WO2015028694A1

WO2015028694A1 - Method for the screening of molecules useful in the treatment and/or prevention of alzheimer's disease

Info

Publication number: WO2015028694A1
Application number: PCT/ES2014/070671
Authority: WO
Inventors: Miguel RODRIGUEZ MANOTAS
Original assignee: GALLAR RUIZ, Juan Carlos
Priority date: 2013-08-26
Filing date: 2014-08-26
Publication date: 2015-03-05
Also published as: WO2015028694A4; ES2530141A2; ES2530141R1; ES2530141B1

Abstract

The invention proposes blocking the channel of the γ-secretase enzymatic complex attributed to the formation of the Αβ42 peptide located in the structure of presenilin, preferably using molecules exhibiting specificity in relation to peptide determinants bordering the entry region to the channel from the extracellular space. Thus, the invention relates to the use of the presenilin channel _ϒ42 as a pharmacological target for screening molecules that are suitable for improving cognitive performance and/or promoting cognitive development, preferably for the treatment and/or prevention of Alzheimer's disease.

Description

METHOD OF SCREENING OF USEFUL MOLECULES FOR THE TREATMENT AND / OR PREVENTION OF ALZHEIMER'S DISEASE

DESCRIPTION

The present invention falls within the field of neurology and medicine, specifically within the methods of screening molecules useful for improving cognitive performance and promoting cognitive development, preferably for the treatment and / or prevention of diseases or syndromes that produce cognitive impairment, such as Alzheimer's disease.

STATE OF THE PREVIOUS TECHNIQUE

The genes coding for presenilins 1 and 2 (PSEN1 and PSEN2, respectively) and the amyloid precursor protein (ΑβΡΡ) are the only molecules known so far involved in Alzheimer's disease (AD) of monogenic cause (Albert Lleó et al., 2002, Archives of Neurology; 59: 1759-1763). The relationship between these proteins and the disease can be understood as an association between enzyme / substrate in which the enzyme is presenilin and ΑβΡΡ is the substrate.

Presenilin is part of a large enzyme complex called γ-secretase, which is the catalytic nucleus (Bart De Strooper et al., 2003, Neuron; 38: 9-12). In vertebrates there are two presenilin genes: PSEN1, which contains the genetic information for presenilin 1 (PS1) and PSEN2, which contains the genetic information for presenilin 2 (PS2). Both genes are highly conserved among different vertebrate species. In the human species the genes coding for these two proteins have a high homology to each other (80%). The γ-secretase complex consists of a total of four proteins that are present in the same stoichiometry: presenilin, nicastrin, gene associated with the defect in the anterior nasopharynx of drosophila (APH 1) and enhancer of presenilin 2 (PEN2).

First, in the sequential proteolytic processing of the ΑβΡΡ protein, a cleavage by the a- or β-secretase takes place. The α secretase leads to the C83 fragment (aCTF). Excision by β-secretase (BACE1) results in two potential fragments: C99 and C89 (pCTFs). All these fragments may subsequently undergo a first proteolytic cleavage (ε cleavage) by the γ-secretase complex. This process releases the intracellular domain of amyloid (AICD or yCTF) into the cytoplasm (mainly two: AICD51 and AICD50); while on the opposite side (the N-terminal end) leaves a peptide remnant in the transmembrane zone (amyloid peptides: Αβ48 and Αβ49 respectively), attached to the γ-secretase complex:

1. a- or pCTF- »Αβ48 + AICD51

2. a- or pCTF- »Αβ49 + AICD50

Subsequently, the transmembrane remnants yβ48 and maneβ49 can undergo progressive sequential cleavage by the γ-secretase complex in the transmembrane domain (γ cleavage), releasing three or four residues at each step of the cleavage sequence, resulting in two distinct cleavage sequences in Function of the starting peptide:

1. Αβ48 ^ Αβ45 ^ Αβ42 ^ Αβ38.

2. Αβ49 ^ Αβ46 ^ Αβ43 ^ Αβ40 ^ Αβ37.

The cleavage sequence (cleavage represented by the arrow) could be interrupted at any step of the proteolytic process, finally releasing the substrate that has not been cleaved. For the sequence that begins in Αβ49 the most likely final product would be Αβ40 and for which the Αβ42 peptide begins in Αβ48.

A functional drug should treat the disease and stop or delay the cellular damage that leads to worsening of symptoms. In this sense, the efforts of the researchers have focused on finding new ways of treating AD, since current medications only help to mask the symptoms of the disease without treating it.

From the point of view of its action, three types of drugs in AD are studied: those that act on the possible cause of the disease, modulating the production of the Αβ peptide, those that try to prevent the consequences of the disease or neuroprotective drugs and those who try to alleviate the consequences of the disease acting on the symptoms (Hugo Geerts et al., 2013, Frontiers of Pharmaco \ ogy; 4:47).

The current trend towards the development of therapies against AD, which aim to reduce Αβ levels in the brain, is supported by the evidence that supports the amyloid cascade hypothesis (1. the deposition of the Αβ peptide in the parenchyma cerebral in the form of plaques; 2. the fact that the three causal genes of monogenic EA, ΑβΡΡ as a substrate and PSEN1 -2 as enzymes, are involved in the formation of peptide Αβ) and points to the crucial role of the Αβ peptide in disease. According to this hypothesis, the deposition of Αβ constitutes the initial pathological trigger of the disease. Therefore, drugs designed based on this hypothesis try to modify Αβ levels from several fronts (Martin Citrón, 2010, Nature Reviews. Drug Discovery; 9 (5): 387-398): i. Acting on the production of the Αβ peptide. Compounds called "lowerβ lowering compounds": γ-secretase inhibitors and modulators (GSI, GSM) and β-secretase inhibitors (BACE1 inhibitors).

I. Increasing the clearance of the Αβ peptide. It is mainly done by antibodies.

i. Decreasing aggregation of the Αβ peptide.

Apart from the discrete results obtained in the clinical trials conducted with the drugs developed from the amyloid hypothesis, there are two major paradoxes regarding the objectives they pursue and the behavior of the mutations associated with the monogenic cause AD, namely, a) most mutations reduce the overall amount of the Αβ peptide produced and b) all mutations lead to an increase in the ratio between the two main forms of Αβ peptide: Αβ42 / Αβ40 (Eñe Karran, et al., 201 1, Nature Reviews Drug Discovery, 10, 698-712). Although GSM manages to reverse this ratio, they do so at the expense of a change in the production of Αβ42 by Αβ38 (in the same split line), which translates into an increase in toxicity in vivo (Lucia Chávez-Gutiérrez et al., 2012, EMBO Journal; 31 (10): 2261-74). Therefore, although the exact mechanism by which AD occurs remains unclear, the objective of any drug designed to combat this disease should be that of decrease the Αβ42 / Αβ40 ratio without decreasing the levels of the Αβ peptide produced, selectively attacking the ε cleavage, which would condition the products of the subsequent excision sequence (γ cleavage).

Therefore, there is a need to develop new approaches, such as the creation of models of kinetic behavior of the γ-secretase enzyme complex for the different cleavage products of the ΑβΡΡ peptide. The correlation between these kinetic models and the established three-dimensional structural models would help to understand the molecular dynamics of the proteolytic processing of the ΑβΡΡ peptide to generate the Αβ42 and Αβ40 peptides and would allow the identification of molecular targets, within the γ-secretase enzyme complex, whose inhibition can be interesting from the clinical point of view for the treatment and / or prevention of AD.

DESCRIPTION OF THE INVENTION

The present invention provides molecular targets within the γ-secretase enzyme complex, specifically located in the presenilin molecule that constitutes the catalytic subunit of said complex, which allow identifying and / or designing molecules, preferably antibodies, whose binding selectively blocks the entry of the substrate ΑβΡΡ to one of the two channels that lead to the active site of the enzyme responsible for the production of the Αβ42 peptide. This channel would be delimited by a hole that connects the extracellular space with the cytoplasm (Fig. 1). Thus, the binding of said molecules to the molecular targets described herein leads to the interruption of the production line of the Αβ42 peptide product against the production line of the Αβ40 peptide product, thus causing a reduction in the ratio Αβ42 / Αβ40, which is desirable from the point of view of the treatment and / or prevention of AD.

The inventor of the present invention has developed a model of kinetic behavior for the γ-secretase enzyme complex that includes preseniline as a catalytic subunit, proposing the existence of two possible channels or entry routes (Fig. 1), which would lead to catalytic site in presenilin, for the same amyloid substrate (ΑβΡΡ), which is the most abundant and relevant substrate of the complex in diseases that affect performance and cognitive impairment, such as EA. In the proposed model, these channels, called channel γ40 and channel γ42, should physically diverge towards the luminal space (due to steric conditions), but they would converge in the active center in the direction towards the cytoplasm, and each of them would be associated with the respective cleavage lines given that the substrate would have different steric conditions with respect to the cleavage ε depending on the input channel (channel γ40 and γ42 would give rise to different cleavages ε, which produce Αβ49 and Αβ48 respectively). So the γ40 channel would give rise to the Αβ40 peptide and the γ42 channel to the Αβ42 peptide. In summary, through two access routes or channels of entry to preseniline, within the enzyme complex, the model allows to explain the kinetic mechanism that gives rise to the formation of these two peptides (Αβ42 and Αβ40) and their quantitative distribution.

Therefore, the present invention proposes the luminal blockade of the entrance channel to the γ-secretase enzyme complex attributed to the formation of the Αβ42 peptide, which is located in the structure of the presenilin and which in this invention has been called the γ42 channel of the presenilina ", by means of molecules that present specificity against antigenic determinants that border the zone of entrance to said channel from the extracellular space. In this way, the presenilin enzyme subunit, which forms the catalytic core of the γ-secretase enzyme complex, is partially inaccessible to the substrate that is to be cleaved, ΑβΡΡ, as long as the molecule remains attached to the γ42 channel exerting a steric hindrance on it. This causes a selective blockade of the γ42 channel and consequently, as shown in the examples of the present invention, a reduction of the Αβ42 / Αβ40 ratio, which is desirable from the point of view of treatment and / or prevention of pathologies associated with processing proteolytic substrate ΑβΡΡ. Therefore, the present invention is based on the utility of the γ42 channel of preseniline as a molecular target and on the therapeutic functionality of any molecule identified or designed from said target that is capable of blocking the entrance area to the γ42 channel from the extracellular space preventing, partially or totally, the entrance of the substrate to the enzyme.

The γ42 channel of presenilin has so far been associated with the entry of water and ions, so it has not been proposed so far as a possible channel of entry of the amyloid substrate that will subsequently be cleaved to produce the Αβ42 peptide. Structurally, the γ42 channel is flanked by the domains transmembrane 2, 3, 5 and 7. The transmembrane domains, as the name implies, cross the cell membrane and contact, on the one hand, with the lumen and, on the opposite side, with the cytoplasm, specifically some of the amino acids that constitute these transmembrane domains will be in contact with the lumen or extracellular space (this region will be called in the present invention "luminal end of the transmembrane domain"). Between any two transmembrane domains there is a loop that connects two domains to each other and that projects to the lumen or the cytoplasm. The conjunction of the luminal part of the transmembrane domains 2, 3, 5 and 7 with the luminous loops with which they connect (LL1, LL2, LL3 and LL4 respectively; TM-II with LL1, TM-lll with LL2, TM-V with LL3 and TM-VII with LL4) configure the access of amyloid molecules into the γ42 channel of presenilin 1 (Fig. 2a). These transmembrane-loop structural combinations will be referred to below as "luminal regions, RL," of access to the γ42 channel. This structural pattern is extrapolable to presenilin 2 (Fig. 2b).

Therefore, a first aspect of the invention relates to the use of the γ42 channel of presenilin that is part of the γ-secretase enzyme complex as a pharmacological target for the screening of molecules useful for the treatment and / or prevention of AD.

The "γ-secretase enzyme complex" is the complex consisting of four proteins: presenilin, nicastrin, gene associated with the defect in the anterior nasopharynx of drosophila (APH1) and presenilin 2 enhancer (PEN2). The complex performs proteolytic processing of the substrate ΑβΡΡ within the plasma membrane to give rise to the two peptides Αβ40 and Αβ42. The catalytic subunit of the γ-secretase enzyme complex is presenilin, presenilin 1 or presenilin 2 being present in the complex. These presenilins are transmembrane proteins whose genes, in human, have a high homology between them (80% identity at nucleotide level). In the present invention, "presenilin" is thus understood as presenilin 1 or presenilin 2. In a preferred embodiment, presenilin 1 is the human amino acid sequence protein SEQ ID NO: 21. In another preferred embodiment, presenilin 2 is the human amino acid sequence protein SEQ ID NO: 22. The amino acid sequences of the presenilins may contain mutations, such as insertions, substitutions, additions or deletions of one or more amino acid residues. Therefore, within the scope of the The present invention includes presenilins 1 and 2 whose amino acid sequence is identical or homologous to the sequences described in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

The term "pharmacological target", as used herein, refers to the γ42 channel of presenilin that is part of the γ-secretase enzyme complex, preferably to any of the luminal regions that delimit said channel described later in the present invention, which are useful for the study of the biochemical effect of molecules capable of binding to them. The molecules of interest are those that exert a steric hindrance that prevents the entry of the amyloid substrate into the enzyme complex through this channel. These molecules can be without limitation, antibodies, aptamers, interfering RNAs or chemical agents, which are selected by screening in which the presence of said steric hindrance and / or the reduction of the ratio Αβ42 / Αβ40 is analyzed.

The term "Alzheimer's disease" or "EA", as used in the present invention, refers to a neurodegenerative disease that presents cognitive impairment and / or behavioral disorders. It is characterized in its typical form by a progressive loss of memory and other mental abilities, as nerve cells degenerate and / or die and different areas of the brain atrophy.

The term "γ42 channel", as used herein, refers to the channel shown in Fig. 1, which allows the entry of the amyloid substrate necessary for subsequent proteolytic processing to generate the generarβ42 peptide, or any of peptides of the same cleavage line, such as Αβ45 or Αβ38. The luminal access to said channel will be delimited, preferably, by the luminal regions (RL) as indicated below and which are represented in Fig. 2.

In another preferred embodiment, said γ42 channel is delimited by: a) a first luminal region (RL1) comprising the luminal end of the transmembrane domain 2 (TM-II) of the presenilin and the luminal loop 1 (LL1) of the presenilin which binds to said luminal end of the TM-II domain, b) a second luminal region (RL2) comprising the luminal end of the transmembrane domain 3 (TM-lll) of the presenilin and the luminal loop 2 (LL2) of the presenilin that links with said luminal end of the TM-lll domain, c ) a third luminal region (RL3) comprising the luminal end of the transmembrane domain 5 (TM-V) of the presenilin and the luminal loop 3 (LL3) of the presenilin that links with said luminal end of the TM-V domain, and d) a fourth luminal region (RL4) comprising the luminal end of the transmembrane domain 7 (TM-VII) of the presenilin and the luminal loop 4 (LL4) of the presenilin that links with said luminal end of the TM-VII domain.

The term "luminal region" (RL), as used herein, refers to a luminal outward region of the γ42 channel of presenilins 1 and 2 (Fig. 2) and composed of the defined elements previously in points a), b), c) or d), where the domains TM-II, TM-lll, TM-V and TM-VII are transmembrane domains of presenilin; and LL1, LL2, LL3 and LL4 are luminous loops of presenilin. The luminal regions a) to d) described above are arranged so that they form the luminal access to the γ42 channel of the presenilin, which is part of the γ-secretase enzyme complex.

"Luminal end of the transmembrane domain" means the amino acid region of any of the transmembrane domains 2, 3, 5 or 7 (TM-II, TM-lll, TM-V and TM-VII) of presenilins 1 or 2 arranged towards the luminal end of the γ42 channel (Fig. 2), within the γ-secretase enzyme complex, which, as indicated above, form part of the γ42 channel of presenilin. These amino acid regions consist of the last 3 to 4 amino acids of the C- or N-terminal end of each transmembrane domain (Fig. 2).

In a more preferred embodiment, the luminal end of the transmembrane domain 2 (TM-II) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 1, the luminal end of the transmembrane domain 3 (TM-lll) of presenilin 1 consists in the amino acid sequence SEQ ID NO: 2, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane domain 7 (TM-VII) of Presenilin 1 consists of the LVG amino acid sequence. "Luminal loop"("luminalloop", LL) means that amino acid region of presenilins 1 or 2 (Fig. 2) arranged entirely towards the lumen and presenting a loop structure or "loop" because it connects two domains transmembrane, contiguous in the primary sequence, with each other.

In a more preferred embodiment, the luminal loop 1 (LL1) of the first luminal region (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 6, the luminal loop 2 (LL2) of the second luminal region (RL2 ) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 7, the luminal loop 3 (LL3) of the third luminal region (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the fourth luminal region (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 9.

In an even more preferred embodiment, the first luminal region (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 13, the second luminal region (RL2) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 14 , the third luminal region (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 15 and the fourth luminal region (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 16.

In another preferred embodiment, the luminal end of the transmembrane domain 2 (TM-II) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 4, the luminal end of the transmembrane domain 3 (TM-lll) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 5, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane domain 7 (TM-VII) of the Presenilin 2 consists of the amino acid sequence LVG.

In a more preferred embodiment, the luminal loop 1 (LL1) of the first luminal region (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 10, the luminal loop 2 (LL2) of the second luminal region (RL2 ) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 1 1, the luminal loop 3 (LL3) of the third luminal region (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the fourth luminal region (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 12. In an even more preferred embodiment, the first luminal region (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 17, the second luminal region (RL2) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 18 , the third luminal region (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 15 and the fourth luminal region (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 19.

The luminal regions defined in the present invention can be used in their isolated form as pharmacological targets for the screening of molecules capable of exerting a steric hindrance on the γ42 channel of the presenilin that prevents the substrate from entering said channel. Therefore, in a second aspect, the present invention relates to a luminal region isolated from the γ42 channel of the presenilin that is part of the γ-secretase enzyme complex, hereafter referred to as the "luminal region of the invention", selected from the list consisting of: a) luminal region 1 (RL1) comprising the luminal end of the transmembrane domain 2 (TM-II) of the presenilin and the luminal loop 1 (LL1) of the presenilin that links with said luminal end of the transmembrane domain 2 (TM-II), b) luminal region 2 (RL2) comprising the luminal end of the transmembrane domain 3 (TM-lll) of the presenilin and the luminal loop 2 (LL2) of the presenilin that links with said luminal end of the domain transmembrane 3 (TM-lll), c) luminal region 3 (RL3) comprising the luminal end of the transmembrane domain 5 (TM-V) of the presenilin and the luminal loop 3 (LL3) of the presenilin that links with said luminal end of the transmembrane domain 5 (TM-V), and d) lum region inal 4 (RL4) comprising the luminal end of the transmembrane domain 7 (TM-VII) of the presenilin and the luminal loop 4 (LL4) of the presenilin that links with said luminal end of the transmembrane domain 7 (TM-VII). The usefulness of these molecular targets is based on their strategic situation within the area that delimits the γ42 channel of presenilin, so their inhibition or blockage leads to a steric impediment of the γ42 channel for the entrance of the substrate ΑβΡΡ, which finally translates into a reduction in the ratio Αβ42 / Αβ40.

The luminal region of the present invention can be synthesized, for example, but not limited to, in vitro. For example, by solid phase peptide synthesis or by recombinant DNA approaches. The luminal region of the invention can be produced recombinantly, not only directly but as a fusion polypeptide together with a heterologous polypeptide, which may contain, for example, but not limited to, a signal sequence or other polypeptide having a cut-off site by a protease, for example, but not limited to, at the N-terminal end of the mature protein or polypeptide. The recombinant production of the luminal region of the invention comprises the amplification of a polynucleotide encoding said region, the cloning of the amplified polynucleotide into an expression vector, the transformation or transfection of a competent cell with said vector, the cultivation of said cell. under conditions that promote the expression of the luminal region of the invention and the isolation and purification of the luminal region of the invention produced by the cell.

The luminal region of the invention may have variants. These variants refer to limited variations in the amino acid sequence, which allow the maintenance of the functionality of the peptide. This means that the reference sequence and the variant sequence are similar as a whole, and identical in many regions. These variations are generated by substitutions, deletions or additions. Such substitutions are, for example, but not limited to conserved amino acids. The conserved amino acids are amino acids that have similar side chains and properties in terms of, for example, hydrophobicity or aromaticity. These substitutions include, but are not limited to, substitutions between glutamic acid (Glu) and aspartic acid (Asp), between lysine (Lys) and arginine (Arg), between asparagine (Asn) and glutamine (Gln), between serine (Ser) and threonine (Thr), and / or among the amino acids that make up the alanine (Ala), leucine (Leu), valine (Val) and isoleucine (lie) group. Variations can be artificially generated variations, such as by mutagenesis or direct synthesis. Therefore, within The scope of the present invention also includes peptides or polypeptides whose amino acid sequence is identical or homologous to the sequences described in the present invention.

In a preferred embodiment of the luminal region of the invention, the luminal end of the transmembrane domain 2 (TM-II) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 1, the luminal end of the transmembrane domain 3 (TM-lll ) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 2, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane domain 7 (TM-VII) of presenilin 1 consists of the amino acid sequence LVG.

In a more preferred embodiment, the luminal loop 1 (LL1) of the luminal region 1 (RL1) of the presenilin 1 consists of the amino acid sequence SEQ ID NO: 6, the luminal loop 2 (LL2) of the luminal region 2 (RL2 ) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 7, the luminal loop 3 (LL3) of the luminal region 3 (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the luminal region 4 (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 9.

In an even more preferred embodiment, the luminal region 1 (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 13, the luminal region 2 (RL2) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 14 , the luminal region 3 (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 15 and the luminal region 4 (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 16.

In another preferred embodiment of the luminal region of the invention, the luminal end of the transmembrane domain 2 (TM-II) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 4, the luminal end of the transmembrane domain 3 (TM-lll ) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 5, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane domain 7 (TM-VII) of presenilin 2 consists of the amino acid sequence LVG

In a more preferred embodiment, the luminal loop 1 (LL1) of the luminal region 1 (RL1) of the presenilin 2 consists of the amino acid sequence SEQ ID NO: 10, the luminal loop 2 (LL2) of the luminal region 2 (RL2 ) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 1 1, the luminal loop 3 (LL3) of the luminal region 3 (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the luminal region 4 (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 12.

In an even more preferred embodiment, the luminal region 1 (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 17, the luminal region 2 (RL2) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 18 , the luminal region 3 (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 15 and the luminal region 4 (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 19.

A third aspect of the invention relates to the use of the luminal region of the invention as a pharmacological target for the screening of molecules useful for the treatment and / or prevention of AD.

In a fourth aspect, the present invention relates to a method of screening molecules useful for the treatment and / or prevention of AD, hereinafter "method of the invention", comprising: a. Contacting a molecule to be studied with the γ42 channel of presenilin that is part of the γ-secretase enzyme complex, ex vivo, or with the luminal region of the isolated invention,

b. Analyze the interaction between the molecule to be studied and the luminal regions of the invention, and

C. Select the molecule from step (a) for the treatment and / or prevention of AD when it has joined at least one of the luminal regions of the invention.

It is understood as "contacting a molecule to study with the γ42 channel of the presenilin that is part of the γ-secretase enzyme complex, or with the luminal region of the isolated invention ", the incubation of the molecule to be studied with the presenilin that is part of the γ-secretase enzyme complex, or with the luminal region of the invention isolated, under conditions that allow pharmacological molecule-target binding, the target being the γ42 channel or the luminal region of the isolated invention.If the molecule to be studied is an antibody, incubation is performed under conditions that allow the formation of antigen complexes -antibody and is maintained for a suitable period of time for an immune response to take place.

The term "analyze the interaction", as used in the present description, preferably refers to the study of the change operated or response after incubation of the molecule to be studied, object of screening in step (a), in the concentration of the substrates / products related to "cleavage and" performed by preseniline, belonging to the γ-secretase enzyme complex. A more preferred embodiment would be when the substrate / product refers to an amyloid substrate / product or a derivative / analog thereof.

If the molecule to be studied is an antibody, the presence of an immunological response can be determined, in a preferred embodiment, by monitoring the substrate binding (previously labeled with a fluorescence donor) by FRET ("Fluerescent Resonance Energy Transfer"). to the enzyme preseniline, which is part of the γ-secretase enzyme complex, when the enzyme is bound to the antibody to be studied (previously labeled with a fluorescence acceptor).

The presence of a response can also be determined, in another preferred embodiment, by studying the products (derivatives of "cleavage and" performed by preseniline, belonging to the γ-secretase enzyme complex) by conventional methods known in the state of the art. Therefore, in a preferred embodiment, the presence of response is determined by assays such as Western blot, electrophoresis gels, immunoprecipitation, protein arrays, immunofluorescence, immunohistochemistry, ELISA or any other enzymatic method; by incubation with a specific ligand; by NMR or any other imaging technique; or, for example, by chromatographic techniques combined with mass spectrometry. The "products / substrates related to" cleavage and "performed by presenilin" preferably refer to an amyloid substrate / product or a derivative / analog thereof, more preferably peptides Αβ48 and Αβ49, as substrates, and Αβ45, Αβ42, Αβ38, Αβ46, Αβ43, Αβ40 and Αβ37 peptides, as products of the γ cleavage. In an even more preferred embodiment, the substrate is Αβ48 and the products are Αβ45, Αβ42 and / or Αβ38.

The criteria for selecting molecules in step (c) must be the following: once the molecule has joined the γ42 channel of the presenilin that is part of the γ-secretase enzyme complex (preferably one or more of the regions of the invention), it should be able to reduce the ratio between the amyloid products Αβ42 and Αβ40 (Αβ42 / Αβ40). The measurement of this ratio can be performed in vitro by techniques well known to those skilled in the art, such as, but not limited to, by incubation with a specific antibody for Αβ42 and another for Αβ40, in assays such as Western blot, gels of electrophoresis, immunoprecipitation, protein arrays, immunofluorescence, immunohistochemistry, ELISA or any other enzymatic method; by incubation with a specific ligand; by NMR or any other imaging technique; or, for example, by chromatographic techniques combined with mass spectrometry.

The luminal region of the invention has antigenic capacity, so it can be used for the immunization of an individual, preferably mammal. Once the immunization has occurred, the serum of the immunized animal is extracted. The extraction of the serum can be carried out by means of, for example, but not limited to, blood extraction, subsequent coagulation of the latter, elimination of the fibrin clot and other components and isolation of the serum. To obtain the serum, anticoagulant is not applied to the blood, but is allowed to clot and centrifuged. The term "serum", "blood serum" or "blood serum" means the resulting blood component after allowing blood clotting and eliminating the fibrin clot and other components, which contains numerous biological effectors, such as the derived growth factor of platelets, segregated by the form elements when said coagulation occurs. It is a yellow color, a little more intense than plasma. Subsequently, the specific antibodies are purified against the luminal region of the invention employed in the immunization present in the serum obtained. Various antibody purification methods that could be carried out for this purpose are known in the state of the art, such as, but not limited to, electrophoretic or chromatographic methods.

The luminal region can also be used for the immunization of a bird. Preferably, once the immunization has occurred, the eggs of the immunized animal are collected, the yolks are separated, the lipids are removed and the antibodies precipitated / purified by various methods known in the state of the art.

"Electrophoresis" is an analytical separation technique based on the movement or migration of dissolved macro-molecules in a medium (electrophoresis buffer), by means of a matrix or a solid support as a result of the action of an electric field. The behavior of the molecule depends on its electrophoretic mobility and this mobility depends on the charge, size and shape. There are numerous variations of this technique based on the equipment used, supports and conditions for carrying out the separation of the molecules. Electrophoresis is selected from the list consisting of, but not limited to, capillary electrophoresis, paper electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis, isoelectric focusing or two-dimensional electrophoresis.

The "chromatographic methods" allow the separation of molecules by, but not limited to, their charge, size, molecular mass, by their polarity or by their redox potential. The chromatography technique is selected from, but not limited to, partition chromatography, filtration, adsorption chromatography, molecular exclusion chromatography, ion exchange chromatography, hydrophobic chromatography or affinity chromatography, and can be performed both in column, and on paper or on plate.

The sequence peptide SEQ ID NO: 20 is an 8 amino acid peptide corresponding to residues 14 to 21 of the amino acid sequence of the luminal loop 1 (LL1) of the first luminal region (RL1) of presenilin 1 (SEQ ID NO : 6) or 2 (SEQ ID NO: 10). As shown in the examples of the present invention, this peptide of SEQ ID NO: 20 has antigenic ability and, therefore, can be used as explained above for the immunization of an animal and the consequent Obtaining antibodies. As the examples show, when at least one specific antibody against said peptide is bound to it in the γ-secretase enzyme complex, said interaction exerts a steric hindrance that prevents the entry of the ΑβΡΡ substrate into the γ42 channel of presenilin, thus causing a reduction in the ratio Αβ42 / Αβ40.

Therefore, another aspect of the invention relates to an isolated peptide consisting of the amino acid sequence SEQ ID NO: 20, hereinafter "peptide of the invention". Said peptide can be synthesized, for example, but not limited to, in vitro. For example, by solid phase peptide synthesis or by recombinant DNA approaches. The peptide of the invention can be produced recombinantly, not only directly but as a fusion polypeptide together with a heterologous polypeptide, which may contain, for example, but not limited to, a signal sequence or other polypeptide having a protease cleavage site. , for example, but not limited to, at the N-terminal end of the mature protein or polypeptide. The recombinant production of the peptide of the invention comprises the amplification of a polynucleotide encoding said peptide, the cloning of the amplified polynucleotide into an expression vector, the transformation or transfection of a competent cell with said vector, the cultivation of said cell under conditions that promote the expression of the peptide of the invention and the isolation and purification of the peptide produced by the cell.

Another aspect of the invention relates to an isolated polynucleotide encoding the peptide of the invention, hereinafter "polynucleotide of the invention". Another aspect of the invention relates to a gene construct, preferably an expression vector, comprising said polynucleotide. Another aspect of the invention relates to a cell comprising the polynucleotide of the invention or the gene construct of the invention.

Another aspect of the invention relates to the use of the peptide of SEQ ID NO: 20 as a pharmacological target for the screening of molecules useful for the treatment and / or prevention of AD.

Another aspect of the present invention relates to a specific antibody against the luminal region of the invention, hereafter referred to as "antibody of the invention". In A preferred embodiment, this antibody of the invention is specific against the peptide consisting of SEQ ID NO: 20.

The term "antibody" refers to immunoglobulin molecules or immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the luminal region of the invention, preferably with the peptide of SEQ ID NO: 20. Examples of portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ') 2 fragments, which can be generated by treating the antibody with an enzyme such as pepsin or recombinantly.

The antibody of the invention can be polyclonal (typically includes different antibodies directed against different determinants or epitopes) or monoclonal (directed against a single determinant in the antigen). The term "monoclonal antibody" refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of the antigen. The monoclonal antibody may be biochemically altered, by genetic manipulation, or it may be synthetic, possibly lacking the antibody in whole or in part, from portions that are not necessary for recognition of the luminal region of the invention, preferably of the peptide of SEQ ID NO: 20, and being substituted by others that communicate additional advantageous properties to the antibody. In a preferred embodiment the antibody of the invention is a polyclonal antibody.

The antibody of the invention can also be recombinant, humanized, chimeric, synthetic or a combination of any of the foregoing. A "recombinant antibody" (rAC) is an antibody that has been produced in a host cell transformed or transfected with a polynucleotide encoding the luminal region of the invention or with the polynucleotide encoding the peptide of SEQ ID NO: 20, with a nucleic acid encoding the antibody of the invention, or producing the antibody of the invention or the luminal region of the invention or the peptide of SEQ ID NO: 20 as a result of homologous recombination. Said host cell includes a cell in an "in vitro" cell culture as well as a cell in a host animal. The antibody of the invention can also be chimeric. Thus, a region of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies from a given species or belonging to a particular class or subclass of antibodies, while the remaining chain (s) ( s) is (are) identical, or homologous (s), to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as to fragments of said antibodies, so as to exhibit the Desired biological activity

As shown in the examples below, an antibody generated against any of the luminal regions of the invention, preferably against the peptide of SEQ ID NO: 20, is capable of blocking the entrance of the substrate ΑβΡΡ to the γ42 channel of the presenilin which It is part of the γ-secretase complex when it is bound to its antigen, which translates into a decrease in the Αβ42 / Αβ40 ratio and thus said antibody is useful for the treatment of diseases or pathological conditions in which the proteolytic processing of the substrate ΑβΡΡ is involved, such as, but not limited to, EA. Therefore, another aspect of the present invention relates to the use of the antibody of the invention for the manufacture of a medicament. Or alternatively, to the antibody of the invention for use as a medicament. In a preferred embodiment, the medicament is for the treatment and / or prevention of AD.

The term "medicine", as used herein, refers to any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in organisms, preferably humans, or that may used or administered to organisms, preferably humans, in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action. The medicament of the invention can be used both alone and in combination with other medicaments or compositions to improve cognitive performance and / or promote cognitive development as a combination therapy, and can be administered at the same time or at different times. The term "treatment" as understood in the present invention refers to combating the effects caused as a result of the disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:

(i) inhibit the disease or pathological condition, that is, stop its development;

(ii) alleviate the disease or the pathological condition, that is, cause the regression of the disease or the pathological condition or its symptomatology;

(iii) stabilize the disease or pathological condition.

The term "prevention" as understood in the present invention is to prevent the onset of the disease, that is, to prevent the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition, but has not yet been diagnosed as having it.

In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody of the invention, hereinafter "pharmaceutical composition of the invention". In a preferred embodiment the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier and / or other active ingredient.

The "pharmaceutically acceptable carrier" is a substance that is used in the composition to dilute any of the components included therein to a certain volume or weight. The pharmaceutically acceptable carrier is an inert substance or action analogous to any of the elements included in the composition of the present invention. The function of the vehicle is to facilitate the incorporation of other elements, allow a better dosage and administration or give consistency and form to the composition.

In addition, the pharmaceutical composition of the invention may comprise one or more adjuvants and / or excipients. The term "excipient" refers to a substance that helps the absorption of the elements of the composition of the invention, stabilizes said elements, activates or aids the preparation of the composition in the sense of giving it consistency or providing flavors that make it nicer. So therefore, the excipients could have the function of keeping the ingredients together, as in the case of starches, sugars or cellulose, for example, the sweetening function, the dye function, the protection function of the composition, for example, for isolate it from air and / or moisture, the filling function of a tablet, capsule or any other form of presentation, the disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.

Preferably, the composition of the invention comprises the antibody of the invention in a therapeutically effective amount, "therapeutically effective amount" being understood as the level, amount or concentration of antibody of the invention that produces the desired effect by improving cognitive performance and / or promoting Cognitive development, preferably, treating and / or preventing AD, without causing adverse effects. The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, the age, weight, sex or tolerance of the individual to whom the composition of the invention is to be administered.

The composition of the present invention can be formulated for administration in a variety of ways known in the state of the art. Examples of preparations include any solid composition (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) for oral, topical or parenteral administration. The composition of the present invention may also be in the form of sustained release formulations of drugs or any other conventional release system, so it may be contained, but not limited to, in nanoparticles, liposomes or nanospheres, in a polymeric material, in a polymeric material. Biodegradable or non-biodegradable implant or in biodegradable microparticles, such as biodegradable microspheres.

Such a composition and / or its formulations can be administered to an animal, and therefore to man, in a variety of ways, including, but not limited to, intraperitoneal, intravenous, intradermal, intraspinal, intrastromal, intraarticular, intrasynovial, intrathecal, intralesional, intraarterial. , intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular, topical, using transdermal patches, percutaneous, nasal spray, surgical implant, internal surgical paint or pump infusion.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Representation of the γ-secretase complex from a luminal perspective. The two channels house two cylindrical structures that represent the amyloid substrate: the white one is located in the γ42 channel, and the black one in the γ40.

Figure 2. Representative scheme of the structures that configure the luminal access to the γ42 channel of the PS1 (Fig. 2a) and PS2 (Fig. 2b). The different luminous regions are represented collected within the rectangles (RL1, RL2, RL3 and RL4). The concatenated circles represent amino acid residues. The residues of the light loops LL1, LL2, LL3 and LL4 are identified with the corresponding amino acid letter and not filled. The residues of the luminal ends of the TM-II, TM-lll, TM-V and TM-VII transmembrane domains are identified with the corresponding amino acid letter and filled.

Figure 3. Graph representing the percentage of amyloid Αβ42 and Αβ40 and the corresponding ratio Αβ42 / Αβ40 in the supernatant collected in 24 of a cell culture of the SHSY5Y-APP695 neuronal line cultured in the presence of polyclonal antibodies generated against the peptide of SEQ ID NO: 20, with respect to a parallel control culture grown in the absence of antibodies.

EXAMPLES

The invention will now be illustrated by tests demonstrating the effectiveness of the use of the γ42 channel of presenilin, and specifically of the peptide of SEQ ID NO: 20, as a pharmacological target for the screening of molecules useful for treatment and / or prevention of AD. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described below show the invention without limiting its scope.

Example 1. Production of polyclonal antibodies against the peptide of SEQ ID NO: 20.

The antigenic peptide was synthesized by solid phase techniques and purified by HPLC (high pressure liquid chromatography) reaching a purity of 97.21%. The original peptide sequence of SEQ ID NO: 20 was modified, adding a cysteine residue to the N-terminal end of the peptide. The peptide was covalently linked by disulfide bonds to BC ("Blue Carrier Immunogenic Protein" of Pierce). A total of two hens were immunized with the BC-peptide complex and Freund's adjuvant (from Sigma) and reinforcements were injected at intervals of 14, 28 and 56 days. Eggs were collected from each chicken between days 40 and 71 after the start of immunization. The yolks were separated, lipids removed and the antibodies precipitated / purified with the Pierce Chicken ("Pierce Chicken IgY Purification Kit" from Thermo Scientific). Two batches of antibody corresponding to each of the immunized animals were obtained: Polyclone H and Polyclonal2.

Example 2. Therapeutic action associated with blocking the γ42 channel with the generated antibodies.

The therapeutic action associated with the blockade of the γ42 channel was not associated with a reduction in the levels of total aumentoβ, since the increase in the production of Αβ40, through the channel that is not the object of the blockade (γ40), was accompanied by increase in the intracellular fragment of the corresponding ΑβΡΡ (AICD50 or C50) responsible for an increase in the expression of the ΑβΡΡ substrate in 8.7 times its value (Sébastien Hébert et al., 2006, EMBO Reports, 7 (7), 739-745). The net result was an increase in the total amount of Αβ, at the expense of a significant increase in the amount of Αβ40, and a lower increase of Αβ42 with respect to the increase in Αβ40, which led to a decrease in the ratio Αβ42 / Αβ40. This result, far from being a problem due to the paradoxical increase of Αβ42, corrects the pathological phenotypic characteristics of most of the mutations of patients with monogenic cause AD, described in the previous section (Eric Karran, et al., 201 1, Nature Reviews Drug Discovery, 10, 698-712):

1. Decrease in the total amount of amyloid produced, total Αβ.

2. Increase in the ratio Αβ42 / Αβ40.

In this sense, there is evidence that there is a positive regulation of the ratio Αβ40 / 42β42 (or otherwise: negative regulation of the ratio Αβ42 / Αβ40) with an increase in the total amount of Αβ, (increase in the amount of Αβ40 and increase lower than 42β42 with respect to the Αβ40 increase) in studies of synaptic plasticity that allow the spectrum of the proposed treatment to be extended to patients with sporadic AD (Iftach Dolev et al., 2013, Nature Neuroscience, 16 (5), 587-595).

The blockade of the γ42 channel of the presenilin which is part of the γ-secretase enzyme complex was evaluated by polyclonal antibodies developed against the peptide SEQ ID NO: 20 described in example 1. SHSY5Y-APP695 neuronal line cells cultured to a confluence of 80-90% in DMEM medium (from Invitrogen) supplemented with 10% fetal bovine serum (from Invitrogen-Gibco) were exposed. The cells were detached from the plate using only mechanical means. They were then seeded in Opti-MEM medium (from Invitrogen-Gibco) in an approximate concentration of 3.5 x 10 ⁴ cells / well. The cells were cultured for 24 hours (at 37 ° C, 5% C0 ₂ ) in the presence (final concentration: 6,000 nM) and in the absence of polyclonal antibody (control culture). The levels of Αβ40 and Αβ42 were measured, both of the supernatants at 24 hours and those of the Opti- MEM culture medium (which was considered as baseline level, reference), using the ELISA kits "Colorimetric BetaMark ™ Beta-Amyloid x-40 ELISA kit "(from Covance) and" Colorimetric BetaMark ™ Beta-Amyloid x-42 ELISA kit "(from Covance) respectively.

The results are shown graphically in Figure 3. These results are able to reverse the phenotypic characteristics presented by the majority of the mutations of patients with AS of monogenic cause, already mentioned. Thus, what was observed is: Increase in the overall amount of amyloid produced, total Αβ, compared to the control without exposure to the antibody.

Descent of the ratio Αβ42 / Αβ40, with respect to the control without exposure to the antibody.

Claims

1. Use of the γ42 channel of presenilin that is part of the γ-secretase enzyme complex as a pharmacological target for the screening of molecules useful for the treatment and / or prevention of Alzheimer's disease (AD).

2. Use of the γ42 channel of the presenilin that is part of the γ-secretase enzyme complex according to claim 1, wherein said channel is delimited by: a) a first luminal region (RL1) comprising the luminal end of the transmembrane domain 2 (TM) -II) of the presenilin and the luminal loop 1 (LL1) of the presenilin that links with said luminal end of the transmembrane domain 2 (TM-II), b) a second luminal region (RL2) comprising the luminal end of the transmembrane domain 3 (TM-lll) of the presenilin and the luminal loop 2 (LL2) of the presenilin that links with said luminal end of the transmembrane domain 3 (TM-lll), c) a third luminal region (RL3) comprising the luminal end of the transmembrane domain 5 (TM-V) of the presenilin and the luminal loop 3 (LL3) of the presenilin that links with said luminal end of the transmembrane domain 5 (TM-V), and d) a fourth luminal region (RL4) comprising the luminal end of transmembrane domain 7 (TM-V II) of the presenilin and the luminal loop 4 (LL4) of the presenilin that links with said luminal end of the transmembrane domain 7 (TM-VII).

3. Use according to any of claims 1 or 2, wherein the preseniline is presenilin 1 or 2.

4. Use according to any of claims 2 or 3, wherein the luminal end of the transmembrane domain 2 (TM-II) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 1, the luminal end of the transmembrane domain 3 (TM- III) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 2, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane domain 7 (TM-VII) of presenilin 1 consists of the amino acid sequence LVG.

5. Use according to any of claims 2 or 3, wherein the luminal end of the transmembrane 2 (TM-II) domain of presenilin 2 consists of the amino acid sequence SEQ ID NO: 4, the luminal end of the transmembrane domain 3 (TM- III) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 5, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane domain 7 (TM-VII) of presenilin 2 consists of the amino acid sequence LVG.

6. Use according to any of claims 2 to 4, wherein the luminal loop 1 (LL1) of the first luminal region (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 6, the luminal loop 2 (LL2) of the second luminal region (RL2) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 7, the luminal loop 3 (LL3) of the third luminal region (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO : 8 and the luminal loop 4 (LL4) of the fourth luminal region (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 9.

7. Use according to any of claims 2, 3 or 5, wherein the luminal loop 1 (LL1) of the first luminal region (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 10, the luminal loop 2 ( LL2) of the second luminal region (RL2) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 1 1, the luminal loop 3 (LL3) of the third luminal region (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the fourth luminal region (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 12.

8. Use according to claim 2, wherein the first luminal region (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 13, the second luminal region (RL2) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 14, the third luminal region (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 15 and the fourth luminal region (RL4) of Presenilin 1 consists of the amino acid sequence SEQ ID NO: 16.

9. Use according to claim 2, wherein the first luminal region (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 17, the second luminal region (RL2) of presenilin 2 consists of the amino acid sequence SEQ ID NO : 18, the third luminal region (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 15 and the fourth luminal region (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 19.

10. Luminal region of the γ42 channel of the presenilin that is part of the γ-secretase enzyme complex selected from the list consisting of: a) luminal region 1 (RL1) comprising the luminal end of the transmembrane domain 2 (TM-II) of the presenilin and the luminal loop 1 (LL1) of the presenilin that links with said luminal end of the transmembrane domain 2 (TM-II), b) luminal region 2 (RL2) comprising the luminal end of the transmembrane domain 3 (TM-lll ) of the presenilin and the luminal loop 2 (LL2) of the presenilin that links with said luminal end of the transmembrane domain 3 (TM-lll), c) luminal region 3 (RL3) comprising the luminal end of the transmembrane domain 5 (TM -V) of the presenilin and the luminal loop 3 (LL3) of the presenilin that links with said luminal end of the transmembrane domain 5 (TM-V), and d) luminal region 4 (RL4) comprising the luminal end of the transmembrane domain 7 (TM-VII) of preseniline and luminal loop 4 (LL4) of the p resenilin that links with said luminal end of the transmembrane domain 7 (TM-VII).

eleven . Luminal region according to claim 10, wherein the preseniline is presenilin 1 or 2.

12. Luminal region according to any of claims 10 or 1, wherein the luminal end of the transmembrane domain 2 (TM-II) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 1, the luminal end of the transmembrane domain 3 ( TM-II I) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 2, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane 7 (TM-VII) domain of presenilin 1 consists of the LVG amino acid sequence.

13. Luminal region according to any of claims 10 or 1, wherein the luminal end of the transmembrane 2 (TM-II) domain of presenilin 2 consists of the amino acid sequence SEQ ID NO: 4, the luminal end of the transmembrane domain 3 ( TM-II I) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 5, the luminal end of the transmembrane domain 5 (TM-V) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 3 and the luminal end of the transmembrane 7 (TM-VII) domain of presenilin 2 consists of the amino acid sequence LVG.

14. Luminal region according to any of claims 10 to 12, wherein the luminal loop 1 (LL1) of the luminal region 1 (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 6, the luminal loop 2 (LL2 ) of the luminal region 2 (RL2) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 7, the luminal loop 3 (LL3) of the luminal region 3 (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the luminal region 4 (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 9.

15. Luminal region according to any of claims 10, 1 1 or 13, wherein the luminal loop 1 (LL1) of the luminal region 1 (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 10, the luminal loop 2 (LL2) of the luminal region 2 (RL2) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 1 1, the luminal loop 3 (LL3) of the luminal region 3 (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 8 and the luminal loop 4 (LL4) of the luminal region 4 (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 12.

16. Luminal region according to claim 10, wherein the luminal region 1 (RL1) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 13, the luminal region 2 (RL2) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 14, the luminal region 3 (RL3) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 15 and the luminal region 4 (RL4) of presenilin 1 consists of the amino acid sequence SEQ ID NO: 16.

17. Luminal region according to claim 10, wherein the luminal region 1 (RL1) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 17, the luminal region 2 (RL2) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 18, the luminal region 3 (RL3) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 15 and the luminal region 4 (RL4) of presenilin 2 consists of the amino acid sequence SEQ ID NO: 19.

18. Use of the luminal region according to any of claims 10 to 17 as a pharmacological target for the screening of molecules useful for the treatment and / or prevention of AD.

19. Method of screening molecules useful for the treatment and / or prevention of AD, comprising: a. Contacting a molecule to be studied with the γ42 channel of the presenilin which is part of the γ-secretase enzyme complex, ex vivo, or with the isolated luminal region according to any of claims 10 to 17,

b. Analyze the interaction between the molecule to be studied and the luminal regions according to any of claims 10 to 17, and

C. Select the molecule from step (a) for the treatment and / or prevention of AD when it has joined at least one of the luminal regions according to any of claims 10 to 17.

20. Specific antibody against the luminal region according to any of claims 10 to 17.

twenty-one . Antibody according to claim 20, wherein the antibody is polyclonal.

22. Use of the antibody according to any of claims 20 or 21 for the preparation of a medicament.

23. Use of the antibody according to claim 22, wherein the medicament is for the treatment and / or prevention of AD.

24. Pharmaceutical composition comprising the antibody according to any of claims 20 or 21.

25. Pharmaceutical composition according to claim 24, further comprising a pharmaceutically acceptable carrier and / or other active ingredient.