Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-04-24
2001-01-30
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C514S002600, C514S04400A, C530S300000, C530S350000, C536S023500, C536S024300, C536S024310
Reexamination Certificate
active
06180362
ABSTRACT:
The present invention relates to new peptide and nucleotide sequences and their pharmaceutical use. More particularly, the present invention relates to new peptides which are capable of at least partly inhibiting the transformation activity of ras proteins.
Various genes, called oncogenes and suppressor genes, are involved in the control of cell division. Among these, the ras genes, and their products generally called p21 proteins, play a key role in control of cell proliferation in all eukaryotic organisms where they have been researched. In particular, it has been demonstrated that certain specific modifications of these proteins cause them to lose their normal control and leads them to become oncogenic. A large number of human tumours have thus been associated with the presence of modified ras genes. Furthermore, excessive expression of these p21 proteins may lead to a dysregulation in cell proliferation. Understanding of the exact role of these p21 proteins in the cells, their mode of functioning and their characteristics thus constitutes a major part in the understanding of and therapeutic approach to carcinogenesis.
In vivo, the precise nature of the events responsible for activation of p21 proteins and transduction of the signal which they carry is still not known. It is known that they perform their function by oscillating between two states of conformation: an inactive form bonded to GDP and an active form bonded to GTP. The activity of these proteins is thus controlled by factors which govern the equilibrium between these two forms, that is to say the transition p21-GDP→p21-GTP and vice versa.
As regards activation of the p21-GDP complexes, recent works report physiological situations in the course of which the proportion of ras proteins bonded to GTP increases in the cell. These are activation of T lymphocytes and stimulation of 3T3 fibroblasts by growth factors such as EGF and PDGF. This increase in the proportion of p21-GTP may be explained at least in part by the action of a protein which plays a role analogous to that of a receptor for the G transduction proteins. In this respect, certain proteins which are capable of promoting the exchange of GDP on p21 proteins have been identified, originating from the brains of cattle (West et al., FEBS Lett. 259 (1990) 245) and rats (Wolfman and Macara, Science 248 (1990) 67). The distinct cell location of these factors and the very different experimental conditions under which they have been obtained suggests that the proteins are different. They are active both on normal ras proteins and on those which are oncogenic. These activities have been classified under the name GEF: Guanide nucleotide Exchange Factor.
As regards the inactivation of the p21-GTP complexes, a cytosol protein having the power greatly to accelerate hydrolysis of the GTP bonded to the p21 protein has been discovered (Trahey and McCormick, Science 238 (1987) 542). This protein, called GAP, interacts with the p21 proteins in a catalytic manner and multiplies the rate of hydrolysis of GTP by 100 to 200, measured in vitro for the normal p21 protein. Various works have demonstrated that the catalytic domain of this protein of about 1044 amino acids was situated in the carboxy-terminal region (residues 702-1044), and that this region was responsible for the interaction of the GAP protein with the p21 proteins (cf. WO91/02749).
However, the role of this GAP protein has not yet been elucidated clearly. In particular, the elements and factors which allow transduction of the activation signals of the p21 proteins to the cell are not known. The present invention results from the discovery by the Applicant that the GAP protein is not simply a regulator having as its sole role deactivation of p21, causing it to pass into the inactive state as the result of hydrolysis of GTP, but that it is also the effector of p21 proteins triggering the cell response. The present invention more particularly results from the identification and characterization of particular regions (so-called effector regions) of the GAP protein which are involved in the transduction of activation signals of p21 proteins. The discovery of the existence of such a region and its characterization enable new peptides which can be used pharmaceutically to be prepared.
The invention thus first relates to peptides which are capable of at least partly inhibiting the transformation activity of activated p21 proteins. It is understood that the term p21 protein designates any expression product of a normal or oncogenic ras gene.
More particularly, the invention relates to peptides which are capable of at least partly inhibiting the transformation activity of p21-GTP-GAP complexes. It is furthermore known that p21 proteins are necessary for expression of the transformation power of oncogenes acting upstream, such as src, HER1, HER2 and the like. As a result, the peptides according to the invention and any pharmaceutical composition comprising them can also be used for treatment of tumours having these activated genes.
The peptides according to the invention are preferably derivatives of the GAP protein.
In the context of the present invention, the term derivative designates any molecule obtained by modification of a genetic and/or chemical nature, the required inhibition capacity being preserved. Modification of a genetic and/or chemical nature may be understood as meaning any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives may be generated for various purposes, such as, in particular, that of increasing the affinity of the peptide for its interaction site, that of improving its production levels, that of increasing its resistance to proteases or of improving its passage through the cell membranes, that of increasing its therapeutic efficacy or of reducing its secondary effects, or that of conferring on it new pharmacokinetic and/or biological properties.
Peptides derived from the protein GAP which may be mentioned are, in particular, any peptide which is capable of bonding the p21 protein, if appropriate complexed with GTP, but carrying an effector region rendered non-functional. Such peptides may be obtained by deletion, mutation or disruption of this effector region on the protein GAP.
The peptides according to the invention more preferably comprise all or part of the peptide sequence SEQ ID No. 2 or of a derivative thereof.
The term derivative has the same meaning as above.
The peptides according to the invention may thus have the structure of the peptide of sequence SEQ ID No. 2, of a fragment thereof, or a structure derived therefrom (for example a peptide incorporating the peptide SEQ ID No. 2). Such peptides may be generated in various ways. In particular, they may be synthesized by a chemical route, on the basis of the sequence SEQ ID No. 2, using peptide synthesizers known to the expert. They may also be synthesized by a genetic route, by expression of a nucleotide sequence coding for the required peptide in a cell host, possibly followed by chemical or enzymatic modifications. In the case of synthesis by a genetic route, the nucleotide sequence may be prepared chemically using an oligonucleotide synthesizer, on the basis of the peptide sequence given in the present Application and of the genetic code. The nucleotide sequence may also be prepared from the nucleotide sequence given in the present Application (SEQ ID No. 1), by enzymatic cutting, ligation, cloning and the like, in accordance with techniques known to the expert. These peptides may also be modified by addition of sequences allowing them a precise cell location. In particular, sequences of the type CAAX, where C is a cysteine, A is an aliphatic amino acid and X is any amino acid, enabling determination of whether a peptide is or is not modified after transduction by a cell farnesyl transferase, may be added (Cancer Cells Vol. 3(9) 1991, 331).
The present invention thus allows generation of peptides derived from the protein GAP and, more particularly, from the sequence SEQ ID No
Duchesne Marc
Schweighoffer Fabien
Tocque Bruno
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Horlick Kenneth R.
Rhone-Poulenc Rorer S.A.
LandOfFree
Peptides which inhibit ras protein activity, their... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Peptides which inhibit ras protein activity, their..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Peptides which inhibit ras protein activity, their... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2551175