96-well plates by overnight incubation at a protein concentration of 5ng/ ⁇ l in 0.1 M carbonate buffer (pH 9.5).
- a His 6 -tag M2PK-luciferase fusion protein is expressed in E. coli.
- human M2PK cDNA Teani, K. et al., Gene 73, ( 1 988), 509 - 1 6) tagged at the N-termi- nus with a stretch of 6 histidines is fused in frame with full length luciferase cDNA of P. pyralis (de Wet, J. et al., Mol. Cell. Biol.7 ( 1 987), 725-737).
- the recombinant DNA molecule obtained is used for the transformation of E. coli strain BL21 .
- the expressed fusion protein is purified by Ni 2 + agarose chromato- graphy (Zerank-Thome, K. et al., Oncogene 1 3, ( 1 996), 2323-2330).
- the His 6 -tag M2PK-luciferase fusion protein ( 1 00 ng/ml in PBS; pH 7.2) is added to the GST-E7 fusion protein coated 96-well plates. The plates are washed with PBS (PH 7.2) and the retention of the M2PK-luciferase protein using a lumino- meter equipped for 96 well plates is measured.
- the luciferase signal obtained for a standard incubation due to the tight interaction of E7 protein with M2PK, will be set 100 %.
- Substances from a substance bank are added individually to the standard incubation and the luciferase signal retained in the plates is measured.
- Substances which reduce the luciferase signal by at least 90 % are considered as primary hits. These substances are included in a second screening procedure.
- Example 2 In vivo identification of substances intervening the binding of E7 protein to a cell regulator protein
- U2-0S cells (Beijersbergen, R. et al., Genes Dev. 9, ( 1 995), 1 340-1 353) are transfected with expression vectors.
- a first vector codes for a fusion protein comprising a DNA binding domain of Lex A and the C terminal part of E7 protein (amino acids 39-98) .
- a second tetracyclin inducable expression vector codes for a fusion protein comprising full length human p27 (KIP1 ) and a B42 transactiva- tion domain.
- a third vector codes for E. coli ⁇ -galactosidase under the control of a LexA-inducible synthetic promoter (Zwerschke, W.
- the transformed cells obtained do not produce any significant ⁇ -galactosidase activity in the uninduced state, but high ⁇ -galactosidase activity upon induction of expression of the LexA-E7 fusion protein, obtained through withdrawal of tetracyclin.
- the cells are lysed and ⁇ -galactosidase activity is measured by a standard colorimetric assay, (Zwerschke, W. et al. see above).
- the induced value for ⁇ -galactosidase activity is set 100 %.
- the cells are grown in 96 well plates.
- Substances from a substance bank are added individually to the standard incubation and the ⁇ -galactosidase signal retained in the plates is measured. Substances which reduce the ⁇ -galactosidase signal by at least 90 % are considered as primary hits. These substances are included in a second screening procedure.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to a system comprising an E7 protein of HPV and a cell regulator protein bound by the E7 protein. Furthermore, this invention relates to a vector and a cell as well as the use of the aforementioned subject matters for the determination of substances active against HPV-associated diseases, particularly genital warts or carcinomas.
Description
Systems for determining substances active against HPV-associated diseases
This invention relates to systems which are suitable for the determination of substances active against HPV-associated diseases, particularly genital wards or carcinomas, and to the use of such systems.
It is known that many humans suffer persistent infections due to human papillo- ma viruses (hereinafter called HPVs) . It is also known that more than 95 % of all ano-genital carcinomas, particularly cervical cancer and a considerable percentage of carcinomas of the mouth/pharyngeal cavity are associated with persistent infections due to HPVs. Furthermore, there are indications that for the occurrence and/or manifestation of carcinomas in cells which exhibit persistent infection by HPVs, an uncontrolled expression of HPV genes, particularly of E7, is necessary.
Many investigations have been undertaken aimed at finding substances active against HPV associated carcinomas. However, these investigations have hitherto not been successful.
The underlying object of the present invention is thus to provide a means with which substances active against HPV-associated diseases, particularly genital wards or carcinomas, can be found.
This is achieved according to the invention by the subject matter in the claims.
The present invention thus relates to a system comprising an E7 protein of HPV and a cellular regulator protein bound by the E7 protein.
The present invention is based on the recognition by the Applicant that an E7 protein of HPV is capable of binding cellular regulator proteins (hereinafter called cell regulator proteins) and can thus intervene in cell regulation. The cell cycle regulator p27KIP1 and the glycolysis control enzyme M2PK are examples of cell
regulator proteins such as these.
The term "E7 protein of HPV" comprises an E7 protein of any HPV type, particularly of HPV1 , 5, 6, 1 1 , 1 6 or 1 8. An E7 protein may comprise a wild type sequence or a sequence which differs therefrom by one or more amino acids. A wild type sequence of an E7 protein from HPV1 6 is given by Seedorf, K., et al., in Virology 145, ( 1 985), 1 81 -5, for example. Furthermore, wild type sequences of E7 proteins from HPV 1 , 5, 6, 1 1 or 18 are given by the following GeneBank Accession numbers: PAPAPI (HPV1 ) No: V01 1 1 6, PPH5CG (HPV5) No: M 1 7463, PAPA6B (HPV6) No: X00203, PPH 1 1 (HPV1 1 ) No: M 141 1 9, or PAPHPV1 8 (HPV1 8) No: X0501 5. Moreover, an E7 protein may exist in a shortened form, i.e. it may be present only in the form of that fragment which is necessary for binding to a cell regulator protein. Such a fragment may be a C- terminal fragment of E7 protein, particularly a fragment comprising amino acids 39-98 of E7 protein. The protuberant fragment can also exist in multiple copies within a polypeptide molecule. Furthermore, an E7 protein or a protuberant fragment thereof may exist in the form of a fusion protein. In a fusion protein such as this, for example, the E7 protein or a protuberant fragment thereof may be combined with a polypeptide which allows binding to a solid support. Such a polypeptide is a His-tag sequence, a glutathione S-transferase (GST) fraction, a sequence tag derived from SV40 T-antigen, a DNA binding domain (DBD) of the bacterial LexA repressor or a trans activation domain (TAD) of the bacterial protein B42, for example. It may be advantageous if the E7 protein or the protuberant fragment thereof is combined with a DNA binding domain (DBD) of the bacterial LexA repressor.
The term "cell regulator protein" comprises any regulator protein of a cell, particularly a cell derived from mammals, e.g. human, mouse or rat, which can be bound by an E7 protein of HPV. The cell cycle regulator p27K1 P1 (see Polyak, K. et al., Cell 78, ( 1 994), 59-66) and the glycolysis control enzyme M2PK (see Tani, K. et al., Gene 73 ( 1988), 509-1 6) are examples of cell regulator proteins such as these. A cell regulator protein may comprise a wild type
sequence or a sequence which differs therefrom by one or more amino acids. A wild type sequence of p27KIP1 is given, for example, by Polyak, K., et al. (see above). In addition, a cell regulator protein may exist in shortened form, i.e. it may be present only in the form of that fragment which is necessary for binding to an E7 protein of HPV. This fragment can also exist in multiple copies within a polypeptide molecule. Furthermore, a cell regulator protein or the above fragment thereof may exist in the form of a fusion protein. In a fusion protein such as this, for example, the cell regulator protein or the above fragment thereof may be combined with a polypeptide which allows binding to a solid support. Such a polypeptide is a His-tag sequence, a glutathione S-transferase (GST) fraction, a sequence tag derived from SV40 T-antigen, a DNA binding domain (DBD) of the bacterial LexA repressor or a trans activation domain (TAD) of the bacterial protein B42, for example. It may be advantageous if the cell regulator protein or the above fragment thereof is combined with a trans activation domain (TAD) of the bacterial protein B42.
The above system is suitable for the determination of substances which intervene in binding of an E7 protein to a cell regulator protein. Particularly, the above system is usable for the determination of substances active against HPV- associated diseases, especially genital wards or carcinomas. The preparation of the above (fusion) proteins can be effected by customary methods.
If a recombinant preparation is selected, for example, it is advantageous to insert the DNA which codes for the (fusion) protein in an expression vector and then to use the latter for the transfection or transformation of host cells. It may be advantageous if the expression vector contains DNA which codes for the E7 protein and the cell regulator protein as mentioned above. According to the invention, a DNA is preferred which codes for the fusion protein B42 (TAD)- p27KIP1 . A DNA such as this was deposited as pB42-p27::TRP1 at the DSMZ (German Collection of Microorganisms) on Nov. 26th, 1 996, under DSM 1 1 307.
For the expression of the above DNA, one skilled in the art falls back on known
expression vectors. For expression in E. coii these are pGEMEX, pUC derivatives, pGEM-T, pGEX-2T, pet3b and pQE-8, for example, pY100, Ycpad l and pREP-L20 can be cited for expression in yeast, for example, pKCR, pEFBOS, cDM8 and pCEV4 can be cited for expression in animal cells, for example, whilst the baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in insect cells.
One skilled in the art is also aware of host cells. Examples of host cells such as these comprise the E. coli strains HB1 01 , DH 1 , x 1 776, JM 1 01 , JM 1 09, BL21 and SG 1 3009, the yeast strains Saccharomyces cerevisiae and Saccharomyces pombe and the animal cells L, NIH-3T3, FM3A, CHO, COS, Vero, Hela, U2-0S and C33A, and also the insect cells sf9.
Furthermore, one skilled in the art is aware of conditions under which host cells can be transformed or transfected with the expression vectors and under which the host cells can then be cultivated. Methods of isolating and purifying an expressed fusion protein are also known.
The above system can be used in the usual manner for the in vitro determination of substances which intervene in binding of an E7 protein to a cell regulator protein. Particularly, the above system is usable for the invitro determination of substances active against HPV-associated diseases, especially genital wards or carcinomas. For example, an E7 protein in the form of a fusion protein, e.g. as a (GST) E7 protein, can be fixed to a support, e.g. to a microtitration plate, to a small tube, to microspheres, or to a microscope slide. The substances to be tested can then be added to the support. Simultaneously or thereafter, a cell regulator protein e.g. p27KIP1 , can be added in the form of a fusion protein, e.g. as His-p27KIP1 . After a washing procedure, it can be determined using a labelled antibody directed against p27KIP1 whether binding has occurred between the E7 protein and the p27KIP1 cell regulator protein or whether this has been prevented by one or more substance. In this manner substances are determined to intervene in binding of an E7 protein to a cell regulator protein.
These substances can be active against HPV-associated diseases, especially genital wards or carcinomas.
According to the invention, a system is also provided which is suitable for the in vivo determination of substances which intervene in binding of an E7 protein to a cell regulator protein. Particularly the above system is usable for the in vivo determination of substances active against HPV-associated diseases, especially genital wards or carcinomas. A system such as this preferably comprises an E7 protein in the form of a lexA (DBD) E7 protein fusion protein and a cell regulator protein in the form of a B42 (TAD) cell regulator protein fusion protein, wherein the fusion proteins are present in the form of DNAs which are expressed together with a DNA coding for a reporter protein, and wherein the expression of the reporter protein-DNA is under the control of a LexA (DBD)-dependent promotor.
The term "DNAs are expressed" indicates that the DNAs which code for the said fusion proteins and the reporter protein can be expressed jointly in one cell. One skilled in the art is aware of expression vectors, host cells and methods for an expression such as this. Reference is made to the above statements in this respect.
The term "reporter protein" comprises any reporter protein, the expression of which may be under the control of a lexA (DBD)-dependent promotor. LacZ, "green fluorescent protein" or luciferase are examples of a reporter protein such as this.
Due to the expression of the DNAs which code for the said fusion proteins, i.e. due to the provision of the fusion proteins comprising the lexA (DBD) E7 protein and B42 (TAD) cell regulator protein, these fusion proteins can bind to each other, and can bind via lexA (DBD) to the lexA (DBD)-dependent promoter of the DNA which codes for the reporter protein and can activate this promoter. B42 (TAD) of the B42 (TAD)-cell regulator protein-fusion protein is particularly important for the latter.
In a preferred embodiment, one to all of the said DNAs are present in one cell in the above system for the in vivo determination of substances as mentioned above. It is advantageous if this presence is a stable one. The present invention also relates to cells which contain one to all of these DNAs. One of these cells which contains all the said DNAs in stable form was deposited as 1 6 E7/p27 at the DSMZ on November 26th 1 996, under DSM 1 1 308.
The application of the above system for the in vivo determination of substances as mentioned above will be described with reference to the cell line DSM 1 1 308 by way of example. This cell line expresses, in stable form, the fusion proteins lexA (DBD) E7 protein and B42 (TAD)-p27KIP1 , as well as the reporter protein lac Z. The latter expression is indicated by the detection of β-galactosidase activity. After the addition of substances which prevent binding of an E7 protein to the cell regulator protein p27KIP1 , a reduced β-galactosidase activity is measured, or β-galactosidase activity can even no longer be measured at all. In this manner, substances as mentioned above are determined.
The above system can be present in the form of a kit. This kit comprises the E7 protein and the cell regulator protein as mentioned above, and optionally other components, e.g. a support as mentioned above.
With the present invention, it is possible to determine substances which intervene in binding of an E7 protein to a cell regulator protein. Particularly, the present invention is usable for the determination of substances active against HPV-associated diseases, especially genital wards or carcinomas. This can be effected in a rapid and simple manner. In particular, it is very advantageous that the determination can be made both in vitro and in vivo. Mutual confirmation or verification of the data obtained is thus possible. This provides reliable information on the effectiveness of substances or combinations of substances. The present invention thus constitutes a considerable step in the search for possible therapies for HPV-associated diseases, particularly genital wards or carcinomas.
The present invention is illustrated by the following examples.
Example 1 : In vitro identification of substances intervening the binding of E7 protein to a cell regulator protein
A GST-E7 fusion protein is expressed in E. coli. For this an expression vector is used coding for glutathione-S-transferase (GST)tagged full length HPV-1 6 E7 protein. Such an expression vector is described by Ciccolini, F. et al., Oncogene 9, ( 1 994), 2342-2348. The expression vector is used for the transformation of E.coli strain σDH5 and the expressed fusion protein is purified as follows: After an induction of 4 hours with 1 mM IPTG at 30°C, bacteria are pelleted, washed once in cold PBS, and sonicated in lysis buffer ( 1 x PBS, 0, 1 % Triton-X-100, 1 mM DTT, 0,2 mM PMSF, 1 mM NaF) in the presence of glass beads. Lysates are centrifuged at 5000 rpm in a SS34 rotor and clarified supernatants are then incubated for 30 min at room temperature with glutathione Sepharose 4B beads (GSH-beads) preequilibrated with lysis buffer. GSH-beads-coupled GST-E7 fusion protein is obtained. After washing with lysis buffer (five times 10 bed volumes) GST-E7 fusion protein is eluted from the GSH-beads using 50 mM Tris-HCI (pH 8.0), 1 0 mM red. glutathione. The GST-E7 fusion protein is adsorbed to 96-well plates by overnight incubation at a protein concentration of 5ng/μl in 0.1 M carbonate buffer (pH 9.5).
A His6-tag M2PK-luciferase fusion protein is expressed in E. coli. For this human M2PK cDNA (Tani, K. et al., Gene 73, ( 1 988), 509 - 1 6) tagged at the N-termi- nus with a stretch of 6 histidines is fused in frame with full length luciferase cDNA of P. pyralis (de Wet, J. et al., Mol. Cell. Biol.7 ( 1 987), 725-737). The recombinant DNA molecule obtained is used for the transformation of E. coli strain BL21 . The expressed fusion protein is purified by Ni2 + agarose chromato- graphy (Zerfass-Thome, K. et al., Oncogene 1 3, ( 1 996), 2323-2330).
The His6-tag M2PK-luciferase fusion protein ( 1 00 ng/ml in PBS; pH 7.2) is added to the GST-E7 fusion protein coated 96-well plates. The plates are washed with
PBS (PH 7.2) and the retention of the M2PK-luciferase protein using a lumino- meter equipped for 96 well plates is measured.
The luciferase signal obtained for a standard incubation, due to the tight interaction of E7 protein with M2PK, will be set 100 %. Substances from a substance bank are added individually to the standard incubation and the luciferase signal retained in the plates is measured. Substances which reduce the luciferase signal by at least 90 % are considered as primary hits. These substances are included in a second screening procedure.
Example 2: In vivo identification of substances intervening the binding of E7 protein to a cell regulator protein
U2-0S cells (Beijersbergen, R. et al., Genes Dev. 9, ( 1 995), 1 340-1 353) are transfected with expression vectors. A first vector codes for a fusion protein comprising a DNA binding domain of Lex A and the C terminal part of E7 protein (amino acids 39-98) . A second tetracyclin inducable expression vector codes for a fusion protein comprising full length human p27 (KIP1 ) and a B42 transactiva- tion domain. A third vector codes for E. coli β-galactosidase under the control of a LexA-inducible synthetic promoter (Zwerschke, W. et al., Oncogene 1 2,- ( 1 996), 21 3-220). The transformed cells obtained do not produce any significant β-galactosidase activity in the uninduced state, but high β-galactosidase activity upon induction of expression of the LexA-E7 fusion protein, obtained through withdrawal of tetracyclin. The cells are lysed and β-galactosidase activity is measured by a standard colorimetric assay, (Zwerschke, W. et al. see above). The induced value for β-galactosidase activity is set 100 %. The cells are grown in 96 well plates. Substances from a substance bank are added individually to the standard incubation and the β-galactosidase signal retained in the plates is measured. Substances which reduce the β-galactosidase signal by at least 90 % are considered as primary hits. These substances are included in a second screening procedure.
Claims
1 . A system comprising an E7 protein of HPV and a cell regulator protein bound by the E7 protein.
2. A system according to claim 1 , wherein the E7 protein is derived from HPV1 , 5, 6, 1 1 , 1 6 or 1 8.
3. A system according to claim 1 or 2, wherein the cell regulator protein is the cell cycle regulator p27KIP1 .
4. A system according to claim 1 or 2, wherein the cell regulator protein is the glycolysis control enzyme M2PK.
5. A system according to any one of claims 1 -4, wherein the E7 protein and/or the cell regulator protein are present in the form of fusion proteins.
6. A system according to claim 5, wherein the E7 protein is present in the form of a LexA (DBD) E7 protein fusion protein and the cell regulator protein is present in the form of a B42 (TAD)-cell regulator protein-fusion protein.
7. A system according to claim 6, wherein the fusion proteins are present in the form of DNAs which are expressed together with a DNA coding for a reporter protein, wherein the expression of the reporter protein-DNA is under the control of a LexA (DBD) dependent promoter.
8. A system according to claim 7, wherein one to all of the said DNAs are present in one cell.
9. A system according to claim 8, wherein a cell containing all of the said DNAs was deposited on November 26, 1 996 under DSM 1 1 308.
10. A vector, comprising DNAs coding for an E7 protein of HPV and a cell regulator protein bound by the E7 protein.
1 1 . A cell, comprising one to all of the DNAs coding for a HPV E7 protein, a cell regulator protein and a reporter protein, wherein the expression of the DNAs is under the control of promoters being heteroiogous to the DNAs, and wherein the expression of the reporter protein-DNA is dependent on the binding of the E7 protein to the cell regulator protein.
1 2. A cell, according to claim 1 1 , wherein the DNAs code for a LexA (DBD) E7 protein fusion protein, a B42 (TAD)-cell regulator protein-fusion pro- tein, and a reporter protein, wherein the expression of the reporter protein-DNA is under the control of a LexA (DBD) dependent promoter.
1 3. The use of the system according to any one of claims 1 -5 for the in vitro determination of substances which intervene in binding of an E7 protein to a cell regulator protein.
14. The use of the system according to any one of claims 6-9 for the in vivo determination of substances which intervene in binding of an E7 protein to a cell regulator protein.
1 5. The use according to claim 1 3 or 14, wherein the substances are active against HPV-associated diseases, especially genital wards or carcinomas.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19649606 | 1996-11-29 | ||
DE19649606A DE19649606C1 (en) | 1996-11-29 | 1996-11-29 | System for identifying substances effective against HPV-associated carcinoma |
PCT/EP1997/006703 WO1998023753A1 (en) | 1996-11-29 | 1997-12-01 | Systems for determining substances active against hpv-associated diseases |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0942986A1 true EP0942986A1 (en) | 1999-09-22 |
Family
ID=7813191
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97952025A Withdrawn EP0942986A1 (en) | 1996-11-29 | 1997-12-01 | Systems for determining substances active against hpv-associated diseases |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0942986A1 (en) |
DE (1) | DE19649606C1 (en) |
WO (1) | WO1998023753A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0969013B1 (en) * | 1998-06-30 | 2003-03-26 | Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts | Peptides for inhibiting HPV E7 proteins |
US20060035301A1 (en) * | 2004-01-26 | 2006-02-16 | University Of Massachusetts | Method of identifying protein kinase modulators and uses therefore |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05310784A (en) * | 1991-09-04 | 1993-11-22 | Merck & Co Inc | Peptide inhibitor of human papilloma virus protein binding to retinoblastoma gene protein |
US5525490A (en) * | 1994-03-29 | 1996-06-11 | Onyx Pharmaceuticals, Inc. | Reverse two-hybrid method |
-
1996
- 1996-11-29 DE DE19649606A patent/DE19649606C1/en not_active Expired - Fee Related
-
1997
- 1997-12-01 WO PCT/EP1997/006703 patent/WO1998023753A1/en not_active Application Discontinuation
- 1997-12-01 EP EP97952025A patent/EP0942986A1/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO9823753A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO1998023753A1 (en) | 1998-06-04 |
DE19649606C1 (en) | 1997-10-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Diefenbach et al. | Herpes simplex virus tegument protein US11 interacts with conventional kinesin heavy chain | |
Sakai et al. | Targeted mutagenesis of the human papillomavirus type 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions | |
Huibregtse et al. | Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins | |
Hughes et al. | E1 protein of human papillomavirus is a DNA helicase/ATPase | |
Chen et al. | Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein | |
Chang et al. | Characterization of the DNA binding properties of polyomavirus capsid protein | |
Bream et al. | Characterization of human papillomavirus type 11 E1 and E2 proteins expressed in insect cells | |
NO303287B1 (en) | DNA, DNA fragment, recombinant DNA, DNA hybridization probe, host cell, recombinant peptide and immunogenic composition | |
CN102497880A (en) | Novel human papillomavirus (HPV) protein constructs and their use in the prevention of HPV disease | |
US5914389A (en) | E6 associated protein | |
Rawls et al. | Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation | |
US7279306B2 (en) | Stable (fixed) forms of viral capsid proteins, and viral capsid protein fusions, preferably papillomavirus L1 proteins, and uses thereof | |
Bodaghi et al. | Human papillomavirus type 16 E2 and E6 are RNA-binding proteins and inhibit in vitro splicing of pre-mRNAs with suboptimal splice sites | |
Park et al. | Interactions among four proteins encoded by the human cytomegalovirus UL112-113 region regulate their intranuclear targeting and the recruitment of UL44 to prereplication foci | |
Schäfer et al. | DNA binding of L1 is required for human papillomavirus morphogenesis in vivo | |
Görnemann et al. | Interaction of human papillomavirus type 16 L2 with cellular proteins: identification of novel nuclear body-associated proteins | |
GB2259705A (en) | Polypeptide inhibitor of viral replication | |
AU732254B2 (en) | A human cytomegalovirus combined antigen and its use | |
US5925516A (en) | Medicaments for the treatment of papillomavirus diseases | |
McMahon et al. | Interactions between human cytomegalovirus helicase–primase proteins | |
US5821048A (en) | Methods, kits, and compositions for diagnosing papillomavirus infection | |
Nelson et al. | The L1 major capsid protein of human papillomavirus type 11 interacts with Kap β2 and Kap β3 nuclear import receptors | |
US6610473B1 (en) | Peptides for inhibiting HPV E6-proteins | |
EP0942986A1 (en) | Systems for determining substances active against hpv-associated diseases | |
Palková et al. | The polyomavirus major capsid protein VP1 interacts with the nuclear matrix regulatory protein YY1 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19990623 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB IT LI NL SE |
|
17Q | First examination report despatched |
Effective date: 20030228 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20030711 |