Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-08
2003-11-25
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007500, C435S007800, C530S300000, C530S350000
Reexamination Certificate
active
06653088
ABSTRACT:
The present invention relates to novel peptide and nucleotide sequences. More particularly, the present invention relates to novel polypeptides capable of inhibiting at least in part the interaction between presenilin 1 or presenilin 2 on the one hand and the precursor of the &bgr;-amyloid peptide and/or the &bgr;-amyloid peptide on the other hand. The present invention also relates to the development of in vitro tests for the demonstration of molecules and in particular of small molecules capable of inhibiting this interaction.
The A&bgr; amyloid peptide, of 37 to 42 amino acids, is the principal protein component of characteristic senile plaques of Alzheimer's disease. This peptide is produced by cleavage of its precursor, the precursor protein of amyloid peptide (APP). Mutations in the gene of the APP are responsible for certain early familial forms of Alzheimer's disease. However, the majority of these forms are connected with the presence of mutations on two genes, presenilins PS1 (initially called S182) and PS2 (initially STM2), recently identified by positional cloning (Hardy, 1997). These forms are dominant and these anomalies all correspond to mis-sense mutations with the exception of one involving the deletion of an exon. The presenilins are hydrophobic membrane proteins of molecular mass of approximately 45-50 kDa and which have 67% identity between them. They are homologous with two proteins of
C. elegans
, SPE4 and Sel-12, which are respectively involved indirectly in intracellular transport and in the signalling of the Notch receptors. However, the physiological function of the presenilins is still unknown. Involvement in Alzheimer's disease of two so closely related proteins suggests that the presenilins contribute to an essential physiological route in the aetiology of this pathology.
The protein PS1 comprises 467 amino acids and PS2 comprises 448. Both of them have the structure of a membrane protein with 6 to 8 potential transmembrane domains. Each of the presenilins is subject in vivo to precise proteolytic cleavage resulting in two fragments generally called N(amino)- and C(carboxy)-terminal fragments (Thinakaran et al., 1996). This cleavage has been mapped between the residues 291 and 299 of PS1 (Podlisny et al., 1997) and in a homologous region of PS2. N-terminal (N-ter) fragment is thus in general understood as meaning the fragment from position 1 to approximately 291 of PS1 and C-terminal fragment is understood as meaning the remainder. Although the exact topology of the presenilins in the lipid membranes has not been clearly established, it is proposed that their N- and C-terminal[RC1] ends, as well as the large hydrophilic loop, are present in the cytosol compartment (Doan et al., 1996, see Scheme FIG. 1).
It has now been demonstrated that the mutated forms of the presenilins induce the increase in the production of the long amyloid peptide A&bgr; 1-42 with respect to that of A&bgr; 1-40 as much in carrier patients (Scheuner et al., 1996), as in transfected cells (Borchelt et al., 1996) or in transgenic mice (Duff et al., 1996). The amyloid peptide A&bgr;, which forms senile plaques, lesions characteristic of the pathology, and its different forms are derived from the catabolism of the amyloid precursor protein, APP. In particular, two essential forms of the amyloid peptide have been described, one of forty residues, A&bgr;40, and the other having two additional residues in its carboxy terminal, A&bgr;42. In vitro, the peptide A&bgr; has strong aggregation properties which are increased for the form A&bgr;42 and the latter effectively seems to form the first aggregates detectable in the pathology. In addition, the form A&bgr;42 is specifically produced after cranial trauma in man, which constitutes one of the best established environmental risk factors of Alzheimer's disease. Moreover, the early genetic forms of the disease connected with mutations as much on APP (there are six of them) as now on the presenilins 1 and 2, all contribute to an increase in the A&bgr;42/A&bgr;40 ratio. All of these factors seem to point to A&bgr;42 as the key agent of the pathology as much in the genetic forms as in the sporadic forms of the disease and the elucidation of its mechanism of formation has become a fundamental question.
In this respect, complex formation in the same cell envelope between the precursor of the &bgr;-amyloid peptide and PS1 or PS2 has been reported (Weidemann et al., 1997, Xia et al., 1997), however, the precise nature of the events responsible for the production of the &bgr;-amyloid peptide is not known and no link has yet been able to be established between the possible role of these complexes and the production of the &bgr;-amyloid peptide A&bgr;42. It is nevertheless important to note that the peptide A&bgr;42, but not A&bgr;40, seem to be localized in the endoplasmic reticulum in neuronal cells (Hartmann et al., 1997).
The present invention results from the identification and the characterization by the Applicant of particular regions of presenilin 1 (PS1) and of presenilin 2 (PS2), as well as of particular regions of the &bgr;-amyloid precursor peptide (APP) involved in the formation of APP/PS1 and APP/PS2 complexes.
The present invention follows in particular from the demonstration of the capacity of the N-terminal hydrophilic region (amino acids 1-87) of PS2 to recognize different domains of APP. It additionally follows from the demonstration of similar properties for the N-terminal region of PS1 (fragment 1-213). It likewise results from the demonstration of the capacity of the polypeptides derived from the presenilin regions defined above to inhibit the formation of complexes between APP and the presenilins. The presenilins of the present application correspond essentially to presenilin 1 (PS1) and/or to presenilin 2 (PS2).
The present invention additionally results from the demonstration of the unexpected special cellular location of the regions in interaction with respect to the lipid membrane. It results more particularly from the fact that these interactions can take place not only at the membrane level but also at the level of the lumen of the endoplasmic reticulum and in the extracellular compartment. This is unexpected inasmuch as the N-terminal region of the PS (involved in the interaction) is generally considered as being localized in the cytoplasm under standard conditions.
The characterization of the domains of interaction of APP and of the presenilins and the demonstration of the different cellular locations of these interactions allow the preparation of novel polypeptides which can be utilized pharmaceutically to be envisaged.
A first subject of the invention thus relates to polypeptides capable of inhibiting at least in part the interaction between a presenilin and the precursor of the &bgr;-amyloid peptide and/or the &bgr;-amyloid peptide.
In the sense of capable of inhibiting the interaction, it is understood that the presence of the polypeptides of the invention and/or of the ligands and/or molecules demonstrated with the aid of the process of the invention suffice to inhibit at least partially the said interaction between a presenilin and/or its N-terminal end and the precursor of the &bgr;-amyloid peptide and/or the &bgr;-amyloid peptide and preferably the A&bgr;
1-42
peptide.
It is demonstrated in the examples of the present application that the inhibition of this interaction with one of the polypeptides of the invention leads to a decrease in the production of the A&bgr;
1-42
intracellular amyloid peptide. This functional consequence is thus envisaged for any polypeptide of the invention and/or ligands and/or molecules demonstrated with the aid of the process of the invention. To inhibit this interaction and thus to inhibit the production of A&bgr;
1-42
as a consquence represents a therapeutic target of choice in the diseases involving this form of the amyloid peptide.
According to a particular embodiment, the polypeptides according to the invention have at least one part of prese
Czech Christian
Mercken Luc
Pradier Laurent
Reboul-Becquart Soline
Aventis Pharma S.A.
Bunner Bridget E.
Coppola William C.
Kemmerer Elizabeth
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