Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-11-25
2003-04-08
Nashed, Nashaat T. (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S350000, C536S023100, C536S023500
Reexamination Certificate
active
06544948
ABSTRACT:
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a §371 national phase filing of International Application No. PCT/FR96/00802, filed May 29, 1996.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a new polypeptide designated &Dgr;P62, to its variants, to the corresponding nucleic acid sequences and to their therapeutic uses, in particular in anticancer gene therapy.
2. Description of Related Art
Various genes, referred to as oncogenes and suppressor genes, are involved in the control of cell division. Among these, the ras genes and their products, generally designated p21 proteins, perform a key role in the control of cell proliferation in all the eukaryotic organisms in which they have been sought. In particular, it has been shown that certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumours have been associated with the presence of modified ras genes. Similarly, an overexpression of these p21 proteins can lead to a deregulation of cell proliferation. An understanding of the exact role of these p21 proteins in cells, their mode of functioning and their characteristics hence constitutes a most profitable focus of attention for our understanding of carcinogenesis and the therapeutic approach thereto.
In vivo, the precise nature of the events responsible for transduction of the signal initiated by the p21 proteins is not known. However, an increasing. number of results highlight the multiplicity of effectors which interact directly and preferentially with the active form (bound to GTP) of the ras proteins. Among these effectors, the GAP protein has been the first one to have its involvement in the transduction of the signal documented. It is a cytosol protein, present in all eukaryotic organisms, which possesses the faculty of strongly accelerating the hydrolysis of the GTP bound to the normal protein. It possesses two domains providing for different functions. Its carboxy-terminal end carries the catalytic activity which interacts with the p21 proteins and which increases their GTPase activity. At its other end, downstream of the N-terminal portion, there is a juxtaposition of SH2 and SH3 domains which participate in the transduction of the message and interact with other proteins. Among these proteins, there are two, p62 and p190, of 62 kDa and 190 kDa, respectively, in which the tyrosine is strongly phosphorylated. These two proteins form a specific complex with GAP and are immunoprecipitated by antibodies directed against different epitopes of GAP. It is known, in particular, that the SH2 domains of GAP are the regions in which the interactions of p62 with GAP take place. Amino acids 271 to 443 of p62 contain phosphorylated tyrosines and appear to be involved in these interactions. These same phosphorylations appear, moreover, to participate in interactions between p62 and the adapter GRB2. Moreover, along the whole length of the p62 sequence, proline-rich consensus sites are distributed which participate in the binding to the SH3 domains of the tyrosine kinases of the src family, and also of phospholipase C&ggr;.
The p62 (or alternatively Sam68) protein was identified by Wong et al. (Cell 69 (1992) 551). It contains 443 amino acids, the sequence of which has been described in the literature (see SEQ ID No. 2).
In addition to the features mentioned above, the p62 protein displays several features characteristic of hnRNPs (heterogeneous nuclear ribonucleoproteins):
it is rich in glycines
it possesses regions rich in arginines
furthermore, its amino acids 145 to 247 define a region of strong homology with an hnRNP described previously, GRP33. This region contains a consensus binding site for RNAs which is homologous to the one contained in hnRNP K. This consensus site is designated KH domain (KH=hnRNPK Homologue). The conserved residues are essential to the binding to RNAs, and the impact of the non-integrity of this domain in a pathology has been shown for FMR1, which is the product of the gene associated with mental retardation which is observed in fragile X syndrome (Siomi et al., Cell 77 (1994) 33).
SUMMARY OF THE INVENTION
The present invention has its basis, in particular, in the demonstration of the importance of the p62 (Sam68) protein in cell proliferation and death. It is the outcome, more especially, of the demonstration that p62 derivatives are capable of interfering in the process of cell transformation, and in particular of inhibiting the signals transduced by the ras and arc proteins. It is the outcome, in addition, of the especially surprising demonstration that these derivatives are also endowed with apoptotic properties, and hence capable of inducing cell death.
A first subject of the invention hence relates to any p62 derivative capable of at least partially inhibiting the interaction between a GAP protein and p62. Preferably, the derivatives according to the invention are capable of at least partially inhibiting the oncogenic power of the ras and/or arc proteins. Still more preferably, the derivatives according to the invention are capable of inducing cell death by apoptosis. The derivatives according to the invention are also characterized by the loss of the capacity to interact with RNA of p62.
The present invention describes, in particular, the demonstration, cloning and characterization of a natural isoform of the p62 protein. This isoform, designated &Dgr;p62 (or &Dgr;Sam68), possesses a deletion in the zone of homology to the GRP33 protein, which covers the KH domain. As a result of this deletion, &Dgr;p62 does not possess the properties of p62 in their entirety. Thus, &Dgr;p62 possesses a domain of interaction with GAP and intact GRB2, as well as the various proline-rich sequences which are partners of SH3 (FIG.
1
). However, &Dgr;p62 is no longer capable of interacting with nucleic acids as a result of the deletion of the domain of homology to the GRP33 protein. The Applicant also showed that the transfer of &Dgr;p62 cDNA in various normal or tumoral cell models impedes the cooperation between p62 and Ras and inhibits the signals transduced by normal and oncogenic Ras proteins. Hence, when overexpressed, &Dgr;p62 interferes with the processes of proliferation and differentiation and leads, in the different cell models, to cell death by apoptosis.
According to a preferred embodiment, the invention relates more especially to any p62 derivative carrying at least one deletion in the zone of homology to the GRP33 protein. More especially, the derivatives according to the invention contain at least one deletion in the region lying between residues 145 and 247 of the p62 protein as shown in the sequence SEQ ID No. 1, and which covers the KH domain. The deletion advantageously involves more than 10 amino acids, and more preferably involves more than 30 amino acids. It can affect one or several sites within this region, provided the resulting derivative displays the properties described above.
It is especially advantageous for the derivative according to the invention to be a polypeptide comprising all or part of the sequence SEQ ID No. 4 or of a variant of the latter. For the purposes of the invention, the term variant denotes any polypeptide whose structure differs from the sequence SEQ ID No. 4 by one or more modifications of a genetic, biochemical and/or chemical nature. Such modifications can entail, in particular, any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for different purposes, such as, in particular, that of increasing the affinity of the peptide for its interaction site, that of improving its levels of production, that of increasing its resistance to proteases or of improving its passage through cell membranes, that of increasing its therapeutic efficacy or of reducing its side effects or that of endowing it with new pharmacokinetic and/or biological properties. Advantageously, the variants comprise deleti
Schweighoffer Fabien
Tocque Bruno
Aventis Pharma S.A.
Finnegan Henderson Farabow Garrett & Dunner LLP
Nashed Nashaat T.
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