HU231182B1 - Small peptide inhibitors of ß-amyloid toxicity - Google Patents

Abstract

The present invention relates to a peptide or peptide mimetic of formula (I), X-A1-A2-A3-A4-A5-Y (I) where X is absent or any of the following: acetyl, propionyl, succinyl; A1 is absent or, for example, any of the following: Ala, Gly, Aib, Sar, N-methyl-Ala, α-methyl-Ala, Abu; A2 is absent or, for example, any of the following: Pro, Tie, tiaproline, α-methyl-Pro, γ-hydroxy-Pro, Mud, 4-fluoro-Pro, Pip, nipecotic acid, Glu; A3 is absent or, for example, any of Ala, Gly, Aib, Sar, N-methyl-Ala, α-raethyl-Ala, Abu; A4 is absent or, for example, any of the following: Pro, Tie, tiaproline, α-methyl-Pro, γ-hydroxy-Pro, Mud, 4-fluoro-Pro, Pip, nipecotic acid, Glu; A5 is, for example, any of Ala, Gly, Aib, Sar, N-methyl-Ala, α-methyl-Ala, Abu, norvalin, Asp, Glu; Y is absent or any of the following: amide, N-methylamide or N, N-dimethylamide; and pharmaceutically acceptable salts and esters thereof, where certain combinations of meanings are excluded.

Description

Small peptide inhibitors that inhibit the toxicity of β-amxloid

The present invention relates to

The present invention provides peptides and peptide mimetics that reduce the amide peptide. aggregates and the neurethoxic effect of their prion-like (prionoid) protein aggregate. The invention further provides pharmaceutical coosites comprising these peptides and peptide mimetics. Peptides and peptide mimetics can be used to prepare drugs for the prevention and treatment of diseases such as those described in Αβ peptides, III. caused by the formation of amyloid plaques and can be cured by preventing the damaging effects of abnormal A3 ~ 15 aggregates. Peptides and peptide numbing agents are useful in the prevention and treatment of the diseases mentioned above. The object of the invention is to provide a treatment for neurodegenerative diseases, in particular Alzheimer's disease (AK), which is characterized by the accumulation of defective spatial and / or aggregated proteins.

The following abbreviations are used to describe the invention:

Αβ, β-amyloid peptide; AK, Alzheimer's disease; PK, Parkinson's disease; Ao, aoethyl; ACN, aoetonitrile; APP, amyloid precursor protein; a mouse model of APP x PSI, AK APP / presenilin 1 (?); apape, pentapeptide SEQ ID NO 51; Boc, tert-butyloxycarbonyl; BSB, β-sheet breaker; DCC, N, N-dicyclohexylcarbodiimide; DCM, dichloromethane; DXPEA, diisopropylethylamine;

DMF, N, N-dimethylformamide; MDSO, dimethyl sulfoxide; Emoc, fluorenylmethoxycarbonyl; GBCR, - G-protein coupled receptor;

HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HOBt, 1-hydrezi $ ?! ·>. Xs <·· penztriazole; HERE, internally disordered. protein; β-β <intraperitoneal; LTP, long-term potentiation; dT, neurofibrillary tangle; MBNA, 4-methyl-benzhydrylamine; MWM, 35 Morris Water Maze; HHOA, N-methyl-D-aspartic acid; PAM, 4

SZTHH «100MT3S4 hydropresimethylphenylethanol; PBS, phosphate buffered saline; TFA, trifluoroacetic acid; ThiofXavin-T (ThT); EDCxHCL 1-EtX1-3- [3-dimethylamino-propyl] carbodiimide hydrochloride; -NH2, C-terminal amide sweep.

Abbreviations for "standard amino acids" are known to those skilled in the art. Abbreviations for non-standard amino acids and similar compounds are as follows:

Aib, 2-amino-butyric acid; ala, D-aianin; pro, D-proline; glu, Dglutamic acid; asp, D-aspartic acid; Glp, L-pyroglutamic acid;

Mud, sarcosine; Pip, L-pipecolic acid; sue, succinyl; GABA, gamma-amino-butyric acid; Abu, 2-amino-butyric acid; Tie, Tetrahydroi z o k i η ο 1 i n - 3 ~ k a r b on s a v.

It is known to those skilled in the art that chiral amino acids have the L or D configuration. When no configuration is indicated in the description of the invention, both the L and D configurations are meant.

BACKGROUND OF THE INVENTION

According to the World Alzheimer's Report 2, the number of patients with Alzheimer's and other dementias in the world is about 20%.

It was 35.6 million, and that number is expected to double every 20 years. The amount spent on treating demons patients is estimated at $ 315 billion a year. AK, the most common dementia in old age, is causing a global health crisis that is placing a heavy burden on individuals, families and society as a whole. There is a great and urgent need to develop effective and relatively inexpensive treatment methods and prevention strategies that can be used in the early stages of the disease and can replace current 30 medications that only temporarily reduce the symptoms of the disease and slow the progression of the disease but do not provide causal treatment. .

Some diseases (e.g., AK, PK, type II diabetes, h * amyloidosis) are characterized by the conversion of normally soluble proteins to insoluble amyloid fibrils (Dobson, Trends Biochem Sci. 24 (9): 329-). 32, 1999; Síp® and

Cohen, J Struct Bid 130 (2-3): 88-98, 2000). Two different fibrillar protein aggregates were found in AK brain samples: plaques containing the β5 amyloid peptide (Αβ) and neufibrillary stone layers composed mainly of the microtubular protein, tau protein (Selkoe, J Alzheimers Dis 3 (1): 75-80 ^ 2001). Αχ Αβ aggregates consist mainly of amyloid fibrils with a 3-fold layer structure with a cross-beta diffraction pattern and W characteristic Congo red and Thiofiavin T staining (Eanes and

Glenner, J Histochem Cytochem 16 (11): 073-7, 1968; LeVine,

Protein Sci 2 (3): 404-10, 1993; Sipe and Cohen, J Struct Biol 130 (2-3): 88-98, 2000). Tau fibers have different forms of appearance, e.g. paired helical filaments, which consist of β-structured fibrils and appear as twisted structures on the electromicroscopic image, and straight filaments which do not show a twisted structure (Goedert et al., Curr Qpin heurobiol 8 (5): 619-32, Review , 1998). Tau filaments bind the Thioflavin S (ThS) dye, give a fluorescent signal, and show a cross-beta diffraction pattern (Berriman et al., Froc Nacl Acad Sci U.S.A. 100 (15): 9034-8, 2003; Friedhoff et al. , Biochemistry 37 (28): 10223-30, 1998).

AK is characterized by neuronal cell death in the brain, extracellular amyloid plaques, cerebrovascular amide unloading, and intracellular neurofibrillary tangles. NKs consist mainly of the microtubule oxo-associated tau protein (Selkoe, Physiol Rev 81: 741-766, 2001), whereas amyloid plaques contain Αβ peptides consisting mainly of 39-43 amino acids. AB peptides are formed from the much longer polypeptide chain amyloid precursor protein (APP) by proteolytic enzymatic cleavage in neurons (and other cells) throughout life (Haase. Et al. Nature 359: 322-325, 1992). Hypothesis of the AK amyloid cascade The starting point for the pathogenesis of the disease is a disorder in the regulation of APP processing, which causes increased levels and aggregation of extracellular Αβ (βΑβ), especially 42 amino acids 1-42. The oligomerization of Αβ and the formation of fibrils initiate a series of cellular and molecular changes, e.g. dysfunction of axonal transport and synapses, decreased neurotransmitter levels, and neuronal destruction. All of these changes cause a decline, a decrease in synaptic plasticity, and the appearance of tau pathology (Hardy and Higgins, Science 256: 184-185, 1992). Some G-protein coupled receptors (GPCRs) directly affect the amyloid cascade by modulating enzymes involved in APP proteolysis (α-, β-, and γ-secretase) as well as Αβ degradation. In addition, Αβ interferes with the function of GP-linked receptors (Verdier and Penke, Curr Prot & Peptide Science: 1.9-31, 2004; Verdier et al. J Neurochem 94: 617-628 2005). Extracellular Αβ causes signaling disorders, leading to cognitive dysfunction and consequent neurodegeneration in the brain (Patel and Jhamandas Expert Rev Mol Med 14: e2 201; Xu et al. Prog Neurobiol 97: 1-13, 2012). Knowledge of the relationship between GPCRs and AK has transformed these receptors into new therapeutic targets for AK (Thathiah and De Strooper, Nat Rev Neurosci 12 (2): 73-87, 2011; United States Patent Application 0100137207, 2010).

The original amyloid cascade hypothesis has changed radically several times over the last 10 years. Currently, AK researchers are turning their attention to intracellular Αβ (ίΑβ). Today, it is a generally accepted view that there are two Αβ stores in the brain: extra- and intracellular Αβ, and there is a dynamic equilibrium between the two. More and more data suggest that βΑβ is not of paramount importance in the onset of the disease and eAO deposits and plaques may not cause all forms of AK pathology, e.g. apoptotic cell death. As early as 1999, Hartmann published that the bog (and in particular the highly toxic 1Αβ1-42) is formed in various subcellular cellular organs (Hartmann Eur Arch Psychiatry Clin Neurosxci 249: 291-298, 1999). According to the intra-cellular Aβ hypothesis (LaFerla et al. Nat Rev Neurosei 8: 499-509, 2007), the accumulation of 1A8 in the development of AK is the original pathological event. The real eitotoxic substance is iAp, the eAp deposition itself, and plaque formation in the later stages of AK are more associated with neuronal death. The mechanism of amyloid plaque formation also suggests an intra-cellular cycle (Friedrich et al. Pros Back Acad Sci USA 107: 1942-1947, 2010). In the last ten years, several summary publications have presented the accumulation of ΙΑβ and its effects (LaFerla et al. Kat Rév Meurosci 8: 499-509, 2007; Glménez-Llort et al. Neuroscí Biobehav Rév 31: 125-147, 2007; Li et al. Prog Neurobiol 83: 131-139, 2007; Gouras et al Act: Neuropathol 119: 523-541, 2010; Bayer and Wirths Nervenartz 79Supp3: 117, 2010; Summary book on the role of ίΑβ: (Zhang in: Jelinek, R (Ed J, Lipids and cellular membranes in amyloid diseases, Wiley 2011, pp. 143-159.) Several experiments have shown the presence of iAp in neurons and primary human neurons, the same studies have demonstrated the interaction of 1AS with proteins and cellular organelles. in mouse models in which Αβ is formed and accumulated within neurons, ΐΑβ induced cognitive decline characteristic of early AK (Casas et al Am J Pathol 165: 1289-1300, 2004; Billings Neuron 45: 675-688, 2005; Tomiyama J Neurosci 30: 4845-4856, 2010; Abramowski et al . J Heurosci 32: 1273-1283, 2011).

Another hypothesis for the formation of Ak is related to the microtubular tau protein (Iqbal et al. Acta Neumpathoi 109 (1): 25-312, 2009). Tau is a very important protein in AKpatology: the Braak classification of disease stages is based on the location and spread of tau secretions. Like Αβ, hyperphosphorylated tan protein forms toxic intracellular aggregates. This results in the collapse of the microtubular system and the bankruptcy of axonal transport (Iqbal et al. Acta Neuropathol 109 (1): 25-312, 2009; Takashima Corr Alzheimer Res 5 (6): 591-8, 2008), which cause the ornamental function of neurons. and his death ..

Nowadays, the two hypotheses have been linked (Small and Duff Neuron. 60: 534-542, 2008} * AK is considered to be a specific type of tauopathy in which Αβ is in a key position: ίΑβ formation and οΑβ accumulation initiate patellar processes. Both Αβ form initiates tau hyperphosphorylation, whereas the tan protein is required for ββ-induced neurodegeneration (Shipton et al. 0 'Neurosei 31: 1688-1692, 2011).

According to Ittner and Gotz's latest tau axis theory (Nature Rev Neuroscience 12, 67-72, 2011), Αβ is the indite factor, but the tau protein plays a leading role in the dendritic compartment signal of neurons: Αβ toxicity is mediated only by tau . Αβ induces tau hyperphosphorylation and detachment from the microtubular system. In parallel, tau accumulation in dendrites makes the neuron vulnerable to Άβ, which further enhances tau Mperphosphorylation, creating a magic circle. Since Αβ is the initiator of pathological processes, the effects of Αβ should be considered.

For two decades, AK drug research was based on the amyloid cascade hypothesis. Potentially neuroprotective organic compounds that either inhibit Αβ aggregation or increase Αβ clearauce have been involved in drug research and development. In addition to compounds that reduce or completely inhibit Αβ aggregation and toxicity (Kisilevsky et al. Nat Med 1 (2): 143-148, 1995; Wood et al. J Biol Chem 271 (8): 4086-4092, 1996) attempted Αβ-induced neuronal t-death. to prevent. One such approach is to turn off risk factors. Αβ1-42 is not formed in the presence of β- and γ-secretase inhibitors. Unfortunately, these enzyme inhibitors are not specific and therefore cause undesirable side effects. Other drug candidates inhibit the interactions between Αβ1-42 and membranes. These include amyloid surface-associated molecules, certain competitive inhibitors (RGD peptides) and memantine. The use of Αβ for active immunization as an antigen also appeared to be a promising therapy. Clinical studies, on the other hand, have shown that Αβ initiates acute and dangerous inflammatory processes in the brain (Munch and Robinson, J Neural Transm 109 (708): 1081-1087, 2002).

Increasing knowledge of the neurobiology of AK has opened up new avenues in drug development. Such e.g. identification of specific aggregation forms of Αβ (Summary: Broersen et al., Alz. Gap Ther 2/12: 1-14, 2010). We have been able to find a self-replicating konβ with a special conformation that transfers AK from neuron to neuron (Aguzzi and Falig, Nature Neurosci. 15 (7) 936-939, 2012). Recent research shows that the severity of the disease correlates more with the amount of oligomer Αβ than with its fibrillar form. (McLean et al. Ann Neurol 46: 860-866, 1999; Read et al. Am J Pathol 155: 853-862, 1999). The Αβ monomer is non-toxic, does not show a specific conformation in solution, but is readily converted and aggregated during β-amyloid formation. Amylose formation is a polymerization reaction starting from a single core, in the introductory phase of which mainly monomeric Αβ conformations exist, from which low molecular weight water-soluble oligomers (dimer, trimer, hexamer, octamer) are formed. Oligomeric intermediates with a β-layer structure are also referred to as prefibrillar aggregates. (il) bind amyloid-specific dyes, e.g. Congo red and Thieflavin ~ T ~ (ThT); till) are less stable than fibrils.

Amyloid fibrils are threads with a water-insoluble β-structure consisting of 2 to 6 protofilaments.

The key step in the pathogenesis of AK is the formation of soluble, toxic Αβ peptides (Αβ1-42 oligomers and protofibrils). These so-called internally disordered proteins that have a variety of metastable conformational states that cause neurons to interact with a number of proteins (Tompa FESS J 276 (19): 5406-15, 2009; Broersen et al. Alzheimer's Kes &.

Therapy 2 (4): 12-14, 2010). Understanding the exact chemical structure and conformation of toxic Αβ oligomers is a primary goal of research, as their knowledge may help to design specific molecules capable of repairing Αβ-induced disturbances in signal transduction and intercellular communication. Based on the knowledge to date, the researchers have developed a strategy that has enabled the design and synthesis of peptides that inhibit Aβ aggregation and dissolve amyloid fibrils (Summary: FülÖp et al. Research Signpost 57/661 82, 161188, 2009). The first results did not confirm the conformation of Αβ-fox'ma responsible for the spread of the disease, so the first set of designed inhibitory peptides was unsuccessful.

In the AK and PK model systems of the last 5 years, it has been demonstrated that various proteins can be used to generate infectious agents that spread the disease in vitro. These studies have demonstrated that defective conformational proteins are active components of infectious agents. This discovery links AK to the family of prion diseases. According to the protein only (prion) hypothesis, infectious prions consist wholly or at least partially of mismatched proteins (Griffith Nature re re re re

215: 1043-44, 1967). This cellular prion protein (PrPSc) is formed from the normally structured native pxion protein (BrPc) by a simple conformational change. Once this occurs, it is mainly PrPSc that accumulates in the extracellular space.

Based on these, Αβ can be directly toxic to neurons and synapses. In the brains of patients, the internally disordered structures of Αβ form each step of the aggregation cascade. The pathology of AK, the Braak stages (Braak H. and Braak E. Neurobiol. Aging 16: 271-278, 1995), also indicate that the disease spreads from cell to cell. It is now almost forgotten that at the beginning of AK research, in the 1980s and 1990s, the disease was considered a prion disease (Prusiner Biochemistry 23 (25): 5898-906, 1984; Gajdusek Hol Neurobiol 8 (1): 1-13.1994 ). Recently, soluble amyloid extracts have been shown to lead to amyloidosis in mice, although these animals do not otherwise develop amyloid plaques (Morales et al. Mol Psychiatry 17, 1347-1353, 2012). A p í r c g 1 u t am í 1 - A β 3-42 e g e re kb e η ρ r 1 o n. - s z e r û t u 1 a j d on s o g a t (Nussbaum st al Nature 485: 651-655, 2012). The pathological similarity between AK and prion diseases suggests that the formation and proliferation of amyloid-type proteins exhibit a common molecular mechanism — defective conformational formation on the protein template (Eisele et al. Science 330: 980982, 2010, at peak and Walker, Ann Neurol 70: 532-540, 2011).

Experimentally, β-amyloid formation and accumulation in the brain can be induced by externally ingested brain extract containing aggregated Αβ particles (inoculation-nucleation model). Recent research shows that when a clear solution of Αβ is injected into the brain, plaques form throughout the brain within 5 to 6 months (Stohr et al. Proc Natl Acad Sci USA).

27, 11025-30, 2012). If Αβ is injected into only one of the main tissues, plaque formation also begins in both hemispheres. The agent that initiates amyloid formation is certainly A3 itself (Auer et al. Physa Rev Lett 101: 258101, 2008). it is most easily formed in the living brain (Polymenidpu and Cleveland, J Exp Red 209: 889-8 93, 2012}. Infectious prions and prionoids can be prepared by cyclic amplification of defective protein structure formation (PMCA, Soto et al., TINS 25: 390394, 2002, Saá et al. J Biol Chem 281: 05245-35252, 2006; Murayama et al. Neurosci Lett. 413: 270-273, 2007; Soto and Satani Trend Mol Med 17: 14-24, 2011;

Gonzalez-Montalban et al. PLoS Pathog 7 «$ 1001277, 2011; MorenoGonzalez and Soto Semin Cell Dev Biol 22: 482-487, 2011). The conversion of a non-infectious protein molecule to a pathogenic form (e.g., PrPc-PrPSc) is not yet fully understood, but data to date suggest the validity of the inoculation-nucleation model. According to the model, the pathogenic conformer (e.g., PrPSc) is an oligomeric aggregate that acts as a template (seed crystal). It binds native protein (PrPc) and catalyzes its conversion to the defective, pathogenic conformation by incorporating it into the growing protein polymer (Soto et al. TIES 25: 390-394. 2002; Soto et al. TIBS 31: 150-155, 2006).

It is not yet clear whether other components (e.g., cofactor) in addition to pathogenic prion-pionoid proteins are required for the formation of the infectious agent. At such coffees, it can catalyze the conversion to prionoids, help stabilize the hypertoxic conformation, and increase the biological stability of prion-prionoids (Soto TIBS 36 {3): 151-8, 2010).

SUMMARY OF THE INVENTION e.g.

We have previously outlined that there have been some attempts to prevent AB aggregate involving small molecules (e.g., conventional organic compounds), some of which are still in clinical trials (for an overview, see Amijé and Scopes, J Alz Dis 17:

33-47, 2009). Considering the latest findings from AD research, most AD therapies focus on what is a horse peptide. For the treatment and prevention of AD, a new compound must be developed that prevents the formation of toxic Αβ oligomers and their structural conversion to toxic prionoids. Such a novel compound potentially inhibits extracellular and intracellular toxic Αβ interactions with the neuronal membrane, membrane proteins, and intraneuronal and molecular cell organs, respectively. If these interactions are prevented, Αβ does not trigger tan pathology, lysosomal enzyme formation, and apoptotic cell death.

Thus, the central problem of the present invention is that there is currently no effective means of preventing the conversion of Αβ aggregates to toxic oligomers. This would allow 20 prevention and treatment of diseases which result from .Αβ aggregation and which can be treated by controlling the harmful effects of Αβ-peptides,

Therefore, it is an object of the present invention to provide compounds that effectively inhibit the conversion of amyloid aggregates to toxic oligomers and initiate the binding of aggregates to larger, non-toxic moieties. These compounds may be effective in the prevention and treatment of diseases associated with the aggregation of amyloid proteins. This includes all neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Down's syndrome, type II diabetes, amyloidosis, and the like.

Surprisingly, it has been found that members of the new group of compounds behave as B-am idd structure modifiers. THE

Thus, to solve the problem outlined above, the present invention provides a new class of compounds consisting of short peptides and peptide mimetics. These peptides (among other useful properties) inhibit A $ aggregation.

Briefly, the present invention provides peptides and peptide drugs of the general formula (iX):

A1 ~ AZ ~ A 3 ^- A 4 ~ A 5— ¥ (χ) where

A1 is barme.i.y.ike: Ara or Glu;

A2 j e 1 e nt e s e P x o;

A3 is Ala;

A4 is Pro;

A5 is any of Al or Glu;

Y is absent or an amide;

or a pharmaceutically acceptable salt or ester thereof.

The present invention further includes the following peptides and peptide mimetics:

20 EPPPA (SEQ ID NO 29), epppa. ~ am.id (SS : q ID NO 32) apapq (SEQ ID HO 46) _f apapn (SEQ ID NO 4 8), apapq ~ amid í -S ; q ID NO 4 9) 25 apapnamide (NEITHER ; q id no 50)

or a pharmaceutically acceptable salt or ester thereof.

As can be seen from the above formula (I), the present invention relates to peptides comprising 5 amino acids in which, in addition to the naturally occurring amino acids, some artificially produced amino acids and peptide chain ends are also present.

A1, A2, A3, A4, and AS were selected so that the resulting peptides or peptide mimetics would bind to the naturally occurring Αβ of the pond, thereby modifying the aggregating properties of the pond and through structural change. inhibit neurotoxicity.

The present disclosure relates primarily to the peptides and peptide mimetics listed in Table 2 and claim 14, respectively (see below).

More particularly, the present invention provides peptides and peptide mimetics having the formula:

EPPPA (SEQ ID NO 2 9), 10 epppa-amide (SEC ID NO 32), apapq (SEQ ID NO 46), apapn (SEQ ID NO 48), apapq-amide (SEQ ID NO 49), apapn-amide (SEQ ID NO 50), EPAPA {SEQ ID NO 28), 15 APAPE-amide (SEQ ID NO 30), apape-amide (SEQ ID NO 33), apape (SEQ ID NO 51)

and their pharmaceutically acceptable salts and esters.

Note that throughout the specification, SEQ ID NO is a sequence identification number.

The invention further relates to pharmaceutical compositions comprising at least one of the above-mentioned peptides or peptide mimetics, their salts and esters. and at least one excipient accepted in the formulation <This carrier provides controlled release of the active ingredient as far as possible.

The invention encompasses pharmaceutical compositions comprising at least one peptide or peppermint interest of the formula:

EPPPA (EEG ID NO 29), epppa-amide (SEQ ID NO 32), apapq (SEQ ID NO 4 6), te apapn (SEQ ID NO 48), te te fe s * & «« · 'apapq-amide ( SEQ ID NO 49), apapamide (SEQ 10 NO 50), EPAPA (SEQ ID NO 28), APAPE amide (SEQ ID NO 30), apapamide (SEQ ID NO 33), apape (SEQ ID NO 51) and their pharmaceutically acceptable salts and esters.

The patent covers the use of the above-mentioned peptides and / or peptidomimetics and their corresponding salts and esters in the manufacture of a medicament for the prevention and treatment of neurodegenerative diseases having Αβ aggregation, e.g. Down syndrome, a. II. types of diabetes, 15 and amyloidosis.

Abrasive glasses

Figure 1 shows an TEM image of oligoteric and fibrillar Αβ 1-42 from ίζο-Αβ 1-42 (75 gM) peptide after 0 min A), 4 20 h 8) and 24 h C), apape (SEQ ID NO 51 ) (1: 5 molar ratio).

Figure 2 shows the effect of apape pentapeptide on the size distribution of amyloid aggregates (pH 50). dH: hydrodynamic diameter, CG7: control peptide Cys- (Gly) · ?. Apape pentapeptide 25 (1: 5 molar ratio) accelerated the formation of large beta-amyloid aggregates, which were already visible after 4 h of incubation, whereas the control peptide had no similar effect. The presence of fibrillar aggregates was detected in both samples after 24 h.

Figure 3 shows the spectra of Αβ 1-42 and apape-NHxECD

During a 168-hour measurement, a 1: 5 molar ratio of Αβ 1-42 to apape. Subtracting the spectrum of pure apape-NH 3 from the mixed spectrum of Αβ 1-42 and apape-NH 2, the differential spectrum was obtained. to you

Figure 4 shows the alignment of the backbone of the indicated peptides by cluster analysis. The ribbon is always fitted to the largest cluster representation.

Figure 5 shows the time dependence of the signal intensity of the ax apape 1H HMR spectrum in the presence of Αβ 1-4 2 (a). The concentration of ίζο-Αβ 1-4 2 is 250 mM and that of apape peptide is 1.9 mM. The bound apape fraction was 3.5% after 24 hours (diamond). In this case, a 66.5 pH apape bound to Αβ 1-42, representing a molar ratio of 1: 4. Apape signal intensity was used as a control without Αβ 1-42 and indicated by a circle (b): After one week, the final value of the bound apape fraction was 9% (diamond). In this case, an apape molecule binds to a Αβ 1-42 molecule. The control signal intensity (without apape Αβ 1-42) was 100 ± 0.3% during the study period (circle). (c): 1H NMR metal signal of free apape (dark gray) and apape + Αβ 1-42 samples after 24 h incubation (light gray) (d): assignment of apape signals; (e): detail of apape 1H NMR spectrum. Shifting of the β-terminal Ala protons was observed upon exposure to ίζο-Αβ 1-42. The lower spectrum is the control apape, the upper 24 hours after administration of ίζο-Αβ 1-42.

Figure 6 shows that the papé peptide inhibits ooz Αβ 1-42 induced LTP decrease in mouse hippocampal slices in vitro. LTP was induced by theta-burst stimulation (TBS) and followed for 75 min (A). Panel B shows the mean amplitude of the excitatory postsynaptic field (fEPSP) 85–90 min after TBS. * p <0.05 versus control; one-way Dunnett test after AHOVA.

THE /. shows the effect of intraperitoneal apape and pentaglycine (GGGGG) on the potentiating effect of Αβ 1-42 NMDA (first gray bar). The apape peptide had a protective effect at −50 and −250 min; the control pentaglycine did not affect the Αβ 1-42-induced effect. Mean maximal NMDA-evoked effects were 1 SEM, normal to control (during excitation episodes before total spike strain «ο Αβ 1-42), *: significant difference, p <0.05, n-4.

Figure 8 shows the neuroprotective effect of apape in a rat Morris water maze test. The bar graphs show the mean ± SEM of the results. Platform access latency was given in seconds during the 5-day measurement. The animals were divided into the following 4 groups: bicarbonate buffered saline (HCBS) treated with saline (white), apape (10 mg / bwkg, lpJ and HCBS treated (light gray), oligomer Αβ 10 1-42 (75 μΜ, 136 hours aggregated, icv) and apape (10.0 mg / bwkg, ip) injected (black), oligomer Αβ 1-42 (75 pb, 136 hours aggregated, icv) and injected with physiological salt (ip), (dark gray). There is a significant difference between the groups on the third day, one-way AHOVA, Γ _{48. 3} ™ 3, 449, p « ^: 0.024. *:

significant (p <0.05} difference in LSD post-hoc test between groups on day 3.

. figure. Neuroprotective effect of apape in Morris water maze test in APP x PS1 tg mice. The bar graphs show the mean latency (± SEM) of platform access over 20 seconds during the 5-day measurement. The animals were divided into the following 4 groups: placebo injected wild type (white); apape injected wild type (light gray); apape injected APPxPS1 mouse (black); placebo-injected APPxPS1 mouse (dark gray). There are significant differences between multi-passengers

AHOVA (^ 30.4 ^ 10.480 p * = 0.0001), in the LSD post-hoc test, the wild-type placebo-treated and AFPXPS1-placebo-treated groups on day 4 (p-0.001), the apape-treated wild-type and APPXPS1 group and the transgenic placebo-injected group on day 4 (p = 0.0001).

Figure 10 shows the neuroprotective effect of apape in a Morris water maze test in APP x PS1 mice during swimming 1. Bar graphs show the mean ± SEM of platform access latency per second over a 5-day measurement. The animals were divided into the following 4 groups: placebo injected wild type (white); apape injected wild type (light gray); apape pond pond injected APPxPSl mouse (black); placebo-injected APPxPS1 mouse (dark gray). Repeated AHOVA analysis found a significant difference between experimental groups (555, p-G, 002). Based on the LSD post-hoc test, the difference was significant between the 5 wild-type placebo and APPXPS1 placebo-injected groups on day 4 p = 0.002, the apape-injected wild-type and APPXPS1 placebo-injected group p <0.01, and the transgenic placebo group. injected and transgenic apape injected group p-ü, 037.

Figure 11 shows tau histology in a 50 micrometer thick hippocampal slice from female APPxPS1 transgenic mouse brain. The vertical axis shows the number of tan immunoplastic cells, placebo-injected wild type (white); placebo injected APPxPS1 mouse (light gray);

apape injected wild type (black) and apape injected APPxPSl in mouse (dark gray) brain. Based on one-way AHOVA analysis, there are significant differences between experimental groups (F ^ y-lS, 642, p <0.0001). In the post hoc LSD test, we found a significant difference between the APPxPS1 transgene-placebo Injected and the other three groups. Average ± none.

Figure 12 shows tau histology in a 50 micrometer thick hippocampal slice from male APPxPS1 transgenic mouse brain. The bar graph shows the mean ± SEM of the number of tan immunopositive cells. The other details are in 11.

shown in the figure. Based on one-way AHOVA analysis, there are significant differences between experimental groups. (F, ₆ « _12,756 , p <10.0). In the post hoc LSD test, we found a significant difference between the transgene-placebo-injected and wild-type placebo-injected and wild-apape-administered groups (p-0, ö01). In addition, the number of tan-positive cells was significantly higher in the APPxPS1 transgene placebo group than in the APPxPS1-BFR-106 injected group.

Figure 13 shows cresyl-violet staining in a 50 micrometer thick to to to »hippocampal slice of female pj APPxPS1 transgenic mouse brain. The bar graph shows the mean ± SEM of the number of tau immunopositive cells. White: wild-placebo injected; light gray: APPxPS1 transgene-placebo injected; black: wild-dad injected; and dark gray: APPxPS1 transgene5 apape injected group. Based on the one-way AHOVA analysis, there is a significant difference between the experimental groups.

Also, the wild-placebo-injected group differed significantly from the APPxPS'-apape-treated group (p ~ '0.003), and the transgenic-placebo group from the APPxPS'1 + apape-injected group (pp ^:::: u _f v 2 i).

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DETAILED DESCRIPTION OF THE INVENTION

The structures of the peptides described herein are proteins that interact with Αβ 1-42 (Verdier et al. J. Neurochem. 94, 617-28, 2005). (Table 4) were designed based on computer space analysis of common, overlapping binding sequences. Based on proteomic analysis, the Αβ-binding region of these proteins contains very similar amino acids, primarily hydrophobic amino acids, proletic, and aminodicarboxylic acids. Based on this, proline has been incorporated into various sites in the designed compounds, as it is well known that this amino acid has a greater β ™ structure-destroying effect than the others (Chou and Fasman, Annu Rev Bicehem 47: 251-276, 1978). It is also known that the incorporation of proline into short peptide homologues of Αβ eliminates amyloid formation (Soto et al. Biochem Biophys Rés Commun 226 (3): 672-680, 1996).

We also designed two peptides in which the L-amino acids were replaced with D because they are resistant to proteases and do not increase the antigenic nature of the peptides. (Poduslo et al. J Neurobiol 39 (3): 371-382, 2003: Dintis et al. Proteins 16 (3): 306-308, 1993). The suitability of the peptides as drug candidates was increased by making appropriate chemical modifications (e.g., incorporation of peptide mimetics at the N- and C-termini) into the molecule (Tables 1 and 2).

Due to the complex nature of the disease, a multidisciplinary approach is needed in drug discovery. This means that both the acute (synoptic activity, long-term potentiation) and late {cytotoxic} effects of Αβ should be measured at the cellular level and combined with the pathophysiological and cognitive effects associated with AK. This is the only way to assess disease progression and the effectiveness of treatments.

The neuro- and synaptoxic forms of Αβ-peptides (Αβ.Ι-42 oligomers and protofibrils) do not have a well-defined, stable conformation, only a few secondary structural elements (eg β-layer structures; re re re re re belong to a family of internally disordered (.T.DF) proteins, and such polypeptide chains can bind to a large number of proteins that play a key role in cells in a non-specific manner, resulting in loss of neuronal function.

The compounds described herein are β-amyloid structural modifiers. These substances bind to soluble, toxic Αβ-oligomers and protofibrils by salt bonding or apolar interaction and transform their structure into a non-toxic conformation. In the subsequent step, the same compounds catalyze the formation of large, non-toxic Αβ aggregates. At the same time, the group of compounds described herein stimulates the formation of dendrites and improves communication between neurons.

The structures of the peptides and peptide mimetics of the present invention can be represented by the following general formula:

A - A r Aj-A r Á ₅ ~ Y where Y is an organic functional group; Amino acids Α1 to Ά5 (encoded and unencoded, R or S) or allyl (see claims 1-4).

The present disclosure also relates to amino acid sequences that are analogs of the sequences described and that have biological effects analogous to those of the pedeptide sequences described herein. It will be apparent to those skilled in the art that certain side chain modifications or amino acid substitutions made in the peptides in question do not affect their biological function. Such modifications are based on the side chain similarity of the amino acids (e.g., size, electrical charge, hydrophobicity, proximity to hydrophilicity). The possible purpose of these modifications is, on the one hand, to increase the stability of the peptides against these drugs and, on the other hand, to improve certain pharmacokinetic parameters.

The present disclosure also encompasses peptides that contain easily detectable elements (fluorescent group, radioactive to-to-atom, etc.).

salt

The present disclosure also encompasses peptides that contain some additional amino acids at the N-, C-, or both terminals of the parent compounds, provided that these changes do not significantly affect the biological activity of the parent peptides. The terminals. such modifications serve to facilitate the immobilization of the original peptides, to allow their binding to other reagents, and to increase their solubility and absorption.

The nomenclature recommended by IUPAC (Nomenclature of αAmino Acids, Recommendations, 1974, 14 (2), 1975) was used to reduce the amino acids in the peptide sequences.

The invention includes Salts of the peptides according to any one of claims 1 to 4 for use in pharmaceutical compositions. By the term is meant salts which do not exhibit in contact with human or animal tissue. toxic, irritant, allergenic or similar effects. The following salts are not intended to be exhaustive: acetate, citrate, aspartate, benzoate, benzenesulfonate, butyrate, digluconate, hydrosulfate, fumarate, hydrochloride, hydroboxide, hydroiodide, lactate, maleate, methanesulfonate, oxalate, propionate, succinate, tartrate, phosphate, glutamate salts.

Non-exclusive examples include salts of the following bases: alkali metal and earth metal salts (lithium, potassium, sodium, calcium, magnesium, aluminum), quaternary ammonium salts, amine cathionics (methylamine, ethylamine, diethylamine, etc.).

The peptides of the invention may be used directly in pharmaceutical compositions which may contain additional additives to elicit the appropriate biological effect. Such pharmaceutical preparations may be e.g. formulations known to those skilled in the art which contain a carrier that allows controlled release of the active drug compound. These are systemic polymers which are e.g. upon entry into appropriate tissues (e.g., blood plasma), they are degraded by enzymatic or acid-base catalyzed hydrolysis (e.g., polylactides, polyglycolides).

The pharmaceutical compositions according to the invention may also contain other excipients, e.g. diluents, pH5 regulators, antioxidants, preservatives, isotonic agents, etc. These are state-of-the-art d a 1 é k a n y a g o k.

According to the invention, the pharmaceutical compositions can preferably be administered parenterally to the body (intravenous, intramuscular, subcutaneous, etc.). preparations which are reconstituted immediately before use. These liquids may include carriers, diluents, or solvents: water, ethanol, various pollen alcohols (e.g., glycerin, propylene glycol, polyethylene, and the like), carboxymethylcellulose, various vegetable oils, organic esters, and mixtures of any of the foregoing,

According to the invention, the preferred formulations of the pharmaceutical compositions are tablets, powders, suppositories, injections, syrups, and the like.

The doses administered depend on the type of disease.

they depend on the sex, age, weight, and severity of the disease in the patient. For oral administration, the preferred daily dose for the active ingredient is e.g. 0.01 mg and Ig, in case of parenteral administration (eg intravenous preparation) e.g. Is between -0.001mg and 100mg

The pharmaceutical compositions can also be used in the latest liposomal or microcapsule formulations. According to the invention, the peptides can also be introduced into the body by the latest gene therapy methods.

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According to the invention, the peptides are particularly useful in the treatment of diseases in which inhibition of aggregated toxic forms of O-amyloid is beneficial, where the target is a β-amyloid peptide having an S-layered sequence capable of spontaneous further aggregation until fibrils are formed. The invention therefore encompasses the use of peptides in the manufacture of a medicament for the treatment of such diseases. As mentioned above, such diseases are primarily neurodegenerations

H) (especially Alzheimer's and Parkinson's disease and type 2 diabetes).

In order to understand the invention, a number of terms are defined below. The definitions provided herein are generally well understood by those skilled in the art.

The term aggregating as used herein refers to the association of peptide chains regardless of whether the resulting structure is in an ordered or disordered, stable or unstable, ordered or disordered native state. Such associations are formed by intermolecular interactions, hydrophobic interactions, hydrogen bonds, van der Waals forces, ionic bonds, or other binding forces that result in the coupling or association of two or more peptides or peptide regions. The term aggregation as used herein includes e.g. the formation of fibrils.

By the term amides is meant different types of protein aggregates that show special behavior in microscopic examinations. The name amyloid comes from a previous misidentification because these substances were incorrectly identified with starch (Latin: amylum) based on their iodine staining reaction. Amyloid is typically identified by the A-change in fluorescence intensity of planar aromatic dyes (e.g., thioflavin T or Congo red). This is usually attributed to the fact that these dyes are incorporated into the β-fibers with an intercalating agent and their immediate environment changes in the process. Amyloid deposits are characterized by misfolding of amyloidogenic proteins, the native secondary structure (often e-helix but also 5 disordered coils) being transformed into a pathological β-fold layer structure, ex allowing aggregation into insoluble fibrils. According to fine structure studies, amyloid fibrillations are composed of two protofilaments with helical conformations. These amyloid deposits show characteristic resistance to digestion with 10 proteases. Polymerization to an amyloid is generally sequence dependent, i.e., mutations in the amino acid sequence may prevent aggregation, especially if the mutation is a β-structure-destroying amino acid, e.g. a 15 proline. Thus e.g. in human type 2 diabetes, an associated amyloidogenic peptide is present, whereas in rodents, proline is present at a critical site of this peptide and no amyloid formation occurs. In man, kh. 25 different proteins are known that can form an amyloid.

The term amyloidosis refers to diseases and conditions in which defective protein folding occurs, resulting in the formation of amyloid or amyloid-like fibrillar deposits. In these, misfolded proteins have a characteristic pathological β-layer structure.

The O-amyloid peptide, amyloid opeptide or O-peptide used in the present invention is a human amyloidogenic peptide derived from APP by proteolytic cleavage. The Άβ-peptide can take various forms: ΑβΙ-42, Αβί-40, 30 A35-42, and the like. (numbers written after Ah are the number of the first and last amino acids in the given peptide, based on the original amino acid sequence). The amino acid sequence of the human ΆβΙ-42 peptide is as follows: DAEFRHDSGYEVHHNKLVFFASDVGSNKGAIIGLMVGGVVIA. (SEQ 10 NO 60). In the case of some familial AK (eg the Swedish mutation) fungi, mutant Αβ 1-42 peptides are found. Unless otherwise indicated, the term β-amyloid peptide includes these mutations.

The protein folding used in the present invention is the physical process in which a polypeptide chain is coiled from a disordered skein into a characteristic, functional three-dimensional structure. The formation of a correct three-dimensional structure is absolutely necessary for biological activity. Folding defects result in the formation of inactive proteins, which are usually toxic. The general opinion is that quite a few neurodegenerat.lv diseases are caused by the accumulation of amyloid fibrillumck formed from defective folding proteins.

The term internally disordered proteins (IDPs) as used herein refers to proteins that do not have a stable secondary and / or tertiary structure in solution and are nevertheless biologically effective.

Proteins can undergo reversible spatial changes during their biological role. The different alternative structures of a given protein are called conformations and the transition between them is called a conformational change.

As used herein, the term peptide refers to compounds that bind to amyloid or amyloid-like deposits and contain 2,3,4,5,6 or more amino acids in the chain, such as D- or Lamino acids in terms of chirality. In this context, the term peptide should not include an antibody or antigen-binding fragment of an antibody (e.g., Fab), nor amyloidogenic peptide sequences, e.g. Αβ1 ~ 40, which aggregates with similar molecules to form a 3-layer structure. As used herein, pepii dm imitium refers to any compound that is composed of amino acids (either D- or L-, natural or synthetic amino acids) and may contain non-amino acid moieties. Your peptide mimetic is designed to show the biological effects of the natural peptide to be mimicked. Thus e.g. Peptide mimetics are compounds that are designed to mimic the structure and binding activity of a particular peptide (e.g., through hydrogen bonds and hydrophobic interactions).

The term pentgpeptld in the present invention refers to compounds consisting of five amino acids, modified or substituted. Such e.g. apape (SEQ ID NO 51) is a pentapeptide consisting of the following amino acids: D-Ala, D-Pro, D-Ala, D-Pro and D-Ala-amide.

As used herein to describe proteolytic digestion or enzymatic enzyme, it is meant that a given spatial polypeptide chain is cleaved at least 25% less than a native polypeptide chain over a period of time when both forms are treated with similar amounts of a progase enzyme under the same conditions. This term is used for peptides and peptide mimetics that reduce the toxicity of amyloid or amyloid-like deposits. A peptide mimetic is resistant to proteases if at least 10%, but more preferably 20,30,40,50,70,80,90 or even 100% less is cleaved under the same conditions by a given amount of protease than a structurally similar peptide. .

The terms reduced or inhibited in the specification mean a reduction of at least 10% of a given parameter in the experiments compared to the untreated control. For example, a reduction in neurotoxicity means that a compound reduces the toxic effects of a substance on neurons or tissue by at least 10% relative to an untreated control neuron or tissue. As used herein, neurotoxicity refers to the ability of a substance to cause or even destroy functional damage to nerve tissue or neurons.

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The bond described herein refers to the physical association of one molecule with another. Binding can occur both covalently and non-covalently, with interactions between atoms and molecules, and can be transient and permanent. Examples include, but are not limited to, ionic bonds, hydrophobic interactions, hydrogen bonds, van de Waals forces, and dipole-dipole bonds. As used herein, the term neural cell refers to cells of the central nervous system and is not limited to the neuron and the gyrus. Examples of these review studies can be found in the description. It will, of course, be apparent to those skilled in the art that other review assays may be practiced without departing from the spirit and scope of the invention.

Examples

In the following section, the invention is illustrated in detail by way of examples, but the applicability of the invention is not limited to the examples listed.

Until recently, it was commercially available synthetic

Samples Αβ 1-42 were not homogeneous in terms of their degree of aggregation and solubility. Consequently, the biological experiments performed with these preparations were not reproducible in all cases. Together with others, we have shown that ζβ forms such as oligomers and fibrils can be reproducibly prepared from the ίζο-Αβ 1-42 peptide with a certain degree of aggregation. (Bozsó et al., Peptides 31; 248-256, 2010). This controlled method of Αβ production greatly facilitates the study of the molecular basis of AD, to to to. Example: Design of peptides that inhibit amyloid toxicity

The oligomerization and aggregation of some polypeptides and proteins (β-amyloid, α-coloruclein, huntingtin, prion protein) play an important role in the neurodegenerative processes of certain disease groups (eg AK). The soluble forms of the neurotoxic and synaptotoxic β-amyloids (AB 1-42 oligomers and profotibrils) have no defined conformation, containing only a few secondary structural elements (e.g., β-fold). H) The Αβ peptide belongs to the family of intrinsically disordered proteins (IDPs). These proteins interact non-specifically with a number of important cellular proteins, causing neuronal dysfunction. Our neuroprotective agents of the present disclosure (Id. Table 2) bind to toxic, soluble Αβ oligomers and protofibrils through 15 salt bridges and apolar interactions. Members of this new family of compounds act as β-amyloid structure-modifying molecules. These compounds alter the conformation of the originally disordered (IDP) Αβ-peptide, thereby initiating the formation of large, non-toxic Αβ-aggregates.

At the same time, members of the family of compounds stimulate the formation of dendrites, improving communication between neurons, which reduces the harmful effects of Αβ-peptide on the brain.

2. Peptide synthesis

Materials

Protected amino acids were obtained from Orpegen (Heidelberg, Germany), Bachem (Bubendorf, Switzerland) and GL Biochem (Shanghai, China). DCC, HOBt and MBHAxHCl resin were purchased from GL 30 Biochem (Shanghai, China). Boo-Ala-PAM resin was obtained from Bachem (Bubendorf, Switzerland). Boc-u-GlnMerrifield resin, Boc-D-Asn-Merrifleld resin, Boc-DGlu (Bzl) -Merrifield resin and EDCxHCl were obtained from IRIS Biotech to GmbH (Marktredwitz, Germany). The solvents and to · to to

DIPEA was purchased from Sigma-Aldrích. HPLC grade TFA was obtained from Pieroe (Rockford, XL, OSA).

AJ ^ -miioid peptide

Oligomers Αβ 1-42 were prepared in situ from isopeptide (Ίζο ~ Αβ 1-42 ') as previously reported (Borsó et al., Peptides 31: 248-256, 2010).

B) Synthesis of Oj inhibitor pulp fi de Jr and peptide sumetics Boc-chemical methods

the. Method for synthesis of the following peptides: EFAPA-NH ₂ , ErPPA-NH 2, APAFE-NH 2, APPPF-NH ₂ , epapa-NH 2, apppa-NH 2, apppe-NH ₄ , EPAP-NH 2 NH ₂ , RPAPA-NH 2, KPAPA-NH ₂ , RPPPA ~ NH ₂ , KPPPA-NH 2, pape-NH 2, pppe ~ NH ₂ , DPAPA-NH ₂ , Succinyl-PAPA-NH ₂ , Propionyl-PAPA-NH 5, β -Ala-PAPA-mh, Xminodiacetyl-PAPA-Nto, ESar-APA ~ WH ₂ , EPA-Sar-A-NH ₂ , E-Pip-APA ~ Nü _2í EPA-Pip-A-NH ₂ , E ~ Sar ~ A-Sar-A-NH ₂ , apapd-NH ₂ , apapq-NH ₂ , apapn-NH 2, propionyl-pape-NH ₂ , propionyl-pap-NH ₂ , apGpe-NH ₂ , ap-N-Me-ala- NH ₂ , α-ticape-NH ₂ , ap-tic-e-NH ₂ , ap-tic-p-HH ₂ , α-Sar-ape-NH ₂ , ap-Sar-e-NH ₂ , a-pip- ape-NH ₂ , ap-pip-e-HH ₂ , Gpap-NH ₂ , (N-Me-ala) -papaKH _2f ap-Aib-pe-NHa, Aib-pape-NH ₂ , apap-OH ₂ , APEPA ~ HH ₂ , ape ~ NH ₂ , Ac-ape-KH ₂ , GABA-pe-NH ₂ .

Pe.pt was synthesized here on two MBHAxHCl polymer resins. After swelling in bCM, the polymer resin was centralized with 5% DIPEA / DCM (2x1 min). After neutralization, the resin was washed three times with DCM. A 4-fold excess of N1- ^Boc protected amine acids was activated with DCC / HOBt in DMF / DCM (1: 1) and the resin was incubated in this mixture for 3 hours. The polymer resin was washed twice with DMF, DCM and methanol, partially dried and the coupling step was checked by a qualitative ninhydrin test. In the case of a positive ninh.idr.in test, the coupling step was repeated. When acylation was complete, the resin was swollen in DCM and the Boc protecting group was removed with 50 TFA / DCM (5 + 25 min).

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The peptide-resin was washed three times with DCM and the same neutralization step was performed before coupling the additional amino acid.

The peptide was cleaved from the resin with HF (cleavage mixture per 1 g of peptide-resin: 10 ml of HE, 0.8 ml of dimethyl sulfide and 0.2 ml of anisole). Cleavage was performed at 0 ° C for 45 minutes. The crude peptide was precipitated with diethyl ether, dissolved in 50% ACN / water and lyophilized.

b. Method for synthesizing the following peptides: EPAPA, EPPPA, apapq, apapn, apape.

Peptides were synthesized on the polymeric resin corresponding to the C-terminal amino acid (Boc-Ala-PAM resin, B © c ~ D ~ Gln ~ Merrifield resin, Boc-D-Asn-Merrifield resin, Boc ~ D ~ Glu (Bel} Merrifield resin)). The polymer resin was swollen in DCM and the Boc group was removed as detailed above, and the neutralization, coupling step, and cleavage from the resin were also performed as described in Section a.

α-chemical process

The following peptides were prepared by Fmoc chemistry:

PPPA-NHs, PAPA ~ NH ₂ , Ac ~ EPAP ~ NH ₂ , EPA ~ NH ₂ , Ac-EPA ~ NH ₂ , EPP-NH ₂ , bPA-NH 2, DP-NH ₂ , Succin.yl ~ PA ~ NH ₂ , Succinyi-pa-NHs and Glp-AspA peptides were synthesized on Fmoc-Rinkamide resin. The polymer resin was swollen in DCM and washed with DMF and treated with 20% piperidine / OMF (5 + 15 min) to remove the Fmoc group. The resin was then washed five times with DMF. A three-fold excess of N ”~ Fmoc protected amino acids

It was activated with OCC / HOSt in DMF / DCM (1: 1) and the resin was incubated for 3 hours. The resin was washed twice with DMF, DCM and methanol, partially dried and the coupling step was checked by a qualitative ninhydrin test. In the case of a positive ninhydrin test, the coupling step was repeated. When acylation was complete, the resin was swollen in DMF and the Fmoc group was removed as directed. The cycles were repeated until the complete sequence was obtained. The peptide was cleaved from the resin with TEA (95%), water (2%), trisopropylsilane (1.5%) and dithiothreitol (1.5%). Cleavage was performed at 0 ° C for 15 minutes and then at room temperature for 2 hours and 45 minutes. The crude peptide was precipitated with diethyl ether, dissolved in 50% ACN / water and lyophilized.

Synthesis of Glp-Gly dipeptide

Glp, Gly-OtBu and EDCxHCl were stirred in 15 ml of 100 mM NaHCO3 for 6 hours. The peptide was extracted from the mixture with ethyl acetate (3x). After evaporation of the ethyl acetate, the residual oil was treated with 50% TFA / DCM for 30 minutes. After evaporation of TFA and DCM, the residual oil was purified by HPLC.

Cleaning

Peptides were analyzed and purified by RP-HPLC. D.i. containing 0.1% TFA as eluent. d.i. containing water (Aeluens) and 0.1% TFA and 80% ACN. water (eluent B) was used. Analytical control was performed on a Hewlett-Packard Agilent 1100 Series HPLC column on a Lana c18 column (100 Å, 5 pm, 250x4.60 π », Phenomenex) at a flow rate of 1.2 ml / min in Table 2. detailed gradients. A Shimadzu HPLC was used for preparative chromatography; and an inna Cl8 column (100 Å, 10 gm, 250x21.2 mm, Phenomenex) was used. The flow rate was 4 ml / min. During the purification, the amount of B-eluent in the A30 eluent was gradually increased from 0% to 30% over 60 minutes.

This spectrum is tria

Mass spectrometry measurements were performed on a FinniganMat TSQ 7000 to to m to to mass spectrometer in ESI-MS mode.

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2. Transmission electron microscopy Oligomeric and fibrillar Αβ 1-32 aggregates were prepared from μΜ ίζο-Αβ 1-42 solution according to a previously reported protocol (Bozsó et al., Peptides 31: 248-256, 2010), 10 μΐ Αβ 1 A 75 μl solution of -42 with or without patent peptide (1: 5 molar) was dropped onto a 400 mesh copper grid coated with formvar-carbpn (Electron Microscopy Sciences, Washington, PA, USA) and then stained negatively with uranyl acetate. Aggregates were examined with a 100 kV Philips CM 10 transmission electron microscope (FEI Company, Hillsboro, Oregon, USA). Microscopic images were obtained at 46,000x and

It was made using a magnification of 64,000 times. Image processing was performed using the Analysis® 3.2 software package (Soft Imaging System GmbH, Münster, Germany).

3. Dynamic light scatter measurement (IDLS)

A solution of the following substances in PBS was placed in a small volume cuvette: Αβ 1-42 (50 μΜ), Αβ 1-42 (50 μΜ) apape (ID No. 51) (250 μΜ}, Αβ 1-42 ( 50 μΜ) r Cys-Glyvamide (250 μΜ) Particle size distribution was performed at 25 ° C in a Malvern 25 Zetasizer Nano ZS (Malvern Instruments Ltd.

Worcestershire, UK) equipped with a He-Ne laser (633 nm) and using a special non-invasive backscatter (NIBS®) technology to detect the intensity of light backscattered at 173 °. Changes in the size distribution of the aggregates were monitored for 22 hours. The correlation function for a given time and the distribution of the apparent hydrodynamic diameter (dH) scattered light intensity were determined from the average of 14 measurements. The re-translational diffusion coefficients of the measured autocorrelation

From the sx re re re function, it was determined using a fitting algorithm built into version 5.1 of the Dispersion Technology Software used by the equipment (Malvern Instruments Ltd. Worcestershire, UK).

4. Chelectronic circular dl chrismism (SCD) spectroscopy

The ECD spectra of Αβ 1-42, apape-NH ₂ and their mixture were recorded on a JASCO J815 (Tokyo, Japan) spectropolarimeter equipped with a Paltier element thermosed 3rd v <2 n® quartz cuvette. The concentration of the Αβ 1-42 solution used for the measurement was 12.5 μΜ, while that of the apapamide was 62.5 μΜ. The spectra of the peptide solutions in the wavelength range 200-250 nm at a scanning speed of 100 nm / s at 37 ^{> 3} C for one week were 0 minutes, 10 minutes, 20 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 24 hours and 48 hours. It was recorded at 72 hours and 168 hours. The spectra presented here were prepared by summing 10 scans and subtracting the spectra of the appropriate solvents.

5. computer bonding exams case

The Αβ-fibril model used was described by Lührs et al. (PDAS 102/48: 17'342-17347, 2005) and contains eight: Αβ 1-42 nionomeric units. Amino acids missing from the terminal terminal were replaced by assuming a β-structure. The improved structure was placed in an explicit aqueous (TIF3P) environment and performed with the AMBERS software package (Case et al. AMBER 8, University of California, San Francisco, 2804; 4 ns molecular dynamics simulation using the EF03 force field {and associated atomic charges). . The binding sites of small molecules on the surface of Αβ 1-42 fibrils were determined by the blind docking method (Hetényi and van der Spoel, Protein Science 11: 1729-1737, 2002), the Autodock program (Morris et al. J. Comput. Chern. 1 »: 1639-1662, 19s8). The ligand set was re-docked using a finer lattice in the possible binding regions, and then the well-binding molecules, those with the highest binding free enthalpy (TFE) in the given environment, were selected. Two types of rankings were used for each of the 5 peptides: on the one hand, the best KS3E at a given binding position, and on the other hand, the average of the 10 best KSEs in a given Uganda. Starting from the docking geometries, 1 ns of explicit aqueous simulation was performed with the selected complexes (fibrilium model ligand) and KEZE values were determined by the MM10 GBSA method (Srinavasan et al. J. Am. Chem. Soc. 120 9401-9409, 1998). calculated.

6. Conformational analyzes

Replica-Exchange Molecular Dynamics (REMD) calculations were performed for each molecule at 24 temperatures between 280 and 400 K in an explicit aqueous (T1P3P) environment. Using the GROMACS software package (Hess et al. J. Chem. Theory Comput. 4: 435-447, 2008), 50 ns of stimulation was performed in each case using the amber99sb force field (http://ambermd.org). For the 10-50 ns section of the trajectory obtained at the lowest temperature, cluster analysis was performed using the ptraj program of the AmberTools package (http://ambermd.org). In each case, 4000 structures were selected from the trajectory and clustered using the 'average linkage * method 25 using two cut-off (ε) values: s ^: “l. 0 for clustering backbone atoms and z »1.5 for heavy atoms.

Conclusions

Based on the results of computer simulations of the common sequences of the amyloid-binding proteins (Table 1), 72 new peptides are designed, 65 of which are part of the present patent. The results of MS and HPLC analysis of the new peptides are shown in Table 2. Physicochemical parameters of Αβ te te te te te te oligomers and fibril atoms were determined both in the presence and absence of new peptides. The morphology of the aggregates is shown on the TEM images (Fig. 2). In our experience, the apape peptide (SEQ ID NO) (used in a molar ratio of 1: 5) promoted the formation of fibrillar aggregates, which in this case appeared as early as 4 hours, whereas in the control sample without apape only protofibrils were present at that time. and large oligomers were identifiable. After 24 h, fibrillar aggregates were present in both samples. These observations, together with the DLS results, suggest that apape (SEQ ID NO 51) does not have an anti-aggregation effect but an aggregation-promoting effect.

The ECD spectrum of Αβ 1-42 shows a strong minimum at 200 nm and another peak at 215 nm, suggesting the formation of a random and p-fold structure after peptide dissolution.

Apape-NHa alone shows a random structure at baseline, which does not change in subsequent recordings either (Fig. 3B). This suggests that the structure of the peptide does not change even over a period of one week. The spectrum of a 1: 5 mixture of Αβ 1-42 and apape-NH _s is a simple combination of the spectra of the two pure components. The aggregation of the isoamyloid β1-42 in the presence of apape-NH _s is less gradual than that of the pure isoamyloid β 1-42. Significant structural change was observed only after 6 hours. However, after the initial unchanging, the same structural transformation occurs as in the experiment without apape ~ NH ₂ . The curve obtained by subtracting the spectrum of apape-NH2 from the spectrum of the mixture of Αβ 1-42 and apape-NH ₂ differs significantly from the spectrum of pure Αβ 1-42. The most striking difference is the blueward shift of the 215 nm minimum, suggesting a slight structural change (Fig. 3 CD). Such a structural change usually occurs during the intermolecular interaction of the two components. The lake

The spectrum shown in Figure 30 suggests that the presence of apape-NH to to may slightly affect the kinetics of Αβ 1-42 aggregation.

This difference in acjgregado behavior, on the other hand, is less pronounced in 3C. and 3D. in the difference5 spectrum of the figures.

Computer binding simulation studies (MM-GB5A-enhanced docking) (Figure 4) revealed the existence of an extended skeletal structure similar to the polyproline II helix, which was detectable in all cases except for the ‘papé’ peptide. The ‘papé’ is too short to form such a structure (Fig. 4), but even the ‘papé’ peptide is characterized by an elongated conformational structure.

Example 2 Investigation of the neuroprotective effect of peptides in 15 in vitro experiments

The source of supply of NaCl, KCl, CaCl ₂ , MgCl ₂ , HEPES, NaHCO 3, D-Glucose, MTT and 96-well culture plates (Costar) was Sigma Aldrich Magyarország Kft. ExViS mini-chamber system development within the institute. The 20 protocols used to treat the animals were licensed by the Institute of Public Health and the University of Szeged, license number I02442 / 001/2006.

Preparation and treatment of acute hippocampal brain slices.

A slightly modified version of the methodology previously reported by Datkí et al. (Datki et al. Brain Res Bull. 74 / 1-3,183-7, 2007) was used in slice survival tests. After anesthesia with chloral bridge wire (5.4 g / kg), 10 ± 1 weeks old male. Wistar rats were decapitated and, after skin removal from the top, were placed in ice-distilled water for 1 min. The brains were quickly removed and placed in a so-called preparative solution at 4 ° C to 1H-ACSF / 1, naoyon low Ca ^! and increased Mo ^{x +} content; to te te te te (Ref. 4), Exact composition of the preparation solution (in mM): NaCl 122.0, KCl 3.0, CaCl ₂ 0.3, MgCl ₂ 3.7, NaHCO 3 25.0, HEPES 5 .0, D-glucose 10.0, ρΗ ^ίκ 7.4. 400 μm thick squamous cell slices were prepared using a McIlwain tissue slicer in ice-cold H-ACSF / 1 solution at 4 ° C and photographed to determine the area of each slice. Slices of approximately 9 mm ² were quickly placed in the ExVis mini-chamber system (maximum 10 slices in 1 ml of liquid) and conditioned for 30 minutes in a carbozgene (CPiCCb-95: 5) preparation solution at room temperature (24 ° O. The carboxygenated H-ACSF / After 30 minutes at room temperature in 1 solution, the slices were placed from the mini-chambers in a 2 ml aliquot of glucose- and carboxygen-free H-ACSF / 2 dispensed into plastic Petri dishes using a cut-off pipette (200 μl) for one hour. Composition of the HACSF / 2 solution (in mM): NaCl 132.0, KCl 3.0, CaCl ₂ 2.0, MgCl 2.0, FaHCCb 25.0, HEPES 5.0, pH 7.4 The Petri dishes were shaken continuously in a modified BIöSAN TS-100 thermostated shaker at 370 rpm After resting in glucose- and oxygen-free H-ACSF / 2, the solution was removed from the Petri dishes and replaced with H-ACSF / 1 solution (2 mL). / cup) The slices were quickly returned to the EzVis mini-chambers (max san 10 slices / 1 ml) for treatment with Αβ peptide. In each chamber, adding 50 μl of Αβ stock solution to 950 μl of slice solution, the final concentration of Αβ was adjusted to 20 μM. The Αβ stock solution (0.4 mM) is all viable! before preparation was freshly prepared in distilled water (pH “5) and used for up to 10 minutes. Peptide mimetics were used at final concentrations of 40 μΜ and 100 μΜ, respectively, or alone with # or Αβ 1-42 peptide, in which case distilled aqueous stock solutions were premixed. With 42β 1-42 and / or the peptide mimetic

Defend

After 4 hours of treatment with Fi, 0.1 ml of MTT stock solution was added to the chambers

IÁ • SSSx

Fi was added (5 mg / ml in H-ACSF / l) and the brain slices were allowed to rest in this solution for 15 minutes without carboxygenation. To stop the reduction of MTT to formazan, the slices were transferred from H-ACSF / I to DMSO (50 DMSO / slice) and incubated for 30 minutes. The optical density of the dissolved form was also determined at 550 and 620 nm. The data were evaluated using the following formula: (00553 OD ^ o) / slice area (mm'j, the value obtained is 100% of the measured value of the slice treated with the control, Αβ 1-42 free solution, the other data are compared to the value.

summary

Table 3 summarizes the MTT results of the various new peptides using 2 concentrations. It can be seen that of the peptides tested, the pape-NIA peptide has the weakest protective effect against the toxic Αβ 1 ~ 42 peptide, while the other peptides tested were able to almost completely prevent Αβ toxicity. Based on these results, the peptide apape (SEQ 10 NO 51) was selected as the lead compound for further biolysis. i k í s é r 1 e t e kh e z.

Example 3. In vivo studies using a rat electrophysiological model

Preparation of Αβ 1-92 sample

The synthetic Αβ 1-42 peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HEIP) and allowed to stand at room temperature overnight. After the HFIP solvent was removed in vacuo, the peptide was dissolved in distilled water and allowed to stand for 3 days. The fibrils were separated by centrifugation (10,000 x g for 15 minutes), and the peptide collected at the bottom of the centrifuge tube was redissolved in distilled water and freeze-dried. Dilute to pH 6.4

At a final concentration of 5 x 10 M, the solution was homogenized by sonication for 15 minutes.

Extra col Ida ri s recordings and iontophoresis

Extracellular single-cell activity was recorded in the hippocampal CA1 region of chloral hydrate-anesthetized (intraperitoneal dose 0.4 g / kg) rats (Wistar male rats weighing 250330 g). The animals were treated and operated in accordance with the following standards:

Communities Council Directives (86/609 / ECC · and the Hungarian Animal Welfare Act (XXVIII.tv. Para. 32) and the ethical permit I74-13 / 2010 We have made every effort to minimize the pain of the animals and the number of animals used.

The animals' heads were captured in a stereotaxic device, and a small hole was drilled in the skull above the hippocampus (ap: 3.8 mm from Bregma; lat: ± 2 mm from the midline) and the dura mater was removed. After waiting for an hour, we began to register neuronal activity. Single-cell activity was recorded extraed from the CA1 region at a depth of 2 44 mm with a low impedance (<1 MQ) 7 gm carbon fiber electrode (Kation Scientific, Minneapolis, MN, USA). The drugs were delivered from the capillaries to the cell surface. Action potentials were amplified with an Ex? \ Mp-20KB extracellular amplifier (Kation Scientific.) And visualized with an oscilloscope. Jd filtration was performed between 300 and 8000 Hz. A window comparator (WD-2, Dagan Corporation, MN, USA) was used to calculate firing. The amplified signals were digitized at 50 kHz and recorded. The action potential number per second was displayed by the computer on a peristired histogram. Iontophoretic drug delivery was controlled by a PCI-1200 dosing card (National Instruments, Austin, TX, USA), controlled by a control program written in the LAbVIEW software package. Minion-16 and BAB-350 pumps (Kation Scientific) were used for iontophoretic delivery. The capillaries of the .multibarrel electrode contained the following solutions: (a) 100 mM NMDA (Sigma-Aldrich, Budapest) dissolved in 1.00 mM NaCl (pH 3.0); 5 (n) 5 ^x 10-5 Μ Αβί-42 and (c) 2.5 χ 10-4 M peptide dissolved in physiological saline (pH 6.4); (d) a mixture of ΑβΙ-42 (5 * 10-5 M) and peptide (2 <5 χ 10-4 M) allowed to stand for one hour, and (e) the 4% Pontamine Sky Blue used to indicate electrode position. (BDH Chemicals, Poole, UK) dissolved in 100 10 mM sodium acetate.

Nerve cells were stimulated with iontophoretically administered NMDA at intervals of 5 seconds every 1 minute. A negative current of 5-100 nA was used to administer NMDA, which stimulated the neuron at 30-80 discharges / sec. A restraining current of 2-21 nA was used for the opposite polarity. A histogram of the neuronal peristimulus was recorded. After obtaining a stable control level, the peptides to be tested were administered by iontophoresis for 3 min using + 100 nA, and then the papaws of Αβ 1-42 were added for 1 min with a current of -0.5 pA. In the second half of the experiments, a mixture of pentapeptides and Αβ 1-42 was administered for 1 min using -0.5 pA. The position of the registered neuron in the brain was indicated by the ej action of PSB dye, which was applied using a current of 10 pA for 10 minutes. At the end of each experiment, the 25 animals were anesthetized with a high dose of chloral hydrate, and the rapidly removed brain was fixed in 4% paraformaldehyde solution. The excised 50 μm thick brain slices were stained with neutral red dye and the position of the recording was checked under a stereomicroscope.

For statistical evaluation, the number of action potentials elicited during each iontophoretic NMDA dose was considered. In some registries, where in principle only cells from one cell were recorded, the numbers of action-potentials elicited during different NMDA stimulation to episodes were compared by eavto to to passenger ANOVA using a post-hoc Dunnet test. For each experiment, the mean pre-dose of Αβ 1-42 was taken to be 10.0%. Data were summarized for statistical analysis. Mean ± SEM is shown in the figures. The statistical significance value was set to p <0.05.

Intraperitoneal administration of peptides and in vivo single cell electrophysiological drainage

Estracellular single-cell drainage was performed from 72 neurons in the CA1 region from a total of 32 anesthetized rats. The pentapeptides were administered i.p. was administered (0.5 mg / 100 g) and a paristimulus histogram was recorded. All 40-min time window results were summed and the percentage mean ± SEM values were calculated. Control firing values before Αβ 1-42 administration were set to -30-80 action potentials / sec. thus, the stimulation elicited by your horses did not depend on the value of the initial firing. Firing values before amyloid administration were taken as 10ft for each recording, and values after Αβ 1-42 administration were calculated as a percentage for statistical evaluations.

summary

These results demonstrate that the apape peptide prevented the NMDA receptor-stimulating effect of Αβ 1-42 in a 50-250 min time window after i.p. after dosing (Figure 7). This suggests that apape crosses the blood-brain barrier.

Example 4. Behavior and learning studies in a rat model

Morris ~ vísiahlrintus test \ Χ · ^: lake m lake lake

The Morris Water Maze is a round pool with a diameter of 1.30 cm and a height of 60 cm. The pool was filled with water (23 ± 1 ° C) to a height of 40 cm and then opaque with milk. The pool was divided into 4 virtual neighborhoods. In the middle of one of the 5 quarters was placed a round platform made of plexiglass with a diameter of 10 cm. The position of the platform remained unchanged during the experiments. The platform was sunk 1.5 cm below the surface of the water, making it invisible from the water level. The pool was separated from the other 10 areas by a black curtain. We placed different colors and shapes on the curtain, helping the animals to orient themselves in space. The experiments are a soundproof, poorly. performed in an illuminated test room.

Behavioral studies were performed for five consecutive days. Two starting points were used in the experiments. One was close to the platform and the other a little further away. Mice were swam twice daily. The animals face the wall. we were placed in the pool. The time interval between the two swims was 30 seconds. The animals. 30 They could stay in the pool for 20 seconds, that’s how much time they had to find the platform, they could be on the platform for another 15 seconds if they found it. Animals that do not. they found the platform, dragged it or put it on the island, which they could also be on for 15 seconds.

After swimming, the animals were placed in their own cages.

During each swim, the time to discovery of the hidden platform was recorded (latency) using a special computer system connected to a video camera (EthoVision 2.3 software, Beggar, Wageningen, The Netherlands). The 30 statistical evaluations were performed by repeated analysis of variance from the mean of the swims, and Fisher's post hoc test was used to compare the groups.

to to to to

Conclusion

Behavioral and learning experiments demonstrated the tendon-carrying neuroprotective effect of the new peptides (Fig. 8–10). The results of the reference memory studies obtained in the Morris water maze confirmed that the new peptides and peptide mimetics significantly reduce the latency of spatial learning and prevent the toxic effects of Αβ1-42 aggregates.

Example 5. Histological examination of rat brain tissue sections

Krezi1-violet painting

After the behavioral experiments, the animals were anesthetized with 5% chloral hydrate (400 mg / kg) and perfused transcardially with physiological saline, followed by paraformaldehyde (0.2 M, pH 7.4) dissolved in 4% phosphate buffer (PBS). The brain was carefully dissected from the skull and postfixed in 4% paraformaldehyde for 1 day. The brains were then placed in phosphate buffer containing 30% sugar and 0.01% sodium azide for 4 days at 4 ° C. Next, sections 50 μm thick were cut from the brain and placed on gelatin slides for cresyl violet staining. Sections were first placed in 0.2 M PBS and stained with 0.5% cresyl violet for 1.5-2 minutes. After staining, sections were resuspended in PBS for 20-30 seconds. This was followed by dewatering in a rising alcohol series (70%, 80%, 95%, 100% alcohol) and sections were placed in 99% butanol solution for 20-30 seconds. Finally, sections on slides were placed in a Hellendahl staining bath containing xylene and covered with DPX. Sections were digitized with the Zeiss MIRAX MIDI section scanner (Carl Zeiss Inc, Germany; 3DHistech Kft, Hungary). Cells were quantified on the digitized sections from the right and left hippocampuses using HistoQuant to to to to to to software (3DRístech Kft, Hungary).

Ta u - 1 mm u n h x s z t ok ém i a

The animals were anesthetized and perfused as described above. The hippocampus was isolated from the brains of the animals and sections 50 g thick were excised with phosphate. Sections were collected in wells of a 96-well culture plate containing PS5 in preparation for immunohistochemical staining. Monoclonal mouse suti-human PHF-tau antibody. (PHF, paired he ilkai, also filaments, clone AT1G0 antibody, 1: 800 dilution) was used as primary antibody (Thermo Scientific, Rockform, IL, U.S.A.), biotinylated anti-mouse IgG (1: 400 dilution) as secondary antibody antibody was used (Vector Laboratiories

Inc.). Immunopositive cells were visualized with Vestastain ABC kit (Vector Laboratories Inc.) and 3,3'-diaminobenzidine tetrahydrochloride (DAB Sigma Aldrich, Co) containing 1% nickel ammonium sulfate based on peroxidase reaction. At the end of the immunohistochemical studies, the free-floating sections were plated, covered, and digitized with a Zeiss MIRAX MIDI section scanner. Quantification of tau immunopositive cells from the right and left hippocampus was performed on the digitized sections using the HistoQuant software (3DHisteoh Kft, Hungary).

Summary

With these studies, we have shown that newly developed peptides and peptide mimetics are likely to influence dendritic formation and improve communication between neurons (Fig. 11-13).

Table 2. The peptides described herein have M. a n aIi tical parameters

I Reteneios ί 1 i i reported 1 numbering [pepik! measuredM * may to , “I time / mm; gradient s i | DP ~ NhJ 229.6 229.23 0-20% / 20 min 12.45 | al EP-NH> 243.66 243.26 0-20% / 20 min in 5.85 '' «''« «'' •••• ~ ^ ™ * ^ AM ** WAWAMAWÍ, AWAWAWA \ WA \ WAV« I 3l Suc-Pro-NH · »| 718 14 J * I 4, Suc-PA-NÍEi 286.14 285.30 0-20% / 20 min 1 12.45 . {í ΐ 5 | Sue-pa-NH ?! 286.14 w CGO 0-20% / 20 min. | 12.45. $ * - '' ^, «V'V.> W ^ W.» «>> **» * »» »» «..« fr ** »« ₁ «« W ^ * ^ ** ^^ * ^ * ^ wS * AAAAAAAAA ^^^ w4 »AAAAAAAAAAAAAAA ^^ 1 6 Glp-Glv 186.4 186.17 0-20% / 20 min 5.85 i : .......................... <...................... ..t ---------- —............... 1? Ae-GI p-G ): 1 8 glp-G 186.15 t; y .................. i .......................... i ... ................... ! ö] Glp-Asp-NIE 486.69 243.22 0-2086 / 20 min 1 4.96 1 l ......................... 10 .......... 8 ^ ~ W ~ NB ₂ I 229.2 । : | 11 EPA-NH ₃ 314.81 314.34 0-25% / 25 min | 6.70 1 12 Ac-EPA-NhJ 356.77 356.37 ..... —1 .... —......! 0-25% / 25 min | 11.21 í I 1 I 13 PPP-Nlh 340.8 340.38 0-20% / 20 min 9.42 | 14 | ape-NHj 314.51 314.34 0-20% / 20 min 6.58 ........ ....., ......................... ¹ 1 ................. ..1 ............................. 1 15 Ac-ape-NH; 356.68 356.37 0-20% / 20 min 5.68 i 16 GABA-pe-NH _? 328.8 w ÍX> se 0-40% / 20 min 12.34 1 ________17 DPA-NIE [_________ 300.31 0-20% / 20 min 8.38 i ! 18 EPAP-NHJ 411.4 411.45 0-25% / 25 min | 10.90 i | 10 Ac-EPAP-Nlfe 453.98 45.3.19 0-2036 / 20 min | 14.40 | 1 20 papéul hl 411.90 411.45 5-15% / IQ min 5.97 i «.A-A-AAA-AAAAAAAAAAAAAAAAAAAAA ^ AAAAAAAAAAAAA-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | 21 pppe-NIE 437.94 437.49 0-25% / 25 min 11.47 | 22 Sue-PAFA-NH ₂ 453.95 453.49 0-25% / 25 min 1 15.88 and pmpionyl-1 i 23 PAPA-NIE 410.2 409.50 5-25% / 20 min | 12.78 | | Iminodiacetyl-1 | 24 PAPA-NH ₂ 468.69 468.52 0-20% / 20 min 11.37 i I Prapionikpape-1 | 251 NIlJ 467.76 467.53 5-25% / 20 min 13.31 j i Propiomkpapq- 1,261 NI 466.84 466.55 0-20% / 20 min। 16.66

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27 apap-NH? 353.82 353.42 0-20% / 20 min 12.49 28 EPAPA 483.97 483.52 0-25% / 25 min 13.93 29 EPPPA 510.2 509.55 0-25% / 25 min 14.31 30 APAPE-NHa 48.20 482.53 0-25% / 25 min 12.63 31 epapa-NHa 483.2 482.53 0-25% / 25 min 12.28 32 ............... WPteNHa 509.2 508.57 0-25% / 25 min 12.35 33 apape-NHg 483.1 482.53 5-25% / 20 min 7.24 34 apppe-NIG 509.20 508.57 0-25% / 25 min 12.34 35 RPAPA-NIE 510.13 509.60 0-25% / 25 min 12.08 36 KPAPA-NHa 482.07 481.59 0-25% / 25 min 10.29 37 rpppa-nh ₂ 534.49 38 KPPPA-NHa 508.15 507.63 0-25% / 25 min 10.88 39 DPAPA-N1E 468.93 468.5 0-20% / 20 min 11.89 40 β-Ah-PAPAnh ₂ 425.05 424.49 0-20% / 20 min 11.88 41 E-Sar-APA-NH ₂ 456.94 456.49 0-20% / 20 min 11.36 42 EPA-Sar-A-NH> 456.95 456.49 0-20% / 20 min 10.83 43 E-Pip-APA-NIÍa 497.0 496.56 0-20% / 20 min 16.81 44 EPA-Pip-A-NH ₂ 496.82 496.56 10-30% / 20 min 11.02 45 E-Sar-A-Sar-A ’ NIE 431.35 430.45 0-20% / 20 min 9.68 46 apapq 482.53 0-20% / 20 min 11.74 47 apapd-NHa 469.00 468.50 0-20% / 20 min 15.69 46 apapn 468.85 468.50 0-20% / 20 min 11.05 49 apapq-NHa 482.41 481.55 0-20% / 20 min 10.58 50 apapn-NHa 467.97 467.52 0-20% / 20 min 10.39 51 apape 484.01 483.51 0-20% / 20 min 13.38 52 $ pGpe-N ^ 469.49 0-2034 / 20 min 1337 53 ap-N-Me-akvpeNHa 497.06 497.54 0-20% / 20 min 1339 54 α-γc-ape-NHa 545.18 545.57 15-3 5% / 20 min 8.86 9.38 55 apatetee-NH? 545.25 545.57 15-35% / 20 min 11.77 11.26 56 ap-iic-pe-NHa 571.26 571.61 10-30% / 20 min 10.28 1.1.29 57 a-Sar-ape-NÍ E 456.95 456.49 0-20% / 20 min 8.49

58 apa-Sar-e-NH ₂ 456.92 456.49 0-20% / 20 min 11.81 59 α-pip-ape-NH ₂ 497.21 496.56 5-25% / 20 min 12.20 60 apa-pip-e-NHj 497.29 496.56 10-30% / 20 min 6.59 61 Gpape NH ₂ 468.94 469.49 0-20% / 20 min 13.30 62 (N-Me-ala) pape-NH? 496.99 497.54 0-20% / 20 min 10.34 63 ap-Aib-pe-NH? 497.24 497.54 10-30% / 20 min 12.39 64 Aib-pape-NHa 497.24 497.54 0-20% / 20 min 6.75 65 APEPA-NH2 483.01 482.53 0-20% / 20 min 7.50

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Neuroprotective effect of the peptides described in rat hippocampal slices as measured by NTT

Numbering Peptides Troubleshooting 5x molar excess A-beta toxicity Dilute 2x molar excess A-beta toxicity % ± SEM % * SEM % ± SE.M % ± SEM 1 DP-NH? 65.7 * 0.453 53.9 * 0.747 EP-NH ₂ • n Suc-Pro-NIE 4 Sue-Pro-Ala-NH ₂ 62 * 0.647 50.3 * 0.597 5 Sac-pa-NH ₂ 93.2 * 1.75 52.2 * 0.97 82.6 * 1,883 6 Glp-Gly 84.5 * 0.312 50.3 * 0.597 f Ac-Glp-Gly 8 glp-Gly 9 Glp-Asp-NH ₂ 79.3 * 0.572 / 56.2 * 0.366 48.8 * 2.187 / 53.9 * 0.747 10 glp-asp-NH ₂ 11 EPA-NH2 40 * 0.57 / 91 * 0.91 / 70 * 1.06 / 91 * 2.71 42 * 1.75 / 45. * 0.78 / 40 * 4 / 47 * 0.87 47.2 * 0.541 / 51.8 * 0.786 / 50.3 * 0.597 12 Ac-EPA-NH ₂ 97 * 0.84 / 94 * 1.33 45 * 0.42 / 45 * 0.78 79.4 * 1.0160 / 85.2 * 0.600 / 50.3 * 0.597 13 EPP-NIE _________ 94 * 0.85 / 95 * 0.83 42x0.70 / 45 * 0.78 88.9 * 0.341 46 * 0.43

Numbering Peptides 5x molar excess A-beta toxicity V lability 2x molar excess A-beta toxicity 14 ape-NIE 95 * 0.1 46 * 0.94 93.00 * 0.92 / 90.40 * 1.005 47 * 0.54 / 15 Ac-ape-NHa 100 * 0.33 47 * 0.87 82.00 * 1.6 / 78.20 * 1.967 47 * 0.54 / 16 GABA-pe-NHa 89.0 * 1.009 53.9 * 0.747 17 DPA-NIB 89.4 * 0.361 / 68.6 * 0.961 48.8 * 2.187 / 53.9 * 0.747 18 epap-nh ₂ 97 * 0.4 44 * 1.27 68.5 * 0.678 48.5 * 1.397 19 A <-EPAPNH _:? 90 * 0.01 45 * 0.00 62.5 * 0.985 46.3 * 0.423 20 ........ 90 * 1.09 42 * 1.25 48.6 * 0.808 46 * 0.43 21 PPpe-NIE 91 * 0.57 42 * 0.70 89.9 * 1.318 46 * 0.43 7? Sw-PAPA-NHa 94 * 1.21 44 * 1.27 63.3 * 1.402 46.3 * 0.423 23 Propionsd -PAPAnh ₂ 82.4 * 2.47 50 * 0.40 57.1 * 0.379 51.4 * 0.44 24 Iminodiacetylfapa-nh / 97.0 * 0.18 50 * 0.40 86.3 * 0.856 50.9 * 0.527 25 PropionyEpape- Nll ₂ 92 * 0.38 46 * 0.94 70.5 * 0.233 53 * 0.529 26 Propionyl -papqNlh 66 * 0.96 46 * 0.94 49.0 * 0.236 52.4 * 0.344 77 apap-NIb 59 * 0.46 42 * 0.46 28 EPAPA 89 * 0.79 42 * 0.4 87.9 * 1.78 47.6 * 0.884 29 ΕΡΡΡΑ 90 * 1.75 42 * 0.4 89.2 * 1.78 47.6 * 0.884 30 APAPE-NHa 95 * 0.42 42 * 0.4 62.8 * 1.386 44.2 * 0.352 31 epapa-NH? 100 * 1.28 45 * 0.42 65.4 * 2.426 47.6 * 0.884 32 cpppa-NlE 96 * 1.23 45 * 0.42 89.8 * 0.984 44.2 * 0.352 33 apape-NH ₂ 94 * 2.07 / 85.1 * 4.12 40 * 2.15 / 53.4 * 1.49 47 * 1.28 / 64.4 * 1.48 / 78.4 * 0.567 47 * 0.54 / 47.6 * 0.884 / 48.8 * 2.187 34 apppe-NHa 89 * 0.60 42 * 0.70 61.5 * 0.566 46 * 0.43 35 RPAPA-NH ₂ 80 * 0.39 43 * 0.65 51.0 * 1.578 50.9 * 0.527 36 kpapa-nie 91 * 0.30 43 * 0.65 65.3 * 0.780 52.4 * 0.344 37 RPPPA-Níh 90 * 0.45 4 3 * 0.65 64.4 * 1.477 52.4 * 0.344 38 kpppa-nh ₂ 79 * 1.05 43 * 0.65 86.0 * 0.990 52.4 * 0.344 39 dpapa-nh ₂ 98 * 0.31 47 * 0.81 64.5 * 5.129 51.4 * 044 40 b-Ala-PAPA-ME 69 * 1.57 47 * 0.81 53.5 * 0.427 51.8 * 0.317

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Numbering Pepid Support 5x molar excess A-beta toxicity Viabilitex 2x molar excess Á-beta taxi tree 41 E-Sar-APA-NHa 95.8 * 1.95 50x0.40 83 * 1998 48.5 * 1.397 42 EPA-Sar-A-NHa 93 * 4.09 45 ^ 0.42 66.8 * 0.614 51.4 * 0.44 43 E-Pip-APA-NHa 97 * 0.49 47M181 87.3 * 1.559 51.4 * 0.44 44 OPA-Pip-A-NFh 63.7 * 0.961 53 * 0.529 45 E-Sar-A -Sar-Anh ₃ 91 * 4.15 54.10.50 91.6 * 1,299 48.5 * 1.397 46 apapq 87.6 * 1.42 54x0.50 89.9 * 1.209 46.3 * 0.423 47 apapd-NHa 92 * 1.62 4014 62.3 * 0.511 44.2 * 0.352 48 apapn 90.3 * 2.00 54.i0.50 91.5 * 0.406 44.2 * 0.352 49 apapq-NFh 33 * 0.52 / 41 ±, 2. 63 / 50.1 * 1,23767.2 00.65 / 48. H0.93Z 55.411.98 42 * 1.75 / 4014 / 50.10.40 / 53.4x1.49 / 52.3 * 0.73 44 * 6.39 / 51.3 * 1.974 47 * 0.54 / 53 * 0.529 50 apapn-NHa 42 ^ 0.72 40x4 48 * 1.529 48.5 * 1.397 51 the priest 91.3 * 2.53 53.4 * 1.49 90.8 * 0.277 / 89.2 * 1.133 53 * 0.529 / 48.8 * 2.187 52 apGpe-NH ₂ 91 * 1.75 / 92 * 0.23 42 * 1.75 / 44 * 1.27 82.1 * 0.366 46.3 * 0.423 53 ap-N ~ Me-ala-penh ₃ 89.3 * 2.36 54 * 0.50 75.6 * 4.136 51.8 * 0.317 54 α-tic-ape-NHi 87 * 0.01 45 * 0.00 54.0 * 0.596 51.8 * 0.317 55 apa-íic-e-NHa 86 * 0.01 45 * 0.00 58.6 * 1.831 51.8 * 0.317 56 ap-tíe-pe-NHa 77.4x0.46 52.2 * 0.97 63.2 * 0.924 52.6 * 1.672 57 a-Sar-ape-Nlh 99.6 * 0.86 52.3 * 0.73 62.9 * 0.723 52.6 * 1.672 58 apa-Sar-e-NHa 84.0 * 3.45 52.2 * 0.97 66.3 * 1.664 52.6 * 1.672 59 .a®ape-NH ₂ . 90.3 * 3.64 51.6 * 1.23 68.0 * 1.745 52.6 * 1.672 60 apa-pip »e-NHa 93.4 * 0.94 51.6 * 1.23 73 * 0.661 50.5 * 0.296 61 Gpape NHa 86/2 * 1.74 53.4 * 1.49 61.7 * 0.663 50.5x0.296 62 (N-Me-alaPpapr- NH- 93.3 * 0.37 52.3 * 0.73 84.7 * 0.895 50.5 * 0.296 63 ap-Aib-pe-NHa 97.ai.30 51.6 * 1.23 71.0 * 1.164 50.5 * 0.296 64 Alb-pape-NHa 85.7 * 0.38 52.2 * 0.97 76.0 * 1.239 50.9 * 0.527 65 APEPA-NHa 9 biO. 3 9 46 * 0.94 63.5x0.657 50.9 * 0.527

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iASsftSmj tws% «« i tototoi '' C'K.-s ss .;

νΧΧΧ “ΛΧΧΧΜ ............................................... .................................................. ............

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SEQUENCE LIST <110> University of Szeged <120> Small peptide inhibitors inhibiting β-amyloid toxicity <130> 12-23 <140>

<141> 2014-05-08 <15h> EN P1300317 <151> 17.05.2013 <160 65 <210> 1 <211> 2 <212> IRT <213> Artificial sequence <220 <22 3> Color peptide d <400 1

000 <210> 2 <211> 2 <212> PRT <213> Artificial sequence <220 <223> Synthetic peptide <400> 2

000 <210> 3 <211> 1 <212> PRT <213> Artificial sequence <400p 3 000 <210p 4 <in> 2 <212> PRT <213> Artificial sequence <220>

<223> Synthetic Peptide <400p%

000 <210> 5 <211> 2 <212> POT <213> Artificial Sequence <220>

<223> Synthetic peptide <400> 5

000 <210> 6 <2U> 2 <212P POT <213> Artificial Sequence <220>

<223> Synthetic peptic <400> S 000 <210> 7 <2IIP 2 <212P RET <213p Artificial sequence <22 0>

<223> Synthetic peptic

AA ftp A 4ft ¹ A <213> Artificial sequence <22 3> Synthetic peptide <400> 16

000 <210> 17 <211> 3 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide <40O> 1?

000 <210> 18 <21I> 4 <212> PRT <213> Artificial Sequence <22ü>

<223> Synthetic peptide <400> 18

Glu-Pro-Ala-Pro

4 «210> 19 <211> 4 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide <400> 19

Giu-Pro-Ala-Pro

1 4 <210> 20 <211> 4 <212> PRT <213> Artificial Sequence <220>

<223> Frol, Ala2, Pro3, Glu4 are D-amino acids <406> 20

F rc-Aia-Pro-Glu

4 <210> 21 <211> 4 <212> FRT <213> Artificial Sequence <220>

<223> Frol, Pro2, Pro3, Glu4 are D ~ amino acids <400> 21

P ro ~ P r o ~ F r c ~ G1 u <210> 22 <211> 4 <212> FRT <213> Artificial sequence <220>

<2 2 3> S z 1 n t e t i ku s p e p t i d <400> 22

P r o ~ Al a ~ F r o - A1 a <210> 23 <2H> 4 <212> FRT <213> Artificial sequence <220>

<223> Synthetic peptide <400> 23

Fro-Ala-Prc-Ala

4 <210> 24 <21I> 4 <212> FRT <213> Artificial Sequence <220>

<223> Synthetic <400 24

P r c · - A1 a ~ P r o ~ A1 a

4 peptides <210 25 <21I> 4 <212> PKT <213> Artificial, sequence <220>

<223> Prol, Ala2, Pre3, Glu4 are D-amino acids <400> 25

P r o - A .1. a - P r c - G1 u <210> 26 <211> 4 <212> PRT <2.13> Artificial Sequence <220>

<223> Frol, Ala2, Pro3, Gln4 are D-amino acids <400> 26

Pro- A1 a - P ro - G1 n <210> 27 <210 4 <212> PKT <213> Artificial sequence <220 <223> Alai, Pro2, Ala3 _z Pro4 are D-amino acids <4 00 27

A1 a ~ P r o ~ A1 a - P r o

I 4 <210> 28 <211> 5 <212> PO <213> Artificial sequence <223> Synthetic peptide <400> 28

G1 u ~ P r ο ~ A1 a - P r o ~ A1 a

1 5 <210> 29 <2U> 5

W <212> PRT <213> Artificial sequence <220>

<223> Synthetic Peptide 15 <400> 29

G1 u ~ P r o - P r ο ~ P r o - A1 a j 5 <210> 30 <211> 5 <212> PRT <213> Artificial sequence 25 <22 0>

<223> Synthetic Peptide <400> 30

A1 a - P r o - A1 a - P r o - G1 u

5 <210> 31 35 <211> 5 <212> PRT <213> Artificial sequence <220> 40 <223> Glul, Pro2, Ala3, Pro4, Ala5 are D-amino acic <400> j1

Giu-Pro-Ala-Pro-Ala 1 6 <210> 32 <211> 5 <212> PRT 50 <213> Artificial Sequence <220>

<223> Glul, Pro2, Pro3, Pro4

Ala5 are D-amino acids <400> 32

Glu-Pro-Prc-Pro-Ala <210> 33 <211> 5 <212> FRT <213> Artificial sequence <223> Alai, Pro2, Ala3, Pro4, Glu5 are D-amino acids <400> 33

Ala-Pro-Ala-Pro-Glu

5 <210> 34 <211> 5 <212> PRT <213> Artificial Sequence <2:23> Alai, Pro2, Pro3, Pro4,

Glu5 are D-aminc acids <400> 34

Ala-Pro-Pro-Pro-Glu

5 <210> 35 <211> 5 <212> PRT <213> Artificial sequence <2 30>

<223> Synthetic peptide <400> 35

Arg ~ P r o ~ A1 a — Pro — A .1 a <210> 36 <211> 5 <212> PRT <2.13> Artificial Sequence from <220>

<223> Synthetic peptide <400 36

Ly s-Pro-AIa-Pro-A1a

1Ö <210> 37 <2U> 5 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide <400> 37

Arg ~ P ro-Pro-P ro-Ala

1 5 <210> 38 <2H> 5 <212> PRT <213> Artificial Sequence <220 <223> Synthetic Peptide 30 <40Ö> 38

Ly s - P r ο - P r o - P r o - A 1 a <210> 39 <211> 5 <212> PRT <213> Artificial sequence 40 <220 <22 3> Synthetic peptide <400 39

Asp-Pro-Ala ~ Pro ~ Ala <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220>

<22 Xaa i is óAia <400> 40

X a a ~ F r o - Al a - P r © -A 1 a

5 <210> 41 <211> 5 'ζ' '0; '/'>

<213> Artificial Sequence 15 <220>

<223> Xaa2 is MeGly <400 41

Glu-Xaa-Ala-Pro-Ala

5 <210 42 <210 5 <212> PRT <210 Artificial Sequence <220>

<223> Xaa4 is MeGly 30 <400 42

G1u ~ Pro-Ala-Xaa-Ala

5 <210> 43 <210 5 <212> PRT <213> Artificial Sequence 40 <z20>

<223> Xaa2 is Pip <400> 43

Glu-Xaa-Ala ~ Pro-Ala

5 <210 44 <210 5 <212> PRT <213> Artificial sequence <220>

<223> Xaa4 is Pip <400> 44

GI u - P r o ~ A1 a -Xa a - A1 a i 5 <210> 45 <211> 5 <212> PRT <213> Artificial sequence <220>

<223> Xaa2 and Xaa4 are MeGly <4O0> 45

G1u-Xaa-Aia-Xaa-Ala

5 <210> 4 6 <211> 5 <212> PRT <213> Artificial Sequence <220>

<223> Alai, Prp2, Ala3, Pro4, Gln5 are D-amino acids <400> 46

Ala-Pre ~ A1a ~ Prp-Gln <210> 47 <211> 5 <212> PRT <213> Structure <220>

<223> Alai, Pro2, Ala3, Pro4, <4O0> 47

A1 a ~ P r o - A1 a - Pro - A s p

5

Asp5 are D ~ amino acids <210> 48 <211> 5 <212> PRT re re

The strong sequence is <220>

<223> Alai, Pro2 _f Ala3, Pro4, Asn5 are D-amino <400> 48

A1 a - P r o ~ A1a ~ Pro - Asn

5 acids <210> 49 <2H> 5 <212> PET <213> Artificial sequence <220>

<223> Alai, Pro2, Ala3, Pro4, Gln5 are D-amino <400 49

A la ~ P r q ~ A1 a ~ P r c - G1 n

5 <210> 50 <2T1> 5 <212> PRT <213> Artificial Sequence <220>

<223> Alai, Pro2, Ala3, Pro4, Asnö are D-amino <4 00 50

Ala-Pro-Ala-Pro-Asn

5 acids acids <210 51 <211> 5 <212> PRT <213> Artificial sequence <220>

<223> Alai, Pro2, Ala3, Pro4, Glut are D-amino <400> 51

Al a - Pro - A1 a - P r o - G1 u

1 5 <210> 52 <211> 5 <212> PRT <213> Artificial Sequence • ^ · lake lake lake lake lake acids <220>

<223> Alai, Pro2, Pro4, Glu5 are D-amino acids <400> 52

Ala-Pro-Gly- P r o ~ G1 u.

5 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220>

<223> Alai, Prc2, Pro4, Glu5 are D-amino acids

Xaa3 is N ~ Methylaine, also D-amino acid <400> 53

Ala-Pro-Xaa-Pro-Glu

1 5

<212> PRT <2.13> Artificial Sequence <220>

<223> Alai, Ala3, Pro4, Glu5 are D-amino acids 30 Xaa2 is Tic, also D-amino acid <400> 54

A .1 a ~ Xa a ~ A1 a ~ P r o — G1 u

5

<213> Artificial sequence <220>

Alai, Pro2, Ala3, Glu5 are D-amino acids

Xaa4 is Tic, also D-amino acid 45 <400> 55

Ala-Pro-Ala ~ Xaa ~ Glu

5 <212> ORT <213> Artificial sequence <223> Alai, Pro2, Pro4, Glu5 are D-amino acids

Xaa3 is Tic, also D-amino acid <400> 56

Ala-Pro-Xaa-Pro-Glu

5 <210> 57 <211> 5 <212> PRT <213> Artificial Sequence <220>

Alai, A.la3, Pro4, Glu5 are D-amino acids

Xaa2 is MeGly, L-amino acid <400> 57

A .1 a - Xa a - A1 a -Pro - G1 u <210> 58 <212> PRT <213> Artificial Sequence <22 0>

<223> Alai, Pro2, Ala3, Glu5 are D-amino acids Xaa4 is MeGly, I »-amino acid <400> 58

Al a - P r o - A1 a ·· X a a - G11 <z 10>

<211>

<213> Artificial sequence <223> Alai, Ala3, Pro4, Glu5 are D-amino acids

Xaa2 is Pip <400> 59

Axa ~ Xaa ~ A i a - Pro — Glu <210> 60 <211> 5 <212> ORT <213> Artificial Sequence.

<220>

<223> Alai, Pro2, Ala3, Glu5 are D ~ amino acids

Xaa4 is Pip: · <400> 60

Aia-Pro-Ala-Xaa-Glu <210> 61 <211> 5 <212> PRT <213> Me s s e e v e ci a <22 0>

<223> Pro2, Ala3, Pro4, Glu5 are D-amino acids 25 <400> 61

G1 y ~ P r o ~ A1 a - P r o - G1 u

1 ’5 <210> 62 <21.1> 5 <21 2> PRT <213> Artificial Sequence <220>

Pro2, Ala.3, Pro4, Glu5 are D-amino acids

Xaal is N-Methylalanine, also D-amino acid <40ö> 62

Xaa-Pro-Ala ~ Pro ~ Glu

5 <210> € 13 <211> 5 <212> PRT <213> Artificial Sequence <220>

<223> Alai, Pro2, Pro4, Glu5 are D-amino acids

Xa3 is Aib, L-amino acid $$ ^ ·· <400> 63

A1 a - P r ο -X a a - P r o - G1 u

5 <210> 64 <211> 5 <212> PET <213> Artificial Sequence <220>

Pro2, Ala3, Pro4, Glu5 are D-amino acids

Xaa1 is Aib, L-amino acid 15 <400> 64

Xaa “Pro — Ala — Pro — Glu

5 <210> 65 <211> 5 <212> PRT <213> Artificial Sequence 25 <223> Synthetic Peptide <400> 65

0 A1a - P r o - G1 u - P r o ~ A1 a

5 <210> 66 <211> 42 <212> PRT <213> homo sapiens

STRAND <400> 66

Asp-A1a-G1u-Phe ~ Arg-His-Asp-Ser-G1y-Tyr-G1u-Va1-H is-H i s-Asn10 b y s - Leu ~ V a 1 ~ Ph e ~ Ph: e ~ A1 a ~ G1 u ~ Asp - Va 1 - G1 y - S e r - As π - L y s -G1 y - A 1 a * 30

Ile-Ile-Gly-Leu-Met-Va1-Gly-G1y-Va1 ~ Va1 ~ 11e-Ala

Claims (10)

♦ The peptide or peptide mimetic of formula (I), A1-A2-A3-A4-A5-Y (I) where Al is any of the following; Ala or Glu; A2 is Pro; A3 is Ala; A4 is Pro; A5 is any of Ala, Glu; Y is absent or an amide; or a pharmaceutically acceptable salt or ester thereof, The peptide or peptide mimetic of claim 1, wherein the sequence is selected from the group consisting of EPAPA (SEQ ID NO 28), APAPE amide (SEQ ID NO 30), apape amide (SEQ ID NO 33), and apape (SEQ ID NO 33). ID NO 51). 3. A peptide or peptide mimetic selected from peptides having the following sequence: EPPPA (SEQ ID NO 29), epppa-amide (SEQ ID NO 32), apapq (SEQ ID NO 4 6), apaph (SEQ ID NO 48), apapq-amide (SEQ ID NO 49), apapn-amide (SEQ 10 NO 50) and pharmaceutically acceptable salts or esters thereof. The peptide or peptide mimetic of claim 1, which is apape (SEQ ID NO 51) or a pharmaceutically acceptable salt or ester thereof. 5. A pharmaceutical composition comprising a) at least one peptide of formula (I) or peptide mimetic, pond pond SZTNH-100287355 ah Al is any of the following: Ala, Gia; A2 is Pro; A3 is Ala; The 4 reports are Pro; A5 is any of Ala, Glu; Y is absent or an amide; or b) QigDf selected from peptides having the following sequence; } cjp t; * EPPPA (SEQ ID NO 29), epppa-amide (SEQ ID NO 32), apapq (SEQ ID NO 46), apapn (SEQ ID NO 48), apapq-amide (SEQ I.D NO 49), apapn-amide (SEQ ID NO 50), and / or a pharmaceutically acceptable salt or ester thereof, and at least one pharmaceutically acceptable substance. Pharmaceutical composition according to Claim 5, characterized in that the at least one pharmaceutically acceptable excipient is a controlled-release matrix. Pharmaceutical composition according to Claim 5 or 6, characterized in that the peptide has the following sequence selected from peptides or peptide mimetics: EPPPA (SEQ ID NO 29), epppa-amide (SEQ ID NO 32), apapq (SEQ ID NO 46), apapn (SEQ apapq-amide apapn-amide ID NO 48), (SEQ ID NO 49), (SEQ ID NO 50), EPAPA (SEQ ID NO 28), APAPE-amide (SEQ ID NO 30), aoape-amide (SEQ ID NO 33), apape (SEQ ID NO 51) or pharmaceutically acceptable salts or esters thereof, preferably apape (SEQ ID NO 51) or pharmaceutically acceptable salts or esters thereof. 8 * The peptide of formula (I) or a peptide mimetic and a pharmaceutically acceptable salt and ester thereof. Al-A2 ~ A3-A4-A5- ¥ (I) where Al is any of Al, Glu; A2 is Pro; A3 is Ala; A4 j e 1 en tése Pro; A5 is any of Ala, Glu; ¥ is absent or is an amide; A peptide or peptide selected from the group consisting of 15 or the following sequences: EPPPA (SEQ ID NO 29), epppa-amide (SEQ ID NO 32), apapq (SEQ ID NO 46), 20 apapn (SEQ ID NO 40), apapq-amide (SEQ ID NO 49), apapn-amide (SEQ ID NO 50) or a pharmaceutically acceptable salt or ester thereof in the manufacture of a medicament for the prophylaxis or treatment of a curable disease by preventing the harmful effects of Αβ-peptides. The use of claim 8, wherein the disease curable by preventing the adverse effects of Αβ-peptides is selected from neurodegenerative diseases and type 2 diabetes mellitus. The use of claim 8, wherein the disease treatable by preventing the adverse effects of Αβ-peptides is selected from the group consisting of familial and late-onset Alzheimer's disease, Parkinson's disease, Down's syndrome, type 2