AU3129500A - Peptides capable of inhibiting the interaction between presenilins and the beta-amyloid peptide or its precursor - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION NAME OF APPLICANT(S): Aventis Pharma S.A.

ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street, Melbourne, 3000.

INVENTION TITLE: Peptides capable of inhibiting the interaction beta-amyloid peptide or its precursor between presenilins and the The following statement is a full description of this invention, including the best method of performing it known to me/us:- The present invention relates to novel peptide and nucleotide sequences. More particularly, the present invention relates to novel polypeptides capable of at least partially inhibiting the interaction between presenilin 1 or presenilin 2, on the one hand, and the P-amyloid peptide precursor and/or the 3-amyloid peptide, on the other hand. The present invention also relates to the development of in vitro tests for detecting molecules, and in particular small molecules capable of inhibiting this interaction.

f The A3-amyloid peptide, of 37 to 42 amino acids, is the main protein component of the characteristic senile plaques of Alzheimer's disease.

This peptide is produced by cleavage of its precursor, the amyloid peptide precursor protein (APP). Mutations in the gene for APP are responsible for certain precocious familial forms of Alzheimer's disease.

However, the majority of these forms are associated with the presence of mutations on two genes, presenilins PS1 (initially named S182) and PS2 (initially STM2), which were recently identified by positional cloning (Hardy, 1997). These forms are dominant and these anomalies all correspond to missense mutations except for one which leads to the 2 deletion of an exon. Presenilins are hydrophobic membrane proteins with a molecular mass of about 45-50 kDa and which show 67% identity with each other.

They are homologous to two proteins of C. elegans, SPE4 and Sel-12, which are indirectly involved, respectively, in intracellular transport and in the signalling of the Notch receptors. However, the physiological function of the presenilins is still unknown. The involvement in Alzheimer's disease of two such similar proteins leads to the notion that the presenilins contribute towards an essential physiological pathway in the aetiology of this :pathology.

S• The protein PS1 comprises 467 amino acids and PS2 448. Both have the structure of a membrane protein with from 6 to 8 potential transmembrane domains. Each the presenilins is subject in vivo to a precise proteolytic cleavage resulting in two fragments generally referred to as the N(amino)-terminal and 20 C(carboxy)-terminal fragments (Thinakaran et al., 1996). This cleavage has been mapped between residues 291 and 299 of PS1 (Podlisny et al., 1997) and in a homologous region of PS2. The expression "N-terminal (N-ter) fragment" thus generally refers to the fragment from position 1 to approximately 291 of PS1, and the expression "C-terminal fragment" generally refers to the remainder. Although the exact topology of the presenilins in lipid membranes is not clearly established, it is proposed that their N-terminal and C-terminal ends [RC1], as well as the large hydrophilic loop, are present in the cytosolic compartment (Doan et al., 1996, see scheme in Figure 1).

It has now been demonstrated that the mutated forms of the presenilins induce an increase in the production of the long AP 1-42 amyloid peptide relative to that of AP 1-40 both in carrier patients (Scheuner et al., 1996) and in transfected cells (Borchelt et al., 1996) or in transgenic mice (Duff et al., 1996).

The AP amyloid peptide, which forms the senile plaques, which are lesions characteristic of the pathology, and its various forms are derived from the catabolism of the amyloid precursor protein, APP. In particular, two 15 essential forms of the amyloid peptide have been described, one of forty residues, AP40, and the other containing two additional residues at its carboxy terminal, AP42. in vitro, the AB peptide has strong aggregating properties which are increased for the A342 20 form, and this latter form appears effectively to form the first aggregates detectable in the pathology.

Moreover, the AP42 form is specifically produced after cranial trauma in man, which constitutes one of the best-established environmental risk factors of Alzheimer's disease. Furthermore, the early genetic forms of the disease associated with mutations both on APP (of which there are six) and now on presenilins 1 and 2 all contribute towards an increase in the AP42/AP40 ratio. This set of factors appears to designate A342 as the key agent in both the genetic and sporadic forms of the disease and the elucidation of its mechanism of formation has become a fundamental issue.

In this regard, the formation of a complex in the same cellular envelope between the P-amyloid peptide precursor 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 P-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 P-amyloid peptide AB42. Nevertheless, it isimportant to note that the peptide Ap42, but not appears to be located in the endoplasmic reticulum in neuronal cells (Hartmann et al., 1997).

The present invention results from the identification and characterization by the Applicant of *20 specific regions of presenilin 1 (PS1) and presenilin 2 (PS2), as well as specific regions of the P-amyloid peptide precursor (APP) involved in the formation of APP/PS1 and APP/PS2 complexes.

The present invention arises in particular from the demonstration of the capacity of the hydrophilic N-terminal region (amino acids 1-87) of PS2 to recognize different APP domains. It also arises from the demonstration of similar properties for the Nterminal region of PS1 (fragment 1-213). It also results from the demonstration of the capacity of polypeptides derived from the regions of the presenilins 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 presenilin 2 (PS2).

The present invention also results from the demonstration of the unexpected specific cellular location of the regions in interaction relative to the Slipid membrane. It arises 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 given that the Nterminal region of the PSs (involved in the interaction) is generally considered as being located in the cytoplasm under standard conditions.

20 Characterization of the domains of interaction of the APP and of the presenilins and the demonstration of the various cellular locations of these interactions make it possible to envisage the preparation of novel polypeptides which can be used pharmaceutically.

A first subject of the invention thus relates to polypeptides capable of at least partially inhibiting the interaction between a presenilin and the -amyloid peptide precursor and/or the -amyloid peptide.

In the sense of being able to inhibit the interaction, it is understood that the presence of the polypeptides of the invention and/or of the ligands and/or molecules detected with the aid of the process of the invention is sufficient to at least partially inhibit the said interaction between a presenilin and/or its N-terminal end and the 3-amyloid peptide precursor and/or the 3-amyloid peptide and preferably the Ai1- 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 intracellular amyloid peptide AP1- 42 This functional consequence is thus envisaged for any polypeptide of the invention and/or ligands and/or molecules detected using the process of the invention. Inhibiting this interaction and thus inhibiting the production of A31- 42 consequently represents a therapeutic target of choice in diseases involving this form of the amyloid peptide.

According to one specific embodiment, the polypeptides according to the invention comprise at least a portion of presenilin 2 (PS2) allowing the interaction with the -amyloid peptide precursor and/or the B-amyloid peptide. Preferably, the polypeptides according to the invention are characterized in that the portion of PS2 corresponds to the hydrophilic Nterminal fragment of PS2. More preferably, the polypeptides according to the invention comprise all or a portion of the sequence corresponding to the sequence SEQ ID No. 1 or of a sequence derived therefrom.

According to another embodiment, the polypeptides according to the invention comprise at least a portion of PS1 allowing the interaction with the O-amyloid peptide precursor and/or the -amyloid peptide. Preferably, the polypeptides according to the invention comprise all or a portion of the sequence corresponding to the sequence SEQ ID No. 2 or of a sequence derived therefrom.

According to another embodiment, the polypeptides according to the invention comprise at least the common homologous regions corresponding to the sequences SEQ ID No. 1 and SEQ ID No. 2.

According to another embodiment, the *polypeptides according to the invention comprise at least one portion of the 3-amyloid peptide precursor (APP) Preferably, the polypeptides according to the invention comprise a portion of the APP other than the region corresponding to the 3-amyloid peptide. Even more preferentially, the polypeptides are characterized in that the portion of the 3-amyloid peptide precursor comprises all or a portion of the fragment 1-596. More preferably, the polypeptides according to the invention comprise all or a portion of a sequence chosen from the 8 sequence corresponding to the fragment 1-596 of the sequence SEQ ID No. 3, or a derived sequence.

For the purposes of the present invention, the expression "derived polypeptide sequence" denotes any polypeptide sequence differing from the polypeptide sequences corresponding to the sequences presented in SEQ ID No. 1 or SEQ ID No. 2, or the designated fragments of SEQ ID No. 3, obtained by one or more modifications of genetic and/or chemical nature, and having the capacity to at least partially inhibit the interaction between presenilin 1 or presenilin 2 and the P-amyloid peptide precursor and/or the 3-amyloid peptide. The expression "modification of genetic and/or -chemical nature" should be understood to mean any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives can be generated for different purposes, such as, in particular, to increase the affinity of the peptide for Sits site of interaction, to improve its level of production, to increase its resistance to proteases, to increase its therapeutic efficacy or to reduce its side effects, or to give it novel pharmacokinetic and/or biological properties.

The invention also provides non-peptide compounds or compounds that are not exclusively peptidic, which can be used pharmaceutically.

Specifically, it is possible, starting with the polypeptide units described in the present application, 9 to prepare molecules which at least partially inhibit the interaction between presenilin 1 or presenilin 2 and the 0-amyloid peptide precursor and/or the Bamyloid peptide, and which are not exclusively peptidic and compatible with a pharmaceutical use. In this regard, the invention relates to the use of polypeptides as described above for the preparation of non-peptide molecules, or molecules that are not exclusively peptidic, which are pharmacologically active, by determination of the structural elements of these polypeptides which are important for their 00 e activity and reproduction of these elements by non- *o o peptide structures or structures that are not so eo exclusively peptidic. A subject of the invention is o o 0 0 also pharmaceutical compositions comprising one or more molecules thus prepared.

o0 0 0..0 The polypeptides according to the invention should comprise sequences allowing a precise cellulose localization, so as to inhibit the interaction between the presenilins and the -amyloid peptide precursor S°o' and/or the -amyloid peptide. Preferably these are the polypeptides derived from SEQ ID No. 1 and SEQ ID No. 2 which comprise exogenous cell localization sequences and even more preferably a polypeptide comprising the N-terminal end of PS1 or PS2. Among the sequences which may be mentioned are signal peptide sequences such as the sequence of the IgkB signal peptide, the APP signal 11 code, obtained by one or more modifications of genetic and/or chemical nature, as well as any sequence which hybridizes with these sequences or fragments thereof and encoding a polypeptide according to the invention.

The expression "modification of genetic and/or chemical nature" can mean any mutation, substitution, deletion, addition and/or modification of one or more residues.

The term "derivative" also comprises sequences homologous to the sequence under consideration, derived from other cell sources and in particular from cells of human origin, or from other organisms. Such homologous sequences can be obtained by hybridization experiments.

.I The hybridizations can be carried out with the aid of a nucleic acid library, using the native sequence or a fragment thereof as probe, under variable hybridization conditions (Maniatis et al., 1982) The nucleotide sequences according to the invention can be of artificial or natural origin. They may be genomic sequences, cDNA sequences, RNA sequences, hybrid sequences or synthetic or semisynthetic sequences. These sequences can be obtained, for example, by screening DNA libraries (genomic DNA library or cDNA library) using probes developed on the basis of sequences presented above. Such libraries can be prepared from cells of different origins by conventional molecular biology techniques known to those skilled in the art. The nucleotide sequences of the invention can also be prepared by chemical synthesis or by mixed methods including chemical or enzymatic modification of sequences obtained by screening libraries. In general, the nucleic acids of the invention can be prepared according to any technique known to those skilled in the art.

Another subject of the present invention relates to a process for preparing the polypeptides of the invention, according to which a cell containing a nucleotide sequence according to the invention is cultured under conditions in which the said sequence is expressed, and the polypeptide produced is recovered.

In this case, the portion encoding the said polypeptide is generally placed under the control of signals oi S" allowing its expression in a host cell. The choice of these signals (promoters, terminators, secretion leader sequence, etc.) can vary depending on the host cell used. Moreover, the nucleotide sequences of the invention can form part of a vector which may be an autonomic or integrative replication vector. More particularly, autonomic replication vectors can be prepared using sequences which replicate autonomously in the host chosen. As regards integrative vectors, these can be prepared, for example, using sequences homologous to certain regions of the host's genome, allowing integration of the vector by homologous recombination.

A subject of the present invention is also host cells transformed with a nucleic acid comprising a nucleotide sequence according to the invention. The host cells which can be used for the production of the peptides of the invention via a recombinant route are both eukaryotic hosts and prokaryotic hosts. Among the eukaryotic hosts which are suitable, mention may be made of animal cells, yeasts or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula. As regards animal cells, mention may be made of COS, CHO, C127, human neuroblastoma, etc. cells. Among fungi, mention may be ee made more particularly of Aspergillus ssp. or .Trichoderma ssp. Prokaryotic hosts which are preferably used are E.coli, Bacillus or Streptomyces bacteria.

According to one preferred embodiment, the host cells are advantageously represented by recombinant yeast strains for the expression of the nucleic acids of the invention as well as the production of the proteins derived therefrom.

Preferably, the host cells comprise at least one sequence or a fragment of a sequence chosen from the sequences SEQ ID No. 1 or SEQ ID No. 2, or the fragments denoted as SEQ ID No. 3 for the production of the polypeptides according to the invention.

The nucleotide sequences according to the invention can be used in the context of gene therapies, in particular by means of the addition of a signal peptide for the derivatives of SEQ ID No. 1 and SEQ ID No. 2, for the in vivo production and transfer of polypeptides capable of at least partially inhibiting the interaction between presenilin 1 or presenilin 2 and the P-amyloid peptide precursor and/or the -amyloid peptide. Specifically, it is demonstrated in the present patent application, unexpectedly, that a signal peptide is required for addressing of the polypeptides of the invention in the lumen of the endoplasmic reticulum and for thus giving biological activity to the polypeptides derived from the sequences SEQ ID No. 1 and SEQ ID No. 2, for the purpose of inhibiting the interaction between the presenilins and the P-amyloid peptide precursor and/or the P-amyloid peptide.

According to another embodiment of the invention, the nucleotide sequences of the invention are used for the construction of an expression cassette which can be used in an expression vector. In particular, the expression vector serves for the production of the polypeptides according to the invention.

The polypeptides of the invention can be obtained by expression in a host cell of a nucleotide sequence as described above, which is incorporated or otherwise into a recombinant DNA, using the techniques known to those skilled in the art, or by a combination of these techniques.

Preferably, the nucleic acid sequences according to the invention form part of a vector which is useful for inducing the in vivo, ex vivo and/or in vitro expression of the polypeptides claimed. The vector used can be of various origins, provided that it is capable of transforming animal cells, preferably human nerve cells. It may be a viral or non-viral vector or a plasmid vector. In one preferred embodiment of the invention, a viral vector is used, which may be derived from adenoviruses, retroviruses, adenoassociated viruses (AAV), herpesvirus, cytomegalovirus (CMV), vaccinia virus, etc. Vectors derived from adenoviruses, retroviruses or AAVs incorporating heterologous nucleic acid sequences have been described in the literature [Akli et al., Nature Genetics 3 (1993) 224; Stratford-Perricaudet et al., Human Gene Therapy 1 (1990) 241; EP 185 573, Levrero et al., Gene 101 (1991) 195; Le Gal la Salle et al., Science 259 (1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208 (1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., New Biol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238; WO 91/18088].

The present invention thus also relates to any recombinant virus comprising, inserted into its genome, a nucleic acid sequence as defined above and encoding a polypeptide of the invention.

Advantageously, the recombinant virus according to the invention is a defective virus. The

U-"

16 term "defective virus" denotes a virus which is incapable of replicating in the target cell. Generally, the genome of the defective viruses used in the context of the present invention thus lacks at least one of the sequences required for the said virus to replicate in the infected cell. These regions may be either removed (totally or partially), rendered non-functional or substituted with other sequences and in particular with the nucleic acid of the invention. Preferably, the defective virus nevertheless conserves the sequences of its genome which are required for encapsidation of the viral particles.

It is particularly advantageous to use the ****nucleic acid sequences of the invention in a form 0 incorporated with a defective recombinant adenovirus, AAV or retrovirus. According to one preferred embodiment, it is an adenovirus.

Different serotypes of adenovirus exist, the structure and properties of which vary somewhat. Among these serotypes, it is preferred in the context of the present invention to use human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see patent application WO 94/26914). Among the adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine (example, Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine, aviary or simian (example: SAV) origin.

Preferably, the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26/61 strain (ATCC VR-800) for example]. Preferably, in the context-of the invention, adenoviruses of human or canine or mixed origin are used. Preferably, in the genome of the adenoviruses of the invention, the El region at least is nonfunctional. The viral gene under consideration can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, partial deletion or addition of one or more bases to the gene or genes under consideration. Other regions can also be modified, and in particular the E3 region (WO 95/02697), the E2 15 region (WO 94/28938), the E4 region (WO 94/28152, WO 94/12649, WO 95/02697) and the L5 region (WO 95/02697). According to one preferred embodiment, the adenovirus comprises a deletion in the El and E4 regions. According to another preferred embodiment, it 20 comprises a deletion in the El region into which are inserted the E4 region and the coding sequence. In the viruses of the invention, the deletion in the El region preferably extends from nucleotides 455 to 3329 on the sequence of the adenovirus Ad5. According to another preferred embodiment, the exogeneous nucleic acid sequence is inserted into the deletion in the El region.

18 The defective recombinant viruses of the invention can be prepared by homologous recombination between a defective virus and a plasmid carrying, inter alia, the nucleotide sequence as defined above (Levrero et al., Gene 101 (1991) 195; Graham, EMBO J. 3(12) (1984) 2917). The homologous recombination takes place after the said virus and plasmid have been cotransfected into a suitable cell line. The cell line used should preferably be transformable by the said elements and (ii) comprise sequences capable of complementing the portion of the genome of the defective virus, preferably in integrated form so as to avoid the risks of recombination. As an example of a line which can be used for the preparation of defective S* 15 recombinant adenoviruses, mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen.

Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome of an adenovirus Ad5 As an example of a line which can be used for the preparation of defective S. recombinant retroviruses, mention may be made of the CRIP line (Danos and Mulligan, PNAS 85 (1988) 6460).

Next, the viruses which have been multiplied are recovered and purified according to the conventional 25 molecular biology techniques.

A subject of the present application is also defective recombinant viruses comprising a heterologous 19 nucleic acid sequence encoding a polypeptide according to the invention.

Another subject of the invention is monoclonal or polyclonal antibodies or antibody fragments. Such antibodies can be generated by methods known to those skilled in the art. In particular, these antibodies can be prepared by immunizing an animal against a polypeptide whose sequence is chosen from the sequences SEQ ID No. 1 or SEQ ID No. 2 or the designated fragments of SEQ ID No. 3, followed by taking a blood sample and isolating the antibodies.

These antibodies can also be generated by preparing hybridomas according to the techniques known to those skilled in the art. The antibodies or antibody 15 fragments according to the invention can be used in particular to at least partially inhibit the interaction between presenilin 1 or presenilin 2 and the -amyloid peptide precursor and/or the 3-amyloid peptide.

Another subject of the present invention relates to a process for identifying compounds capable of at least partially modifying or inhibiting the interaction between presenilin 1 or presenilin 2 and the -amyloid peptide precursor and/or the 3-amyloid 25 peptide. In particular, the process can be used as a test for screening molecules to identify such inhibitory compounds.

This test is based in particular on detecting the inhibition of the general interaction between the presenilins (1 or 2) and the APP or the A3 peptide and particularly between the peptide 42 and the Nterminal end of PS2. Specifically, using marker proteins bound to the presenilins or fragments thereof and suitable revelation systems, in particular by immunoprecipitation or the use of chromophores or fluorophores, it is entirely possible to detect an inhibition in interaction of the proteins or fragments thereof, mentioned above. Such a process thus comprises at least one step of labelling the presenilins and/or the APP or fragments thereof and a step of detecting the inhibition of the interaction either between the 15 peptide A1- 42 and the N-terminal end of the presenilins and preferably PS2, or between the whole APP and S. presenilin proteins.

According to a first embodiment of the process, the detection and/or identification of such compounds is carried out according to the following steps: the peptide AP1-42 is preabsorbed onto a nitrocellulose membrane by incubation; a bacterial extract containing all or a 25 portion of a presenilin (PS1 or PS2) and advantageously the N-terminal end, is then added for incubation with the molecule or a mixture containing various test molecules; after washes, the interaction of the presenilin with the AP1- 42 peptide on the nitrocellulose filter is detected with the aid of presenilin-marker proteins. The desired molecules inhibit the interaction and thus reduce the intensity of the signal from the marker proteins.

The marker proteins used are advantageously a) the S-tag-binding protein, coupled to alkaline phosphatase or to a fluorescent chromophore, or b) an anti-PSNT antibody, i.e. an antibody directed against the N-terminal end of a presenilin.

According to another embodiment of the process, the search for novel compounds is carried out in the following way: S. 15 the AP42 peptide [lacuna] preincubated on a plate containing wells (96-well format or higher), the N-terminal end of a purified recombinant presenilin is then added with the molecule or a mixture containing various test molecules, for incubation, after washes, the interaction of the presenilin with the API- 42 peptide in the plate is detected with the aid of presenilin-marker proteins.

The loss of interaction between the presenilins and the -amyloid peptide precursor and/or the -amyloid peptide is detected by spectrophotometry.

In the specific case of using the S-tagbinding protein coupled to alkaline phosphatase as a marker protein, after revelation with a colorimetric substrate, the signal is detected at 450 nm.

According to one advantageous and preferred embodiment of the process, the detection and/or isolation of compounds capable of at least partially modifying or inhibiting the interaction in general between presenilin 1 or presenilin 2 and the P-amyloid peptide precursor and/or the 3-amyloid peptide, and specifically between the AP1-4 2 peptide and the Nterminal end of PS2, is carried out according to the following steps: a molecule or a mixture containing various molecules with the A31- 42 peptide synthesized is placed in contact with a biotin and an arm of 3 3-alanines (or 15 3 lysines) at its N-terminal end (upstream of position 1), the above reaction mixture is incubated with the N-terminal end of a purified presenilin labelled with a first fluorophore. Advantageously, the fluorophore is europium cryptate, streptavidin (which binds to the biotin of the biot-A 1- 42 peptide), coupled to a second fluorophore, is added, this second fluorophore being capable of being excited at the emission wavelength of 25 the first fluorophore so that it benefits from a transfer of fluorescence if the two fluorophores are close together, the detection of novel compounds which inhibit the interaction is made by fluorometry at the emission wavelength of the first fluorophore and/or by measuring the reduction of the signal at the emission wavelength of the second fluorophore.

According to one specific embodiment, this second fluorophore is XL665, which is allophycocyanin chemically crosslinked to increase its fluorescence at 665 nm (CisBiointernational). The loss of interaction is thus detected by fluorometry at the emission wavelength of the first fluorophore and by the reduction in the signal of XL665 whose emission wavelength is 665 nm.

This process is entirely advantageous since 15 it allows direct detection of the interaction between the presenilins and the APP and/or the AP peptide in a liquid, homogeneous phase, and consequently allows the detection of molecules which inhibit the said interaction. Specifically, this process is based on the transfer of fluorescence between two fluorophores if these two chromophores are physically close together (thus in the case of interaction between A1- 42 and the recombinant protein). According to one preferred variant of the process, the first fluorophore is 25 europium cryptate, carried by the labelled recombinant protein (excited at 337 nm), which reacts with the streptavidin-XL665 bound to the biot-AP peptide, and in particular the biot-Aj 1 42 peptide. Advantageously, the labelled protein consists of the N-terminal end of one or other of the presenilins (PSNT-K). The loss of fluorescence at 665 nm and the increase in fluorescence at 620 nm, characteristic of europium cryptate, indicates an inhibition of the interaction between the presenilins or their N-terminal ends and the APP and/or the A3 peptide by the desired molecules.

According to one variant of this process, the molecule or the mixture containing the various molecules can be placed in contact first with the Nterminal end of a purified presenilin labelled with europium cryptate (PSNT-K) and then with the Apl-40 or APl-42 peptide carrying a biotin and an arm of 3 P-alanines (or 3 lysines) at their N-terminal end. The 15 detection of novel molecules capable of at least partially modifying or inhibiting the interaction Sebetween the presenilin 1 or presenilin 2 and the 3-amyloid peptide precursor and/or the -amyloid peptide will also be made, after adding the XL665- 20 labelled streptavidin, by spectrofluorometry according to the above process and in particular by reading the fluorescence at 665 nm.

According to a final embodiment of the process for detecting compounds which inhibit the interaction in general between the presenilins (1 or 2) and the APP or the A3 peptide, and specifically between the AP1- 4 2 peptide and the N-terminal end of PS2, comprises the following steps: a mixture a) of cell lysates containing all or a portion of a presenilin (PS1 or PS2) and advantageously the N-terminal end, b) of cell lysates containing the APP, lysates obtained from cells infected with viruses and in particular with baculoviruses, and c) the test molecule or a mixture containing various test molecules, is placed in contact, the solubilized proteins corresponding to the presenilins or to the APP or the AP peptide are coimmunoprecipitated with the aid of suitable antibodies which are well known to those skilled in the art, the loss of co-immunoprecipitation of the presenilins and of the APP is revealed by Western 15 blotting with labelling antibodies, indicating that the :test molecules have the desired inhibitory property.

S"In one specific embodiment, the processes of the invention described above are adapted to the detection and/or isolation of ligands, agonists or antagonists of the interaction between the presenilins and the P-amyloid peptide precursor and/or the P-amyloid peptide.

The present invention also relates to the use of the polypeptides defined above for the detection of ligands for the polypeptides, but especially of ligands for the presenilins, for the P-amyloid peptide precursor and/or for the P-amyloid peptide, and preferentially for the AB1- 42 peptide and/or for the Nterminal end of PS2, as well as of compounds capable of at least partially inhibiting the interaction between a presenilin and the 3-amyloid peptide precursor and/or the 3-amyloid peptide.

Another subject of the invention relates to the use of a ligand or of a modifier identified and/or obtained according to the processes described above, as a medicinal product. On account of their capacity to interfere with the interaction between the presenilins and the -amyloid peptide precursor and/or .the 3-amyloid peptide, such ligands or modifiers can thus modify the production of the A31- 42 amyloid peptide and allow the treatment of certain neurological complaints and in particular Alzheimer's disease.

15 Another subject of the invention relates to the development of a test of interaction between a presenilin and the 3-amyloid peptide precursor and/or the -amyloid peptide, and preferentially between the AP1 42 peptide and the N-terminal end of PS2, 20 characterized in that it comprises at least one step of fluorescence transfer between two fluorophores bound to the above molecules and a step of revealing the interaction measured by spectrofluorometry. As mentioned above, this test is also used for the 25 detection of molecules which inhibit the said interaction, according to the process for detecting the inhibition of the interaction, described in the present application.

A subject of the invention is also any pharmaceutical composition comprising, as active principle, at least one polypeptide as defined above.

A subject of the invention is also any pharmaceutical composition comprising, as active principle, at least one antibody or an antibody fragment as defined above, and/or an antisense oligonucleotide, and/or a ligand as defined above. A subject of the invention is also any pharmaceutical composition comprising, as active principle, at least one nucleotide sequence as defined above.

Moreover, a subject of the invention is also pharmaceutical compositions in which the peptides, antibodies, ligands and nucleotide sequences defined 15 above are combined together or with other active principles.

A subject of the invention is also compositions in which the nucleotide sequences according to the invention are incorporated in a viral 20 or non-viral recombinant vector.

*9 The pharmaceutical compositions according to the invention can be used to at least partially inhibit the interaction between a presenilin and the -amyloid peptide precursor and/or the P-amyloid peptide. They 25 are more preferably pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as, for example, Alzheimer's disease.

Another subject of the present invention is the use of the polypeptides described previously to at least partially inhibit the interaction between a presenilin and the 3-amyloid peptide precursor and/or the 3-amyloid peptide, and preferably the use of these polypeptides to obtain a medicinal product intended for the treatment of neurodegenerative diseases and in particular Alzheimer's disease.

For their use according to the present invention, the polypeptides of the invention on the one hand, or any molecule capable of at least partially inhibiting the interaction between a presenilin and the 3-amyloid peptide precursor and/or the 3-amyloid peptide, and the corresponding nucleic acid sequences 15 on the other hand, or alternatively the vectors as described above, are preferably combined with one or more pharmaceutically acceptable vehicles to be S"formulated for the purpose of topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc.

administration. Preferably, they are used in an oral form. Nevertheless, the injectable form can be envisaged and may in particular be formulated with isotonic, sterile saline solutions (monosodium 25 phosphate, disodium phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc. or mixtures of such salts) or dry compositions, in particular freeze-dried compositions, which, on addition of sterilized water or physiological saline, depending on the case, allow injectable solutions to be made up.

The doses of vector and in particular of virus used for the administration can be adapted as a function of various parameters, and in particular as a function of the site of administration under consideration (organ, nerve tissue or muscle tissue), the number of injections, the gene to be expressed or the desired duration of the treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu and preferably 106 to 1010 pfu.

The term "pfu" (plaque forming unit) corresponds to the infectious power of a solution of virus, and is determined by infection of a suitable cell culture and measurement, generally after two weeks, of the number S of plaques of infected cells. The techniques for determining the pfu titre of a viral solution are well documented in the literature.

The present invention offers an effective means for treating diseases for which the interaction between a presenilin and the P-amyloid peptide precursor and/or the P-amyloid peptide is involved, and 25 preferably for the treatment of neurodegenerative diseases and in particular Alzheimer's disease.

The present invention will be described in greater detail with the aid of the examples below, which are considered as illustrative and non-limiting.

List of figures Figure 1: A) Scheme of the truncated PS2 constructs B) Expression in COS1 cells.

Figure 2: Interaction of the truncated forms of PS2 with APP.

Figure 3: A) Interaction of the secreted N-terminus of PS2 (SecPS2NT) but not of its cytoplasmic form (myc-PS2NT) and of extracellular APP.

B) Detection of the SecPS2NT/APP interaction but not of the myc-PS2NT/APP interaction in 15 extracellular medium.

o 0 Figure 4: Interaction of PS2 with the truncated forms of APP: Interaction of PS2 with the SPA4CT form .4 00

S

of APP but not with the cytoplasmic domain of APP S'g B) Interaction of PS2 with the C100 form of

APP.

Figure 5: Interaction of PS1 and PS1DC2 with APP and *S its short form: 25 A) Interaction of PS1 with the whole form of APP and the truncated SPA4CT form.

B) Interaction of the truncated form of PS1 (PS1 AC2) with APP.

Figure 6: Interaction of PS1 and of APP in insect cells.

A) Anti-histidine immunoprecipitates (label on PS1), APP revelation.

B) Anti-PS1 immunoprecipitates, APP revelation.

C) Anti-APP inverted immunoprecipitates, PS1 revelation.

Figure 7: Interaction of the secreted form of PS2NT with the A3 peptide in the extracellular medium.

Figure 8: Reconstitution of the A3/PS2NT interaction in vitro on a nitrocellulose filter.

A) Dose-dependence as a function of the concentration of PS2NT.

15 B) Dose-dependence as a function of the concentration of A3.

Figure 9: Interaction in vitro of the whole forms of .e o.

PS1 and APP.

A) Anti-histidine immunoprecipitates.

20 B) Anti-PS1 immunoprecipitates.

Figure 10: Displacement, by the N-terminal end of PS2 preceded by a signal peptide (SecPS2NT), of the interaction between PS1 and the fragment SPA4CT.

Figure 11: Interaction of PS2NT with the A31- 42 peptide in vitro: detection by the 96-well plate test (ELISA).

Figure 12: Interaction of PS2NT with the AP1- 42 peptide, detected by the HTRF (homogeneous time-resolved fluorescence) fluorescence-transfer test.

Figure 13: Blocking of the APP/PS1 interaction with SecPS2NT leads to inhibition of the intracellular production of A31- 4 2 amyloid peptide.

A) Demonstration that the blocking of the APP/PS1 interaction with SecPS2NT leads to inhibition of the intracellular production of AP1- 42 amyloid peptide.

B) Control that the expression of SecPS2NT has no influence on the expression of the various transgenes.

Figure 14: Detection of the interaction of PS2 with the endogenous APP of COS cells using the pharmacological treatment with lactacystine.

Figure 15: Interaction of PS2 and PS2NT with a second 15 region of APP, different from the A3 peptide.

S...'Materials and methods A/ Materials 1. Constructs expressing the presenilins 20 The production of an expression vector (host vector, pcDNA3, InVitrogen) in mammalian cells for the human proteins PS1 and PS2 has been described previously (Pradier et al., 1996). Several successive deletions by the C-terminal end of PS2 were generated (see Fig. 1A). The numberings are given taking the start codon of PS2 as position 1.

The HindIII restriction fragment of the PS2 vector (from the non-coding 5' end, position -55, to the internal site at position 1080) was purified and ligated with the vector pcDNA3 linearized with HindIII and treated with alkaline phosphatase. The truncated PS2 thus produced (PS2AC1) extends from the N-terminal end to residue 361 plus 7 residues supplied by the 3' end and thus comprises the first six transmembrane domains and a large portion of the hydrophilic loop (Fig. 1A).

PS2AC2 was constructed by digesting the PS2 plasmid with PstI (internal site at position 679) and ligation with the PstI fragment corresponding to the non-coding 3' portion of the PS2 vector. The truncated protein PSAC2 extends from position 1 to residue 228 of PS2 plus 18 additional residues supplied by the 3' end.

15 It includes the first four transmembranes domains of S PS2.

A similar construct was made for PS2 carrying the N141I mutation, PS2AC2*.

The HindIII restriction fragment o. 20 (-55)/MscI(590) of the PS2AC2 construct was then cloned into the pcDNA3 vector treated with HindIII and whose Apal end was made blunt. This construct PS2AC3 extends from the N-terminal end to residue 198 plus 2 additional residues, thus comprising the first three transmembrane domains of PS2.

The HindIII restriction fragment of PS2AC2, (-55)/NcoI(504) made blunt at its NcoI end by treatment with the Klenow fragment of DNA polymerase, was recloned into the same pcDNA3 vector HindIII/(ApaI blunt end) to construct PS2AC4 extending up to residue 168 of PS2 plus 3 additional residues.

The construction of the hydrophilic Nterminal end of PS2 was obtained by amplifying the PS2 sequence with the ext5' oligonucleotide: 5'-CGGAATTCATCGATTCCACCATGCTCACATTCATGGCC-3' (SEQ ID4) (overlapping the initial ATG, in bold, and introducing an EcoRI restriction site, underlined), and the ext3' oligonucleotide: 5'-CCGCTCGAGTCATTGTCGACCATGCTTCGCTCCGTATTTGAGG-3' (SEQ ID5) (introducing a stop codon after residue 90 of PS2 as well as an XhoI restriction site, underlined).

After cloning into the pCRII vector by the TA 15 cloning method (InVitrogen), the conformity of the PCR fragment was checked by sequencing. This fragment (EcoRI/XhoI) was then introduced into a pcDNA3 vector in phase with a sequence corresponding to the myc epitope at its N-terminal end: mycPS2Nter.

20 So as not to compromise the topology of PS2, the same Nter fragment was recloned into the pSectagB vector, in phase with the sequence of the IgkB signal peptide to direct the secretion of the PS2Nter protein: SecPS2-Nter.

The C-terminal end of PS2 was constructed in a similar manner using the HindIII restriction fragment (1080)PstI (non-coding 3' end) recloned into the pSecTagB HindIII/PstI vector, SecPS2Cter extending from residue 361 to the C-terminal end, or into the pcDNA3myc vector in phase with the myc epitope.

A truncated PS1 construct was similarly obtained. The pcDNA3-PSl vector was digested with PflmI (site at position 636 of the coding nucleic acid sequence of PS1) and XhoI in the non-coding 3' end of the sequence of PS1. These sites were transformed into blunt ends by treatment with T4DNA polymerase. The vector fragment, purified on agarose gel, was selfreligated to give a truncated PS1 expression vector extending from the N-terminal end of PS1 to residue Ile213 (after the 5th transmembrane domain) plus 12 additional residues. This construct corresponds to the AC2 chimer and is referred to as PS1AC2.

o* S" 2. Constructs which express APP 2.1 APP constructs The various constructs: whole APP (isoform 695) and SPA4CT (the last 100 residues of APP (amino 20 acid 597 to 695) preceded by a signal peptide for insertion into the membrane) have been described previously (Dyrks et al., 1993). The vectors, for the expression of C100 and of the cytoplasmic domain of APP, were obtained in the following way: the corresponding cDNAs were obtained by enzymatic DNA amplification (PCR (polymerase chain reaction)) using the following oligonucleotides as synthetic primers: for C100: oligonucleotides 8172 and 8181; for the cytoplasmic domain of APP: oligonucleotides 8171 and 8181.

Oligo 8172 5; CAAAGATCTGATGCAGAATTCCGACAT 3' (SEQ ID6) containing: a recognition site for the restriction enzyme BglII (underlined) the coding sequence for amino acids 597-602 of APP (in bold) [APP numbering of 695 amino acids] Oligo 8181 5' CAAGCGGCCGCTCATCCCTTGTCATCGTCGTCCT TGTAGTCTCCGTTCTGCATCTGCTC 3' (SEQ ID7) containing: a recognition site for the restriction enzyme NotI (underlined) 15 the sequence complementary to the coding sequence for amino acids 691-695 of APP (in bold) [APP numbering of 695 amino acids] the sequence complementary to the sequence Asp-Tyr-Asp-Asp-Asp-Asp-Lys corresponding to the FLAG epitope (in italics).

Oligo 8171 5' CAAAGATCTAAGAAACAGTACACATCC 3' (SEQ ID8) containing: a recognition site for the restriction enzyme BglII (underlined) the sequence coding for amino acids 650-655 of APP (in bold) [APP numbering of 695 amino acids] The products of the enzymatic DNA amplification were cloned into the pCRII vector. The 37 nucleotide sequence was checked by the specific DNA terminator method.

The cDNAs were then introduced by ligation into the expression plasmid derived from the pSV2 plasmid and containing, in the same reading frame, an MYC epitope.

2.2 Construction of the soluble forms of APP: a-sAPP and -sAPP The cDNAs corresponding to the secreted forms of APP ending at the a- and P-cleavage sites were obtained by PCR.

The oligonucleotide 1: 5'-ccatcgatggctaCATCTTCACTTCAGAG-3' (SEQ ID 9) 15 introduces: a stop codon (inverted complementary sequence underlined) after position 1788 of APP corresponding to "the 3 cleavage site, and a ClaI restriction site.

The oligonucleotide 2: 5'-ccatcgatggctaTTTTTGATGATGAACTTC-3' (SEQ ID introduces: 000 a stop codon (inverted complementary sequence underlined) after position 1836 of APP corresponding the a cleavage site, and a Clal restriction site.

The oligonucleotide 3: 5'-CCGTGGAGCTCCTCCCG-3' (SEQ ID 11), which is common for the two forms, corresponds to the region 1583 to 1600 of APP including the SacI internal restriction site of APP (underlined).

The cDNA of APP was amplified with PCR using the pairs oligo3-oligol and oligo3-oligo2 for 3-sAPP and a-sAPP, respectively. The amplification products were subcloned as above into pCRII and the sequences checked by sequencing. The SacI-ClaI restriction fragment for each were purified and recloned into the APP expression vector (see above), which was itself digested with SacI-ClaI to replace the C-terminal portion of APP with the C-terminal fragments of P-sAPP and c-sAPP, respectively, and to reconstitute the whole 15 proteins.

3. Baculovirus constructs The production of a baculovirus transfer vector encoding the human PS1 protein was carried out 20 using an expression vector for mammalian cells (Pradier et al., 1996). The cDNA encoding the PS1 protein was extracted by digestion with the restriction enzymes XhoI and NotI, and then cloned into the transfer plasmid pAcHTLB (6-Histidines fusion protein) and pAcSG2 (native protein). The production of recombinant baculoviruses is carried out according to the supplier's (Pharmingen) protocol and consists in cotransfecting 2 x 106 insect cells (sf9) with 1 ug of transfer plasmid containing the gene of interest and gg of viral DNA (Baculogold). After 5 days at 27 0

C,

the cells are scraped off and then centrifuged, the supernatant is used as a viral stock for the amplification and determination of the viral titre, and the protein expression is visualized by Western blotting on the cell pellet.

The production of the baculovirus expressing human APP (695) has been described previously (Essalmani et al., 1996).

For the study of the expression of PS1 and APP, the sf9 cells are co-infected at an M.O.I. of 2 with baculoviruses expressing human APP (695), human presenilin 1 (PS1) protein, or PS1 with an N-terminal 6-Histidine tag (6HisPS1), or the XylE control protein of Pseudomonas putrida with an N-terminal 6-Histidine Stag (6HisXylE), and then solubilized with a 10 mM Tris, 130 mM NaC1, 1% Triton X100, 1% NP40, pH 7.5 buffer.

The solubilized proteins are immunoprecipitated with an antiHistidine antibody an antiPS1 antibody (1805) or an antiAPP antibody (22C11) The presence of APP or PS1 was revealed by Western blotting with the antiAPP antibody aCT43(A), 22C11(B) or the antiPS1 antibody 95/23(C).

25 The solubilized fractions containing the APP, PS1 or 6HisPS1 are mixed and then immunoprecipitated in the presence of anti-Histidine or antiPS1 antibodies overnight at 4 0 C. The co-immunoprecipitation of the APP is revealed after Western blotting with the aCT43 or 22C11 antibodies.

4. The plasmids The plasmids used for the invention are the following: pcDNA is a commercial plasmid (InVitrogen) used for the cloning and expression in mammalian cells of the PS1 and PS2 sequences and of their truncated forms, pCRII is a commercial plasmid (InVitrogen) used for cloning PCR fragments, pSecTagB is a commercial plasmid (InVitrogen) used for the cloning and expression in 15 mammalian cells of cDNAs to which are added the secretion signal (Ig k signal peptide), pSV2 is a commercial plasmid (Pharmacia) used for the cloning and expression of cDNA in mammalian cells, 20 pAcHTLB is a commercial plasmid 9 (Pharmingen) for the insertion of an (His)6 epitope into cDNAs and the homologous recombination with **baculoviruses, pAcSG2 is a commercial plasmid (Pharmingen) 25 for the homologous recombination with baculoviruses, pET29a is a commercial plasmid (Novagene) for the expression of cDNA in bacteria.

41 B/ METHODS 1. Transfection of cells The method established for the COS1 cells or the CHO cells consists in using a lipofectant in a ratio of 1 to 8 (weight/weight) relative to the DNA and a synthetic peptide H1 (sequence: KTPKKAKKPKTPKKAKKP) at the same ratio in order to optimize the compaction of the DNA and the transfection efficacy. This method is based in particular on the neutralization of the DNA phosphate charges with the positive charges of the lipofectant.

COS1 cells are cultured in an incubator at 370C, 95% humidity and 5% C02 in DMEM (Dulbecco's Modified Eagle's Medium) containing 4.5 g/l of glucose 15 (Gibco-BRL) supplemented with 3% L-glutamine, 1% penicillin-streptomycin and 10% foetal calf serum.

On the day before the transfection, the cells are inoculated at a density of 2.5 x 106 cells per 100 mm dish. On the day of the transfection, the cells 20 are rinsed twice with PBS (phosphate-buffered saline) and once with OptiMEM (patented composition: Gibco-BRL) for a residence time of at least 15 minutes in the incubator.

8 pg of plasmid DNA in total are added to 25 300 .l of OptiMEM and 64 pg of peptide H1 per 100 mm dish-equivalent. After vortexing vigorously for seconds, the above mixture is left to stand for minutes and lipofectamine (32 p1, i.e. 64 pg) diluted in 300 1l of OptiMEM is added. The whole is again vortexed vigorously and then left to stand for minutes. Five millilitres of OptiMEM are added per tube and the vortexed mixture is placed on the cells (whose medium has been removed beforehand). The cells are then placed in an incubator for 4 hours, after which the mixture is replaced with whole medium.

2. Lysis of the cells and assay of the proteins The cells are usually lysed 48 hours after the transfection (at the usual expression maximum). The lysis buffer contains 10 mM of Tris pH 7.5, 1 mM of EDTA, 1% Triton X100, 1% NP40 and a cocktail of protease inhibitors (Complete®, Boehringer-Mannheim).

15 For each plaque, after rinsing with PBS, 800 p1 of cold buffer are added. The lysates then undergo a sonication followed by stirring with a magnetic bar at overnight. Centrifugation for 30 minutes at 15000 rpm separates the pellet from the supernatant. The soluble proteins are then assayed according to the BCA kit (Pierce) in order to be able to normalize the following experiments.

3. Immunoprecipitations The antibodies directed against the PS2 Nterminal peptide, 95041, (Blanchard et al., 1997) and against the first twenty amino acids of PS1 (Duff et al., 1996) were obtained in rabbits by immunization 43 with synthetic peptides. For the immunoprecipitation, 100 tg of proteins are diluted in 400 gl of modified RIPA (150 mM NaCI, 50 mM Tris pH 8.0, 1% Triton X100 v/v, 1% NP40 Thirty microlitres of protein A Sepharose suspension m/v dissolved in PBS) and 3 gl of antibodies are added. The suspensions are mixed gently on a rotary shaker at 40C overnight. The protein A Sepharose complex is washed 3 times with 0.5 ml of modified RIPA and once with 0.5 ml of "Wash C" washing buffer (10 mM Tris pH 4. Immunotransfer The samples (cell lysates) are denatured in an equal volume of deposition buffer (125 mM Tris 15 pH 6.8, 4% m/v SDS, 20% glycerol, 0.02% bromophenol blue, 50 mM dithiothreitol) at 950C for 5 minutes. For the analysis of the expression of the presenilins, the samples are denatured in the presence of 8 M urea at 370C in order to avoid the aggregation intrinsic to the 20 presenilins at 95 0

C.

The samples are deposited on Tris-glycine gels (Novex), with a different percentage of acrylamide depending on the molecular weight to be discriminated.

A molecular weight marker is also deposited (Broad Range, BioRad). Migration takes place over about 2 hours at 100 volts constant in SDS 1X final buffer (Novex). The gel is then transferred onto a nitrocellulose or PVDF membrane (1X final transfer buffer (Novex) with 10% methanol) for 2 hours at 150 mA constant.

After transfer, the membrane is blocked for 2 hours at room temperature in 50 ml of PBS-T (PBS with 0.5% Tween) containing 2% skimmed milk (Merck). The first antibody (diluted to an optimum concentration from about 1/1000 to 1/5000 in PBS-T with or without 2% skimmed milk) is left overnight at 4 0 C. After a brief rinsing with PBS-T, the membrane is incubated for minutes in the presence of the second antibody (antimouse or anti-rabbit IgG depending on the case, coupled with horseradish peroxidase) diluted to 1/5000 in a socalled "ECL" buffer (20 mM Tris, 150 mM NaC1, 0.1% Tween).

15 The membrane is then rinsed for 4 times minutes in the "ECL" buffer. It can be revealed with the ECL reagent (Amersham) consisting of 2 buffers to be mixed together in equal volume at the time of use.

Various exposures of a photographic film (Hyperfilm ECL; Amersham) are carried out, followed by developing.

in vitro fixing of PS2 NT with the A1 amyloid peptide 5.1 Production of the PS2 NT recombinant protein in bacteria For the creation of a bacterial expression vector for PS2NT (amino acids 1 to 87), the cDNA of PS2 was amplified by PCR with the oligonucleotides 3' (CCGCTCGAGTCATTGTCGACCATGCTTCGCTCCGTATTTGAGG) and 5' (CCGGAATTCATCGATTCCACCATGCTCACATTCATGGCC). The resulting fragment was cloned into pCRII and the sequence was confirmed. This fragment was then subcloned into the pET29a vector (Novagene) in phase with the S-tag label sequence. The protein was produced in the BL21 bacterium. After induction with IPTG for the bacteria were recovered by centrifugation min at 6000 rpm) and the cell pellet was dissolved in RIPA buffer (volume calculated by multiplying the OD of the culture after induction by the volume of culture divided by 23). The bacteria were lysed by sonication and the lysate was centrifuged at 13000 rpm for 20 min at 4 0 C. The supernatant (total extract) was used for 15 the binding studies.

The PS2NT recombinant protein was also purified from the total extract on a Nickel column (poly-His label supplied in the pET29a vector) as described by the supplier (Novagene).

5.2 Test of PS2NT/A342 binding to **nitrocellulose membrane The synthetic A3 peptide (in solution) was deposited on a nitrocellulose membrane (Schleicher and Schuell) using a 96-well dot-blot machine. After deposition, the filter was blocked (with respect to the non-specific protein binding sites) with the gelatin blocking reagent (Novagen) diluted 10-fold in TBST.

After blocking, the filter was placed back on the dotblot machine and the PS2NT bacterial extract was added to the wells for an incubation of 2h at room temperature. As a control, a bacterial extract containing the empty pET29 plasmid was used on wells in duplicate. The filter was then washed once with RIPA buffer and then removed from the machine and washed three times with PBST (15 min for each washing).

Detection of the S-tag label was then carried out as prescribed by the supplier (Novagen) with a colorimetric substrate. Quantification of the colorimetric reaction (precipitate) was carried out by optical scanning of the filter and quantification of the intensity in each well was carried out using the Tina 2.1 program (Raytest).

o 15 5.3 Test of PS2NT/A 42 interaction in ELISA 0 0 format The synthetic AP peptide (1-40 and 1-42) o o o (100 pl, 2 Gg/ml) is incubated overnight in 96-well plates for binding to the plastic. The plates are 20 rinsed twice with PBS and the non-specific binding sites are saturated by incubation with 5% of o .0 bovine serum albumin in PBS. The purified (according to the protocol described in 5.1) recombinant protein, PS2NT, diluted in buffer (25 mM Tris/HC1, pH 7.5, o o Triton X-100, 0.5% NP40) is added and incubated for 4h at room temperature. After rinsing twice with Tween, the PS2NT protein retained on the plate (by interaction with the AB peptide) is revealed by 48 or for 24 h at 4 0 C, and the plates are read on a Packard Discovery counter which measures the emission of europium cryptate at 620 nm after excitation at 337 nm, on the one hand, and the emission of XL665 at 665 nm after fluorescence transfer at 620 nm by the europium cryptate to XL665, on the other hand. The formation of the XL665-streptavidin/biot-AB42/PS2NTcryptate complex leads to a fluorescence transfer from the cryptate to XL665, which is measured at 665 nm by the counter. In the absence of formation of an XL665streptavidin/biot-A342/PS2NT-cryptate complex, the europium cryptate fluoresces at 620 nm.

EXAMPLES

15 Example 1. Interaction between APP and PS2 and mapping of the zone of interaction on PS2 The aim of this example is to determine the zone of interaction of PS2 and to detect an interaction between the said region and APP.

20 The interaction between the APP and PS2 proteins in mammalian cells is exemplified in Figure 2.

The lysate of COS cells transfected with PS2 and APP is subjected to an immunoprecipitation with an antibody directed against the N-terminus of PS2 (95041, Blanchard et al., 1997). The immunoprecipitate is then analysed by immunotransfer with an anti-APP antibody.

The APP is clearly detected in the immunoprecipitates of the cells co-transfected with APP and PS2, but not in the absence of PS2 (Fig. 2, lane 6 relative to lane 7) as described previously (Weidemann et al., 1997). To map the zone of interaction between these two proteins, several truncated forms of PS2 were constructed. In order to conserve the membrane topology of PS2 determined in general by the N-terminal portion of the membrane proteins, progressive truncations of the Cterminal end of PS2 were produced, ending after different transmembrane domains TM6 (PS2AC1), TM4 (PS2AC2), TM3 (PS2AC3) and TM2 (PS2AC4), scheme, Fig. 1A. The hydrophilic N-terminal end (87 residues) of PS2 was also constructed in cytoplasmic form (native sequence) or in secreted form by insertion of the signal peptide for the Igk chain. The expression of S. 15 these various forms is exemplified in Figure 1B, revealed with the anti-PS2 antibody (95041). The constructs containing hydrophobic domains additionally present bands corresponding to the monomeric forms with expected molecular weights (forming close doublets in this case), dimeric forms and aggregates of high *molecular weight typical of PS2 (Fig. 1B, lanes In particular for whole PS2, only these aggregates are detectable in this figure, while the monomeric form is not detectable (lane The two constructs of the hydrophilic N-terminus of PS2: mycPS2NT and SecPS2NT, give rise to bands of expected molecular weight (Fig. 1B, lanes 1 and The construct SecPS2Nt is also secreted in the extracellular medium (Fig. 3B, lane while the construct mycPS2Nt is itself cytoplasmic.

These constructs were co-transfected individually with APP. The detergent-soluble fraction of the cell lysates was immunoprecipitated with the antibody directed against the N-terminus of PS2 and these immunoprecipitates were analysed by immunoblotting. As with the whole PS2, APP is detectable in the immunoprecipitates with all the truncated forms of PS2, PS2AC2 to PS2AC4 (Fig. 2, lanes demonstrating the interaction between APP and the forms containing the N-terminus of PS2. This interaction with APP is conserved with the PS2 Nterminus construct in its secreted form (Fig. 2, lane 15 demonstrating that the anchoring of PS2Nt in the lipid membrane is not necessary for this interaction.

In contrast, the cytoplasmic form mycPS2NT does not interact with APP (Fig. 2, lane 1) The inverse experiment of immunoprecipitation 20 with an anti-APP antibody and detection with the PS2 Nterminus antibody made it possible to confirm the interaction between APP and SecPS2Nt under different experimental conditions.

In the culture medium for the cells cotransfected with APP and SecPS2Nt, an interaction between these two proteins is also demonstrated by coimmunoprecipitation (Fig. 3A, lane 4 and Fig. 3B, lane The mycPS2NT form does not interact with APP in the medium (Fig. 3B, lane The presence of this interaction in the medium demonstrates that the APP/PS2Nt complex is relatively stable during the secretion process.

Example 2. Interaction between APP and PS2 and mapping of the zone of interaction on APP.

The aim of this example is to demonstrate the zone of interaction, on APP, and to detect an interaction between the said region and presenilin 2.

To this end, truncated forms of APP were used to delimit the zone of interaction on APP. A construct comprising the final 100 residues of APP under the control or otherwise of a secretion peptide (SPA4CT and 15 C100, Dyrks et al., 1993) and a construct comprising 0e° only the cytoplasmic domain (the final 45 residues of APP) were used and their presence was detected using an antibody directed against the cytoplasmic domain of APP (aCT43, Stephens and Austen, 1996). In the cells co- 20 transfected with PS2, an interaction of SPA4CT but not of the cytoplasmic domain of APP with PS2 could be detected (Fig. 4A, compare lanes 4 and The interaction of PS2 with the construct C100 (no secretion signal) could also be demonstrated (Fig. 4B, lane Even by combining the cytoplasmic domain of APP with the membrane in a chimeric construct with the alpha receptor of IL2, no interaction with PS2 could be observed. This example demonstrates that there is an interaction with SPA4CT (residues 597 to 695 of APP) but not with the cytoplasmic domain (residues 651 to 695), thus indicating that on APP, the region of the AP (residues 597 to 637) and the rest of the transmembrane segment (up to residue 650) are sufficient for the interaction with PS2.

Example 3. Interaction of PS1 with APP and initial mapping The aim of this example is to determine the zone of interaction on PS1 and to confirm the interaction between the said region and APP.

By analogy with the results obtained for PS2 (Examples 1 and the study of the interaction of PS1 15 with APP was carried out in the same COS1 cell system.

After co-immunoprecipitation with an antibody directed against the final 20 amino acids of PS1 (Duff et al., 1996), SPA4CT, the C-terminal fragment of APP, could be detected in the precipitates (Fig. 5A, lane APP 20 also interacts with PS1. Similarly, the truncated form of PS1, PS1AC2 (1-213), interacts with APP (Fig. S.lane These first data make it possible to envisage that the regions of interaction between APP and PS1 should be close to those exemplified previously with PS2.

To check the validity and generality of this PS1/APP interaction, a different cell system was used, in which the insect cells were infected with 53 recombinant baculoviruses expressing PS1 with or without a His6 label and APP (cf. Materials and Methods). The study of the cell lysates allowed APP to be detected in the anti-His6 immunoprecipitates (for PS1-His6, Fig. 6A, lane 4) or antiPS1 (for PS1 with or without His6, Fig. 6B, lanes 4 and 5) when the cells are co-infected with the two types of recombinant virus, but not when only one of the proteins is expressed (corresponding lanes 1, 2 and Conversely, in the anti-APP immunoprecipitates, the PS1-His6 and PS1 proteins are detectable (Fig. 6C, lanes 4 and for the double infections. This experiment makes it possible to confirm the interaction in the reverse sense with different antibodies.

4 4 Example 4. Interaction of A0 and PS2 in cells 4* Given that the region of interaction between So4 APP and PS2 involves on APP a region including the amyloid peptide (from 595 to 635) and, on PS2, its 20 hydrophilic N-terminal domain, it is demonstrated in this example that PS2 interacts directly with the amyloid peptide (AP) produced by cells. The expression of SPA4CT (corresponding to the final 100 residues of APP preceded by a signal peptide) in COS cells leads to a strong production of the amyloid peptide, partly because SPA4CT is considered as the biological precursor of AP. The COS cells were transfected with SPA4CT alone, SPA4CT and SecPS2Nt or with SecPS2Nt alone. The corresponding extracellular media were immunoprecipitated with the antiPS2 antibody and the AP peptide was detected by means of the specific antibody W02 (Nida et al. (1996) J. Biol. Chem. 271, 22908- 914)(Fig. The A3 peptide is identified only for the cells co-transfected with SecPS2Nt and SPA4CT, as a band of low intensity (Fig. 7, lane but not with the individual controls (lanes 2 and Moreover, an additional band at approximately 40 kDa is also detected specifically for the doubly transfected cells.

After washing the filter and detection with the PS2 antibody, it appears that a band of the same molecular weight is also PS2-immunoreactive (Fig. 7, lane 4).

This band is also present for the cells transfected 15 with SecPS2Nt as expected. Thus, in the doubly 0Sw* 6 SO transfected cells, this band represents an SDS-stable complex between SecPS2Nt and A3, which can confirm the interaction between these two species. The small difference in mass provided by the A3 peptide (4kDa) :20 would explain why there is no detectable difference in size with the cells transfected with SecPS2Nt alone.

The results of these experiments make it possible to conclude that the secreted form of PS2 (secPS2Nt) interacts in vitro with the AP peptide (residues 597- 637 of APP695).

Example 5. Reconstitution of the interaction of A3 and PS2Nt in an in vitro test on nitrocellulose membranes The aim of this example is to demonstrate the reconstitution of the PS2Nt-APP(Ap) interaction in vitro.

To confirm the interaction between the A3 peptide and PS2Nt (N-terminal end of PS2), a test of in vitro binding was developed. A PS2Nt fusion protein carrying the label/marker peptide S (S-tag) at its Nterminal end was constructed and expressed in bacteria.

The peptides AiP-4 0 and ApI- 42 were deposited on nitrocellulose membranes which were incubated in the presence of a bacterial extract expressing the PS2Nt protein. The S-tag was then revealed with the S-tag- 15 binding protein coupled to alkaline phosphatase and by colorimetric reaction. The S-tag-PS2Nt protein does S•indeed bind to the A3 peptides in this in vitro test (Fig. 8A). As controls, duplicates were incubated in the presence of a bacterial extract expressing only the peptide S which is used as the level of non-specific binding on the AB peptide (forms 1-40 and 1-42). Serial dilutions of the bacterial extract make it possible to establish that this binding is dose-dependent and saturable. In this experiment, the binding appears to be greater on Ai1- 42 than on AIP-4 0 with, however, a certain level of variability. For example, the binding of PS2Nt is dependent on the dose of AB deposited on the membrane, while AiP-40 and A31- 42 show equivalent binding values.

This example thus provides a demonstration of the reconstitution of the PS2Nt-APP (AP) interaction in vitro between synthetic AB and PS2NT of bacterial origin. Given that the pathological mutations of PS2 lead to an increase in the Apl-42/Apl-40 ratio produced in many systems and that, moreover, there is a physical interaction between PS2 and APP, it appears that this physical interaction might be involved in the production of the A1- 42 peptide. Thus, the inhibition of this interaction constitutes an extremely novel therapeutic approach for Alzheimer's disease.

15 Example 6. Test of AP42/PS2NT interaction in 96-well S*.i (ELISA type) format Fig. 11 Example 5 provides results demonstrating the direct interaction between the AP peptide and the PS2NT protein on nitrocellulose membrane. The aim of this 20 example is to confirm the information of Example 5 and to describe the detection of the interaction in a 96o* well-plate format. The AP peptide (Ap40 or AP42) is bound to plastic 96-well plates by incubation. The plates are then incubated with the recombinant PS2NT protein. After rinsing, the detection of the interaction (A3/PS2NT) is carried out by binding the Stag-binding protein to the wells and colorimetric revelation. PS2NT binds in a dose-dependent manner to the A3l-42 peptide (Fig. 11) but not to the peptide or to the peptide of inverted sequence AP40-1, nor to another amyloid-generating peptide, amylin. The amounts of Ap1-42 or A1p-40 peptide bound to the plates are identical, as confirmed by immunodetection. The binding constant for PS2NT onto AP42 is 0.18 JM. The specificity of PS2NT for the AP42 form of the peptide relative to AP40 was merely suggested in Example This example establishes this specificity, which is entirely reproducible in the present test.

This test format of Ap42/PS2NT interaction thus makes it possible readily to envisage a test for screening molecules which inhibit this interaction.

15 Example 7. Test of AP42/PS2NT interaction in HTRF Sformat In Examples 5 and 6, the direct interaction between the AP42 peptide and the PS2NT protein was demonstrated firstly on nitrocellulose membrane and 20 secondly on 96-well plates (ELISA type).

The aim of this example is to confirm these data, and describe the detection of the interaction in liquid/homogeneous phase using the fluorescence transfer technique.

The principle of the test described in Materials and Methods is based on fluorescence transfer. The recombinant protein PS2NT labelled with europium cryptate (PS2NT-K) interacts with the biot-A342 peptide, as shown in Figure 12 (bar No. 3).

This signal is decreased and thus the PS2NT-K/biot-A342 interaction is displaced by an excess of unlabelled PS2NT (IC 50 400 nM). The fluorescence signal detected is stable over time: from 4h at room temperature to 24h at 4 0 C (depending on the conditions chosen, described in Materials and Methods).

This interaction is dose-dependent for the biot-A342 peptide (zone of signal linearity from 0 to 2.5 jM) and for PS2NT-K (zone of signal linearity from 0 to 7.5 nM). As indicated in Figure 12, bar No. there is no interaction of PS2NT-K with the biot-A340 peptide bearing an additional specificity element. The specificity of PS2NT for the AP42 form of the peptide 15 relative to A340, which was merely suggested in Example 5, is thus confirmed in Example 6 and the present example.

This HTRF test of interaction between PS2NT and A342 (reflecting the APP/PS interaction in cells) 20 thus allows the identification of chemical molecules which inhibit this interaction by means of a highthroughput screening.

Example 8. Reconstitution of the APP/PS1 interaction in vitro The aim of this example is to demonstrate that the interaction between the whole APP and PS1 proteins can be recreated using different cell lysates, mixed together only to detect the interaction.

Since the baculovirus expression system allows the expression of large amounts of recombinant proteins, the lysates of cells individually infected with each of the three viruses were used as a source of APP, PS1 and PSl-His6 proteins. The solubilized fractions containing APP, PS1 or 6HisPS1 are mixed together and then immunoprecipitated in the presence of anti-Histidine or anti-PS1 antibodies overnight at 4 0

C.

The APP is clearly detected in the immunoprecipitates (Fig. 9A, lane 3 and Fig. 9B, lane demonstrating that the interaction of APP with PS1-His or PS1 is reconstituted in vitro by incubating the two proteins.

15 APP alone appears to be weakly precipitated by the anti-PS1 antibody (Fig. 9B, lane but not with the anti-His antibody, thus confirming the specificity of the interaction in the latter case. These results allow the development of a test of in vitro interaction of the two whole proteins APP and PS1.

Example 9. SecPS2Nt blocks the interaction of APP and PS1 in transfected cells It has been demonstrated in the preceding examples that APP interacts with PS1 in a similar manner to PS2 and that, for the latter, the SecPS2Nt construct is sufficient for the interaction with APP.

The aim of this example is to evaluate whether or not the binding of SecPS2Nt to APP can block the interaction with PS1 in a crossed (heterologous) manner. In the COS1 system, SPA4CT (corresponding to the final 100 residues of APP preceded by a signal peptide) can be detected in the anti-PS1 immunoprecipitates of the cells expressing SPA4CT and wt PS1 or mutant PS1, PS1* (Fig. 10A, lanes 1 and 2).

In addition, when SecPS2NT is also co-transfected, the SPA4CT signal virtually disappears in the anti-PS1 immunoprecipitates (Fig. 10A, lanes 3 and After the anti-PS1 immunoprecipitation, the supernatants (fraction not linked to protein A sepharose) underwent a second immunoprecipitation with the anti-PS2 antibody. The SPA4CT is clearly detected in the cells 15 co-transfected with PS1 and SecPS2Nt (Fig. 10B, lanes 3 and demonstrating that in these cells, on becoming bound, SecPS2Nt displaced the binding of SPA4CT to PS1.

This experiment thus makes it possible to conclude that SecPS2Nt is a molecule capable not only of binding to 20 APP but also of displacing the binding of APP to PS1 and quite probably to PS2. SecPS2Nt can thus be used in cells as a decoy to block the interaction of APP with the two presenilins, PS1 and PS2. Specifically, the results of the mapping of the PS1/APP interaction confirm that the zones of interaction involved are similar to those of PS2.

Example 10. Blocking of the APP/PS1 interaction leads to inhibition of the production of the intracellular Ap42 amyloid peptide The preceding example demonstrates that the expression of SecPS2NT can block the interaction between APP and PS1 or mutant PS1. The present example analyses the consequences of this inhibition on the production of the amyloid peptide, in particular of these two forms AP40 and AP42. Specifically, it has been described previously in the literature that the pathological mutations of the presenilins (PS1 or PS2) led to an increase in the ratio of the long form of the AP peptide, the AP42 form, to the A340 form, the S. A42/A40 ratio (Borchelt et al., 1996 and, for review, 15 Hardy, 1997).

SPA4CT was co-expressed with wt PS1 (Fig. 13A, lanes 1 3) or with mutant PS1 (Fig. 13A, lanes 2 4) either in the absence (lanes 1 2) or in the presence of SecPS2NT (lanes 3 The cell lysates and the conditioned cell media were analysed for the production of the amyloid peptide. The AB40 and A342 forms were analysed by immunoprecipitation with antibodies specifically recognizing the C-terminal ends of AP40 (FCA3340) or of A342 (FCA3542, Barelli et al., 1997) and the immunoprecipitates were analysed by immunoblotting with an antibody which recognizes the two forms. In the cell lysates, the expression of mutant PS1 does indeed lead to an increase in the production of A342 to 2-fold) and of its multimeric forms relative to wt PS1 and little variation in the levels of Ap40 (compare Fig. 13A, lanes 1 and 2, AP42 and AP40 cell lysate panels) as expected. In the presence of SecPS2NT, the levels of AB42 (and multimers) are considerably reduced (Fig. 13A, lanes 3 and 4) both with wt PS1 and with mutant PS1. In the extracellular medium, the levels of Ap42 also appear to be reduced, but to a smaller extent. There is no variation in the levels of amyloid peptide between the different conditions, which demonstrates that the effect on A42 is specific and is not due to an overall change in the levels of expression. This is confirmed by analysis of the 15 expression of the various genes transfected: SPA4CT, S* SecPS2NT and PS1 (Fig. 13B). In this example, it has thus been demonstrated that inhibiting the PS1/APP interaction with the genetic dominant SecPS2NT leads to a decrease in the levels of production of intracellular AB42 both with mutant PS1 and with wt PS1. With regard to the primordial role accorded to Ap42 in the development of Alzheimer's disease, this example provides the demonstration that inhibition of the APP/PS interaction thus represents a therapeutically important target both for the genetic forms and for the sporadic forms of the disease.

63 Example 11. Detection of the interaction of PS2 with the endogenous APP of COS cells using a pharmacological treatment The aim of this example is to demonstrate that the results obtained in the preceding examples (these results in cells corresponded to the overexpression of the two partners of the interaction: APP and PS (PS1 or PS2)) are also valid with nonoverexpressed or endogenous proteins. Specifically, strong overexpression of the two proteins might lead to an interaction artefact. The detection of the interaction under conditions not involving the simultaneous overexpression of the two partners was investigated in this example.

15 COS cells express APP endogenously, although at low levels. The COS cells were thus transfected with PS2 alone. The cell lysates were analysed by immunoprecipitation with the antibody directed against the PS2 N-terminal peptide and revealed by immunoblotting with the anti-APP antibody, W02. The upper panel in Figure 14 shows that the transfection with PS2 alone does not make it possible to detect any interaction with endogenous APP by coimmunoprecipitation (Fig. 14, lane 1).

Moreover, since the APP/PS interaction leads to the production of the A342 amyloid peptide (preceding example), and thus to the catabolism of APP, in the endoplasmic reticulum, the proteasome which is 64 the proteolytic degradation system in this cell compartment might be involved. The effect of lactacystine, a selective inhibitor of the proteasome, was analysed. After incubating cells transfected with PS2 in the presence of lactacystine, the endogenous APP of the COS cells can be clearly detected in the PS2 immunoprecipitates (Fig. 14, lane 5, band at 110 kDa).

This interaction with the endogenous APP has the same characteristics as previously, since it can be displaced by the genetic dominant SecPS2NT (Fig. 14, lanes 6 and 7) with a dose-dependent effect.

Specifically, lane 7 shows that a moderate dosage of SecPS2NT, a band of low intensity corresponding to the endogenous APP is always visible. At a higher dosage of 15 SecPS2NT (lane the band corresponding to the •0 endogenous APP appears very weakly and demonstrates the dose-dependent nature. A weak residual APP signal is always present (band at about 110 kDa) and is due to the complex between APP and SecPS2NT which is rapidly 00 20 secreted (since SecPS2NT has no more transmembrane anchoring domains) and thus does not accumulate intracellularly.

Figure 14 (middle and lower panels) shows that the treatment with lactacystine does not effect the total levels of APP either cellular or secreted.

Specifically, the bands corresponding to the levels of expression of APP are virtually constant in intensity.

The specificity of the lactacystine effect is thus demonstrated on the sub-population of APP interacting with PS2.

From these results, it has been possible to demonstrate that the APP/PS2 interaction can be detected with the endogenous APP of the COS cells if the proteasome is inhibited. In addition, these results demonstrate that the APP/PS2 interaction can be detected under less artificial conditions. However, this interaction is very labile. Thus, in order to obtain a more pronounced detection, use was made either of an inhibitor of proteolytic degradation in the present example, or of the overexpression of the two partners in the preceding examples. This example thus demonstrates that the results obtained in the preceding examples with cellular overexpression of the two partners of the interaction (APP and PS1 or PS2) are also valid with non-overexpressed or endogenous 4 proteins.

Example 12. PS2 interacts with a second segment of APP, other than AD The aim of this example is to demonstrate that another segment of APP interacts with PS2.

It has been demonstrated previously that SecPS2NT interacts in the extracellular medium with the secreted forms of APP (Fig. 3A, lane The secreted forms of APP are released after cleavage either at the 3 site (position 595), corresponding to the start of the A3 peptide, or at the a site (position 612) within this peptide. These results suggest that PS2NT also interacts with an N-terminal region of APP other than AP. Truncated forms of APP were constructed by inserting a stop codon into the 3 site (0-sAPP) and into the a site (a-sAPP) and were tested. Whole PS2 and SecPS2NT effectively interact with a-sAPP (Fig. lanes 3 and 5) and 3-sAPP (Fig. 15, lanes 4 and 6).

These results establish that a segment of APP between position 1 and 595 (and thus other than A3) is also capable of interacting with PS2 and PS2NT. These results also make it possible to confirm that the PS2NT/APP interaction can take place in the absence of anchoring of the two partners to the membrane and in 15 the luminal (or extracellular) compartment of the cell.

*o eagoe 67 References Doan et al. (1996) Protein topology of presenilin 1.

Neuron 17: 1023-1030.

Thinakaran et al., (1996) Endoproteolysis of Presenilin 1 and accumulation of processed derivatives in vivo. Neuron 17: 181-190 Podlisny et al., (1997) Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue.

Neurobiology of Disease 3: 325-337 Pradier, Czech, Mercken, Revah, F. and Imperato, A. (1996) Biochemical characterization of 15 presenilins (S182 and STM2) proteins. Neurobiol.

Aging 17: S137 Scheuner et al., (1996) Secreted amyloid b-protein •similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and S* 20 2 and APP mutations linked to familial Alzheimer's disease. Nature Med. 2: 864-870 Dyrks, Dyrks, Minning, Urmoneit, B., Turner, J. and Beyreuther, K. (1993) Generation of bA4 from the amyloid protein precursor and fragment thereof. FEES Lett. 335: 89-93 Weidemann, Paliga, Dirrwang, Czech, C., Evin, Masters Beyreuther, K. (1997).

Formation of stable complexes between two Alzheimer's disease gene products: Presenilin-2 and b-Amyloid precursor protein. Nature Medicine 3: 328-332 Blanchard, Czech, Bonici, Clavel, N., Gohin, Dalet, Revah, Pradier, L., Imperato, A. and S. Moussaoui. (1997) Immunohistochemical analysis of presenilin 2 expression in the mouse brain: distribution pattern and co-localization with presenilin 1 protein. Brain Res. 758: 209-217 Borchelt, Thinakaran, Eckman, Lee, Davenport, Ratovitsky, Prada, Kim, Seekins, Yager, Slunt, Wang Seeger, Levey, Gandy, Copeland, Jenkins, Price, Younkin, S.G. and S.

15 Sisodia (1996) Familial Alzheimer's disease-linked Spresenilin 1 variants elevate Abl-42/1-40 ratio in *o vitro and in vivo. Neuron 17: 1005-1013 oo S- Duff, Eckman, Zehr, Yu, Prada, Perez-tur, Hutton, Buee, Harigaya, Y., 20 Yager, Morgan, Gordon, Holcomb, L., Refolo, Zenk, Hardy, J. and S. Younkin (1996) SIncreased amyloid-b42(43) in brains of mice expressing mutant presenilin 1. Nature 383: 710-713.

Hardy, J. (1997) Amyloid, the presenilins and Alzheimer's disease. Trend in Neurosci. 20: 154-159.

Stephens, D.J. and B.M. Austen (1996) Metabolites of the b-amyloid precursor protein generated by bsecretase localise to the Trans-Golgi Network and 69 late endosome in 293 cells. J. Neurosci. Res. 46: 211-225.

Essalmani, Guillaume, Mercken, and Octave, (1996) Baculovirus-Infected Cells Do not Produce the Amyloid Peptide of Alzheimer's Disease from its Precursor. FEBS Lett. 389: 157-161 Barelli et al., (1997), Characterization of new polyclonal antibodies specific for 40 and 42 amino acid-long amyloid P peptides: their use to examine the cell biology of presenilins and the immunohistochemistry of sporadic Alzheimer's disease and cerebral amyloid angiopathy cases. Molecular Medecine 3: 695-707.

*e SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: RHONE-POULENC RORER STREET: 20 Avenue Raymond Aron CITY: Antony COUNTRY: France POSTAL CODE: 92165 FAX: 01.55.71.72.91 (ii) TITLE OF THE INVENTION: Peptides capable of inhibiting the interaction between presenilins and the beta-amyloid peptide precursor and/or the beta-amyloid peptide, and test of interaction to search for molecules which inhibit the said interaction.

(iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER-READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30

(EPO)

INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 261 base pairs TYPE: nucleotide STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..261 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG CTC ACA TTC ATG GCC TCT GAC AGC GAG GAA GAA GTG TGT GAT GAG 48 Met Leu Thr Phe Met Ala Set Asp Ser Glu Glu Glu Val Cys Asp Glu 1 5 10 CGG ACG TCC CTA ATG TCG GCC GAG AGC CCC ACG CCG CGC TCC TGC CAG 96 Arg Thr Sex Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gin 20 25 GAG GGC AGG CAG GGC CCA GAG GAT GGA GAG AAT ACT GCC CAG TGG AGA 144 Glu Gly Arg Gin Gly Pro Glu Asp Gly Glu Asn Thr Ala Gin Trp Arg 40 AGC CAG GAG AAC GAG GAG GAC GGT GAG GAG GAC CCT GAC CGC TAT GTC 192 Ser Gin Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val 55 TGT ACT GGG GTT CCC GGG CGG CCG CCA GGC CTG GAG GAA GAG CTG ACC Cys Sex Gly Val. Pro GJly Arg Pro Pro Gly Leu Giu Glu Glu Leu Thz 70 75 CTC AAA TAC GGA GCG AAG CAT Leu Lys Tyr Gly Ala Lys His INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 243 base pairs TYPE: nucleotide STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: l. .243 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 240 261 ATG ACA GAG TTA CCT GCA CCG TTG TCC TAC Met Thr Giu Leu Pro Ala Pro Leu Set Tyr 1 510 TIC CAG AAT GCA CAG ATG Phe Gin Asn Ala Gin Met 9, 0 S 000 0 .004 .9 £0 S 5* 0

S

5*

S

900905 9 9* 0.

0 0 TCT GAG GAC Ser Glu Asp 7AAC CAC Asn His 20 CTG AGC AAT Leu 5er Asn GTA CGT AGC Val Arg Ser AGA GAA CGG GAG GAG CAC Ac GAC Arg Glu Axrg Gin Glu Hi~s Asn Asp 35 40 AGA CGG AGC CTT Arg Arg Ser Leu GAG AAT GAC AAT Gin Asn Asp Asn GGC CAC CCT GAG Gly His Pro Glu GAG GTC CTC GAG Gin Val Val Glu 96 -4 4 192 240 CCA TTA Pro Leu CAA GAT Gin Asp TCT AAT1 GGA CGA Sem Asn Gly Arg GAG GAA GAA GAT Glu Glu Glu Asp 70 CAG GGT AAC Gin Gly Asn TCC CGG Ser Arg GAG GAG CTG ACA Glu Giu Leu Thr TIC AAA TAT GGC GCC AAG Leu Lys Tyr Gly Ala Lys 75 s0 0 9

S

0600 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 2088 base pairs TYPE: nucleotide STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 72 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION:1. .2088.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG CTG CCC GGT TTG GCA CTG CTC CTG CTG GCC GCC TGG ACG GCT CGG 48 Met Leu Pro Gly Leu A1a. Leu Leu ieu Leu Ala Ala 'rzp Thr Ala Arg 1 5 10 C CTG GAG CTA CCC ACT CAT CCT AAT CCT CCC CTC CTC CCT GAA CC 96 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 CAG ATT GCC ATG T'TC TGT GGC AGA CTG AAC ATG CAC ATG AAT GTC CAG 144 Gin Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 40 AAT GGG AAG TGG CAT TCA GAT CCA TCA GGC ACC AAA ACC TGC ATT GAT 192 Asn Cly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 ACC AAG GAA GGC ATC CTG CAG TAT TGC CAA GAA GTC TAC CCT GAA CTG 240 Thr Lys Glu Gly Ile Leu Gin Tyr Cys Gin Giu Val Tyr Pro Glu iLeu '70 75 CAG A7C ACC AAT GTG GTA GAA GCC AZ CAA CCA GTG ACZ ATC CAG AAC 288 Gin Ile Thr Msn Val Val Giu A la Mn Gin Pro Val Thr Ile Gin Asn 90 **TGG TCC AAG CGG GGC CCC AAG CAC TGC AAG ACC CAT CCC CAC TTT GTG 336 Trp Cys Lys Arg Gly Arq Lys G-In Cys Lys Th r 1-1is Pro His Phe Val 100 .05 110 *ATT CCC TAC CGC TGC TTA GTT GGT GAG TTT GTA AGT GAT GCC CTT CTC 384 Ile Pro Tyr Arg Cys Leu Val dly Giu Phe Val Ser Asp Ala Leu Leu 115 120 125 CTT CCT CAC AAC TCC AAA TTC TTA CAC ZAG GAG AGG ATG CAT CTT TGC 432 *Val Pro Asp Lys Cys Lys Phe Leu His Gin Giu Arg Met Asp Val Cys 130 135 140 GAA ACT CAT CTT CAC TCG CAC ACC GTC GCC AAA GAG ACA TGC ACT GAG 480 Glu Thr His Leu His Trp His Thr Val Aia Lys Ciu Thr Cys Ser Clu 145 150 155 160 AAC ACT ACC AAC TTC CAT CAC TAC CCC ATG TTG CTG CCC TCC GCA ATT 528 Lys Ser Thr Asn Leu His Asp Tyr Giy Met Leu Leu Pro Cys Gly Ile 165 170 175 *GAC AAG TTC CGA GGG GTA GAG TTT GTG TGT TGC CCA CTG GCT GAA GAA 5-76 Asp Lys Phe Arg Gly Val Giu ?he Val Cys Cys Pro Leu Ala Ciu Ciu 180 185 190 ACT GAC PAT GTG GAT TCT GCT GAT GCG GAG GAG CAT GAC TCG GAT GTC 624 Sez Asp Asn Val Asp Ser Ala Asp Ala Glu Giu Aap Asp Scr Asp Val 195 200 205 TGG TCC CCC CCA GCA GAC ACA GAC TAT GCA CAT GGG ACT GAA CAC AA 672 73 Tyr Ala Asp Trp rrp 210 Gly Gly Ala Asp Thr Asp 215 Ser Giu Asp Lys a ao a a a

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AAC

Asn

ACA

Thr

TCC

Ser 655

GAG

Glu 480

AAC

As

AGT

Ser

ACC

Thr

CAG

Gin

AAC

Asn 560

ACC

Thr

TCT

Ser

OTT

Val

AAA

Lys

GTG

Val 640

ATT

Ile

CGC

Azg 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1966 2016 2064 2088 S S

S

S

S

GGC TAC GAA AAT CCA ACC TAC AAG Gly Tyr Glu Asn Thr Tyr Lys INFORMATION FOR SEQ ID NO: 4: oligo SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..38 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGGAATTCATCGATTCCACCATGCTCACATTCATGGCC 38 INFORMATION FOR SEQ ID NO: 5: oligo SEQUENCE CHARACTERISTICS: LENGTH: 43 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA "(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..43 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCGCTCGAGTCATTGT CGACCATGCTTCGCTCCGTATTTGAGG 43 INFORMATION FOR SEQ ID NO: 6: oligo 8172 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CAAAGATCTGATGCAGAATTCCGACAT 27 INFORMATION FOR SEQ ID NO: 7: oligo 8181 SEQUENCE CHARACTERISTICS: LENGTH: 59 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..59 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CAAGCGGCCGCTCATCCCTTGTCATCGTCGTCCTTGTAGTCTCCGTTCTGCATCTGCTC59 INFORMATION FOR SEQ ID NO: 8: oligo 8171 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CAAAGATCTAAGAAACAGTACACATCC 27 INFORMATION FOR SEQ ID NO: 9: oligo 77 SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:- NAME/KEY: LOCATION: 1..29 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ccatcgatggctaCATCTTCACTTCAGAG 29 INFORMATION FOR SEQ ID NO: 10: oligo SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..31 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ccategatggctaTTTTTGATGATGAACTTC 31 INFORMATION FOR SEQ ID NO: 11: oligo S* SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleotide STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: LOCATION: 1..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CCGTGGAGCTCCTCCCG 17 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

e ooo

Claims (39)

1. Polypeptide capable of at least partially inhibiting the interaction between a presenilin and the 3-amyloid peptide precursor and/or the P-amyloid peptide.

2. Polypeptide according to claim 1, characterized in that it comprises at least one portion of PS2 allowing the interaction with the P-amyloid peptide precursor and/or the P-amyloid peptide.

3. Polypeptide according to claim 2, characterized in that the portion of PS2 corresponds to the hydrophilic N-terminal fragment of PS2.

4. Polypeptide according to one of claims 1 15 to 3, characterized in that it is a polypeptide comprising all or a portion of the sequence SEQ ID SNo. 1 or of a sequence derived therefrom. Polypeptide according to claim 1, characterized in that it comprises at least a portion 20 of PS1 allowing the interaction with the P-amyloid peptide precursor and/or the P-amyloid peptide.

6. Polypeptide according to claim c. haracterized in that it is a polypeptide comprising all or a portion of the sequence SEQ ID No. 2 or of a sequence derived therefrom.

7. Polypeptide according to one of claims 1 to 6, characterized in that it comprises at least the homologous regions common to the sequences SEQ ID No. 1 and SEQ ID No. 2.

8. Polypeptide according to claim 1, characterized in that it comprises at least one portion of the 3-amyloid peptide precursor other than the portion corresponding to the 3-amyloid peptide.

9. Polypeptide according to claim 8, characterized in that the portion of the -amyloid peptide precursor comprises all or a portion of the fragment 1-596. Polypeptide according to claim 9, characterized in that it comprises all or a portion of a sequence chosen from the sequence corresponding to the fragment 1-596 of the sequence SEQ ID No. 3, or a derived sequence.

11. Non-peptide compound or compound which is not exclusively peptidic, capable of at least partially inhibiting the interaction between a presenilin and the -amyloid peptide precursor and/or the -amyloid peptide, obtained by reproduction of the active units of the peptides according to claims 1 to *o 10 with non-peptide structures or structures that are not exclusively peptidic.

12. Polypeptide according to one of claims 1 to 11, characterized in that it also comprises a signal sequence.

13. Polypeptide according to claim 12, characterized in that the signal sequence is chosen from the sequence of the IgkB signal peptide, the APP signal peptide and the signal peptides of the muscular and central acetylcholine nicotinic receptor subunits.

14. Polypeptide according to claim 13, characterized in that it is a polypeptide comprising the N-terminal end of PS1 or PS2. Polypeptide according to claim 14, characterized in that it is a polypeptide comprising the first 87 residues of the N-terminal end of PS2 and the IgkB signal peptide.

16. Nucleotide sequence encoding a polypeptide as defined according to one of claims 1 to 15

17. Nucleotide sequence according to claim S16, characterized in that it is a sequence comprising all or a portion of the nucleotide sequence SEQ ID No. 1 or of a sequence derived therefrom.

18. Nucleotide sequence according to claim 16, characterized in that it is a sequence comprising all or a portion of the nucleotide sequence SEQ ID No. 2 or of a sequence derived therefrom.

19. Nucleotide sequence according to claim 16, characterized in that it is a sequence essentially comprising the homologous zones common to the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2. Nucleotide sequence according to claim 16, characterized in that it is a sequence corresponding to the fragment 1-596 (nucleic acids 1 to 1788) of the sequence SEQ ID No. 3, or a derived sequence.

21. Process for preparing a polypeptide according to one of claims 1 to 15, characterized in that a cell containing a nucleotide sequence according to one of claims 16 to 20 is cultured under conditions in which the said sequence is expressed, and the polypeptide produced is recovered.

22. Host cell for producing a peptide according to one of claims 1 to 15, characterized in that it has been transformed with a nucleic acid 15 comprising a nucleotide sequence according to one of claims 16 to

23. Process for detecting or isolating compounds capable of at least partially inhibiting the interaction between a presenilin and the P-amyloid peptide precursor and/or the P-amyloid peptide, :*characterized in that it comprises at least one step of labelling the presenilins and/or the APP or fragments thereof and a step of detecting the inhibition of the interaction either between the peptide A31- 42 and the N-terminal end of the presenilins, or between the whole APP proteins and presenilins. 83

24. Process according to claim 23, characterized in that the compounds isolated by the process are capable of at least partially inhibiting the interaction between the Ai1- 42 peptide and the N-terminal end of PS2. Process according to either of claims 23 and 24, characterized in that the following steps are carried out: the peptide AII- 42 is preabsorbed onto a nitrocellulose membrane by incubation; a bacterial extract containing all or a portion of a presenilin (PS1 or PS2) and advantageously the N-terminal end, is then added for incubation with the molecule or a mixture containing various test S 15 molecules; the interaction of the presenilin with the APi- 42 peptide on the nitrocellulose filter is detected o eeo with the aid of presenilin-labelling proteins. The desired molecules inhibit the interaction and thus 20 reduce the intensity of the signal from the labelling proteins.

26. Process according to claim characterized in that one of the marker proteins is the S-tag-binding protein, coupled to alkaline phosphatase.

27. Process according to either of claims 23 and 24, characterized in that the following steps are carried out: 84 the AP1- 42 peptide is preincubated on a plate containing wells (96-well format or higher), the N-terminal end of a purified recombinant presenilin is then added with the molecule or a mixture containing various test molecules, for incubation, after washes, the interaction of the presenilin with the AP1- 42 peptide in the plate is detected with the aid of presenilin-labelling proteins. The loss of interaction between the presenilins and the -amyloid peptide precursor and/or the 3-amyloid peptide is detected by spectrophotometry, after revelation with a colorimetric substrate.

28. Process according to claim 27, 15 characterized in that one of the marker proteins is the *S S-tag-binding protein, coupled to alkaline phosphatase, and the revelation of the loss of interaction is carried out at 450 nm.

29. Process according to either of claims 23 20 and 24, characterized in that the following steps are carried out: a molecule or a mixture containing various molecules with the Ap1- 42 peptide synthesized is placed in contact with a biotin and an arm of 3 P-alanines (or 3 lysines) at its N-terminal end (upstream of position 1), the above reaction mixture is incubated with the N-terminal end of a purified presenilin labelled with a first fluorophore, streptavidin, coupled to a second fluorophore, is added, this second fluorophore being capable of being excited at the emission wavelength of the first fluorophore so that it benefits from a transfer of fluorescence if the two fluorophores are close together, the detection of novel compounds which inhibit the interaction is made by spectrofluorometry at the emission wavelength of the first fluorophore and/or by measuring the reduction of the signal at the emission wavelength of the second fluorophore. 15 30. Process according to claim 29, characterized in that the first fluorophore is Europium cryptate and the second fluorophore is XL665.

31. Process according to claim characterized in that the revelation of the loss of 20 interaction is carried out at 620 nm and/or by measuring the reduction in the signal at 665 nm.

32. Process according to either of claims 23 and 24, comprising the following steps: a mixture a) of cell lysates containing all or a portion of a presenilin (PS1 or PS2) and advantageously the N-terminal end, b) of cell lysates containing the APP, lysates obtained from cells 86 infected with viruses and in particular with baculoviruses, and c) the test molecule or a mixture containing various test molecules, is placed in contact, the solubilized proteins corresponding to the presenilins or to the APP or the A3 peptide are co- immunoprecipitated with the aid of suitable antibodies, the loss of co-immunoprecipitation of the presenilins and of the APP is revealed by Western blotting with labelling antibodies, indicating that the test molecules have the desired inhibitory property.

33. Test of interaction between a presenilin and the P-amyloid peptide precursor and/or the g* 3-amyloid peptide, and preferentially between the API- 42 15 peptide and the N-terminal end of PS2, characterized ih that it comprises at least one step of fluorescence transfer between two fluorophores bound to the above eeo molecules and a step of revealing the interaction measured by fluorometry. 20 34. Test for detecting molecules capable of inhibiting the interaction between a presenilin and the P-amyloid peptide precursor and/or the 3-amyloid peptide, and preferentially between the Ap1- 42 peptide and the N-terminal end of PS2, characterized in that the inhibition of the said interaction is detected according to the process in claims 23 to 32. 87 Ligand a) for the polypeptides as defined according to claims 1 to 15, b) for the presenilins and/or for the P-amyloid peptide precursor and/or for the -amyloid peptide, and preferentially for the Ai1- 42 peptide and/or for the N-terminal end of PS2, which can be obtained by the process according to one of claims 23 to 32.

36. Defective recombinant virus comprising a nucleotide sequence encoding a polypeptide according to one of claims 1 to

37. Vector comprising a nucleotide sequence according to one of claims 16 to

38. Vector according to claim 37, characterized in that it is a plasmid vector. 15 39. Vector according to claim 37, characterized in that it is a viral vector. Vector according to claim 39, C characterized in that it is a virus which is defective for replication. 20 41. Pharmaceutical composition comprising one or more vectors according to one of claims 36 to

42. Pharmaceutical composition comprising, as active principle, at least one polypeptide according to one of claims 1 to 88

43. Pharmaceutical composition comprising, as active principle, at least one ligand according to claim

44. Composition according to one of claims 41 to 43, intended for at least partially inhibiting the interaction between a presenilin and the P-amyloid peptide precursor and/or the P-amyloid peptide. Composition according to one of claims 41 to 43, intended for the treatment of neurodegenerative diseases.

46. Use of the polypeptides according to claims 1 to 15, to at least partially inhibit the interaction between a presenilin and the p-amyloid e* 0 peptide precursor and/or the P-amyloid peptide. 15 47. Use of the polypeptides according to 0o0 0 claims 1 to 15, to obtain a medicinal product intended for the treatment of neurodegenerative diseases and in. particular Alzheimer's disease.

48. Use of the polypeptides according to 20 claims 1 to 15, for the detection of ligands for the polypeptides, ligands for the presenilins, for the P-amyloid peptide precursor and/or for the P-amyloid peptide, and preferentially for the AP1- 42 peptide and/or for the N-terminal end of PS2, and/or of compounds capable of at least partially inhibiting the interaction between a presenilin and the P-amyloid peptide precursor and/or the P-amyloid peptide. 89

49. Use of the nucleotide sequences according to claims 16 to 20, for the construction of an expression cassette which can be used in a vector according to claims 36 to

50. Expression cassette which can be used in a vector defined according to claims 35 to 39, for the production of polypeptides according to claims 1 to 0 0oo *00* 0o0 4 *o 0* 00 0 0 0

51. The steps, features, compositions and compounds disclosed herein or referred to or indicated in the specification and/or claims of this application, individually or collectively, and any and all combinations of any two or more of said steps or features. DATED this THIRD day of MAY 2000 Aventis Pharma S.A. by DAVIES COLLISON CAVE Patent Attorneys for the applicant(s) S. S S S S S. S 55 S S 55 S 0 oaf, S

55.5 .r .00. S