Peptides for treating alpha-synuclein amyloid-based disorders

Abstract

The present invention relates to cell penetrating peptides and derivatives thereof for inhibiting α-synuclein fibril formation. The present invention specifically relates to peptide-based inhibitors of Parkinson's disease. The present invention discloses the identification of peptides capable of penetrating the blood-brain barrier for inhibition. The formation of α-synuclein fibrils is observed in the presence of peptides as shown in SEQ ID NO:7, SEQ ID NO:8, where SEQ ID NO:7, SEQ ID NO:8 are truncated versions of SEQ ID NO:4 with 83% and 75% homology, respectively. The present invention provides a peptide having an amino acid sequence of Formula 1. The present invention also provides a pharmaceutical composition comprising a peptide of Formula 1 together with a pharma- ceutically acceptable excipient, having inhibitory activity against β-sheet polymerization of amyloidogenic proteins. Analysis showed that the peptides corresponding to SEQ ID NO:7, SEQ ID NO:8 are better inhibitors of α-synuclein fibril formation than SEQ ID NO:4.

Description

[Technical field]

[0001] The present invention relates to peptides for the treatment of α-synuclein amyloid-based disorders, such as Parkinson's disease (PD) and other α-synucleinopathies.

[0002] The present invention relates to the use of small, essentially cell-permeable peptides in inhibiting the process of conversion of intrinsically disordered proteins, such as α-synuclein, into β-sheet-rich amyloid fibrils and can therefore be used as potential lead molecules for the treatment of diseases known as α-synucleinopathies. [Background technology]

[0003] This study focuses on the development of inhibitors of the process of α-synuclein fibril formation, in which cytotoxic fibrils are formed by the intrinsically disordered monomeric protein α-synuclein. α-Synuclein is known to form amyloid fibrils, and the deposition of such protein inclusions in cells mediates the cytotoxicity associated with Parkinson's disease (PD) as well as many other diseases collectively referred to as α-synucleinopathies. α-Synuclein as the main component of Lewy bodies, the defining pathological feature of PD, has been reported by Spillantini, MG et al., [alpha]-Synuclein in Lewy bodies., Nature 388, 839-840 (1997). Spillantini, MG, Crowther, RA, Jakes, R., Hasegawa, M. &Goedert, M., α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies., Proceedings of the National Academy of Sciences of the United States of America 95, pp. 6469-6473 (1998), doi:10.1073 / PNAS.95.11.6469, reports that α-synuclein forms the main filamentous component of Lewy bodies and Lewy neurites. According to the Centers for Disease Control (CDC) report, PD is the 14th leading cause of death in the United States (National Vital Statistics Report, 2014). PD is caused due to the death of dopaminergic neurons involved in controlling muscle activity. The key pathological feature of PD is the formation of abnormal aggregates of proteins known as Lewy bodies.

[0004] α-Synuclein is the main component of Lewy bodies deposited in dopaminergic neurons of PD patients. α-Synuclein is a member of the synuclein family of proteins that is abundant in presynaptic terminals of neural tissues. Synuclein members are characterized by five or six imperfect repeats of the motif KTKEGV at the N-terminus, a central hydrophobic non-amyloid beta component (NAC) domain, and an acidic carboxy-terminal tail. Protofibrils formed during the conformational transition of native α-synuclein into fibrils cause toxicity (Bucciantini, M. et al., Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases., Nature 416, 507-511, 2002), and therefore strategies to block the process of fibril formation are believed to lead to effective therapies for amyloid-based disorders. One of the main challenges in designing such inhibitors is that many of the amyloid-based disorders are neurodegenerative diseases, and therefore potential inhibitors must be able to cross the blood-brain barrier. Designing such molecules with the dual properties of inhibiting fibril formation and the ability to cross the blood-brain barrier remains a challenge.

[0005] Over the last decade, cell-penetrating peptides (CPPs) have emerged as versatile drug delivery systems (Heitz, F., Morris, MC & Divita, G., Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics., British Journal of Pharmacology, 157, pp. 195-206, 2009, doi:10.1111 / j.1476-5381.2009.00057.x). Reported by Lindgren, M. & Langel, U., in Cell-Penetrating Peptides: Methods and Protocols (edUloLangel), pp. 3-19 (Humana Press, 2011), CPPs constitute a family of small peptides (fewer than 30 amino acids), often carrying a net positive charge, and possessing an intrinsic ability to cross the plasma membrane without causing significant membrane damage. Due to their cell-penetrating ability, CPPs have been used as a therapeutic agent for the delivery of proteins (Nasrollahi, SA, Fouladdel, S., Taghibiglou, C., Azizi, E. & Farboud, ES, A peptide carrier for the delivery of elastin into fibroblast cells., International Journal of Dermatology 51, pp. 923-929, (2012), doi:10.1111 / j.1365-4632.2011.05214.x.), peptides (Boisguerin, P., Giorgi, J.-M. &Barrere-Lemaire, S., CPP-conjugated Anti-apoptotic Peptides as Therapeutic Tools of Ischemiareperfusion Injuries., Current Pharmaceutical Design 19, pp. 2970-2978, 2013; Soto-Jara, C., U.S. Patent No. 5,948,673, 1999; Frangione, B., U.S. Patent No. 7,473,482, 2009; El-Agnaf, OMA, U.S. Patent No. 2,004,009,625, 2004), small molecules (Shi, N.-Q., Gao, W., Xiang, B. & Qi, X.-R., Enhancing cellular uptake of activable cell-penetrating peptide-doxorubicin conjugate by enzymatic cleavage., International Journal of Nanomedicine 7, pp. 1613-1621, 2012, doi:10.2147 / ijn.s30104; Nordstedt, C., U.S. Patent No. 6,331,440, 2001), Nucleic acids (Lehto, T., Kurrikoff, K. & Langel, U., Cell-penetrating peptides for the delivery of nucleic acids., Expert Opinion on Drug Delivery 9, pp. 823-836, 201, doi:10.1517 / 17425247.2012.689285 and Margus, H., Padari, K. &Pooga, M., Cell-penetrating Peptides as Versatile Vehicles for Oligonucleotide Delivery., Molecular Therapy. 20, pp. 525-533, 2012, doi:10.1038 / mt.2011.284), Nanoparticles et al. (Dekiwadia, CD, Lawrie, AC &Fecondo, JV, Peptide-mediated cell penetration and targeted delivery of gold nanoparticles into lysosomes., Journal of Peptide Science 18, pp. 527-534, 2012, doi:10.1002 / psc.2430) have been widely used for intracellular delivery of various cargoes (both in vitro and in vivo).

[0006] CPPs have been widely used to deliver various therapeutic agents to treat a variety of diseases including cancer, psoriasis, and microbial infections (Fonseca, SB, Pereira, MP & Kelley, SO, Recent advances in the use of cell-penetrating peptides for medical and biological applications. Advanced Drug Delivery Reviews 61, 953-964, 2009, doi:http: / / dx.doi.org / 10.1016 / j.addr.2009.06.001), but their application in the treatment of neurodegenerative diseases has been less explored.Interestingly, Zou, L.-L., Ma, J.-L., Wang, T., Yang, T.-B. & Liu, C.-B., Cell-Penetrating Peptide-Mediated Therapeutic Molecule Delivery into the Central Nervous System., Current Neuropharmacology. 11, pp. 197-208, 2013, doi:10.2174 / 1570159X11311020006; Xia, H. et al., Penetratin-functionalized PEG-PLA nanoparticles for brain drug delivery., International Journal of Pharmaceutics 436, pp. 840-850, 2012, doi:http: / / dx.doi.org / 10.1016 / j.ihpharm.2012.07.029; Spencer, B. et al., α-synuclein conformational antibodies Several studies, such as fused to penetratin are effective in models of Lewy body disease., Annals of Clinical and Translational Neurology 3, 588-606, 2016, doi:10.1002 / acn3.321 and Lin, T. et al., Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides., Acta Pharmaceutica Sinica.B 6, 352-358, 2016, doi:10.1016 / j.apsb.2016.04.001, have reported the ability of CPPs to cross the BBB, thus making them potential candidates for the delivery of therapeutic agents to access diseased areas of the brain.Penetratin is a well-known CPP that was discovered more than 20 years ago in 1994 (Derossi, D., Joliot, AH, Chassaing, G. & Prochiantz, A., The third helix of the Antennapedia homeodomain translocates through biological membranes., The Journal of biological chemistry 269, pp. 10444-10450, 1994). Penetratin is a 16 amino acid long polypeptide derived from the third α-helix (segment 43-58) of the homeodomain of the Drosophila homeoprotein Antennapedia (Derossi, D. et al., Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent., The Journal of biological chemistry 271, pp. 18188-18193, 1996). Penetratin is rich in positively charged residues and is increasingly being used as a potential vehicle for various drug and biomolecule delivery applications (Fonseca, SB, Pereira, MP & Kelley, SO, Recent advances in the use of cell-penetrating peptides for medical and biological applications. Advanced drug delivery reviews 61, 953-964, 2009, doi:10.1016 / j.addr.2009.06.001 and Stewart, KM, Horton, KL & Kelley, SO, Cell-penetrating peptides as delivery vehicles for biology and medicine. Organic & biomolecular chemistry 6, 2242-2255, 2008, doi:10.1039 / b719950c).It has also been reported by Meade, A., Meloni, B., Mastaglia, F. & Knuckey, N., The application of cell penetrating peptides for the delivery of neuroprotective peptides / proteins in experimental cerebral ischaemia studies., Journal Of Experimental Stroke and Translational Medicine 2, pp. 22-40, 2009 that penetratin can cross the blood-brain barrier. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent No. 5,948,673 [Patent Document 2] U.S. Patent No. 7,473,482 [Patent Document 3] U.S. Patent No. 2,004,009,625 [Patent Document 4] U.S. Patent No. 6,331,440 [Patent Document 5] International Publication No. 2004009625 [Non-patent literature]

[0008] [Non-Patent Document 1] Spillantini, MG et al., [alpha]-Synuclein in Lewy bodies., Nature 388, pp. 839-840 (1997) [Non-Patent Document 2] Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., & Goedert, M. α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proceedings of the National Academy of Sciences of the United States of America 95, 6469 - 6473 (1998), doi:10.1073 / PNAS.95.11.6469 [Non - Patent Document 3] National Vital Statistics Report, 2014 [Non - Patent Document 4] Bucciantini, M. et al. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature 416, 507 - 511, 2002 [Non - Patent Document 5] Heitz, F., Morris, M. C., & Divita, G. Twenty years of cell - penetrating peptides: from molecular mechanisms to therapeutics. British Journal of Pharmacology, 157, 195 - 206, 2009, doi:10.1111 / j.1476 - 5381.2009.00057.x [Non - Patent Document 6] Lindgren, M., & Langel, U. in Cell - Penetrating Peptides: Methods and Protocols (ed Ulo Langel), 3 - 19 (Humana Press, 2011) [Non - Patent Document 7] Nasrollahi, S. A., Fouladdel, S., Taghibiglou, C., Azizi, E. & Farboud, E. S., A peptide carrier for the delivery of elastin into fibroblast cells., International Journal of Dermatology 51, 923 - 929, (2012), doi:10.1111 / j.1365-4632.2011.05214.x.

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[0009] The main objective of the present invention is to provide peptides for treating α-synuclein amyloid-based disorders. The present invention provides inhibitors of the process of fibril formation formed by the intrinsically disordered protein α-synuclein, and thus capable of curing disorders and diseases associated with α-synuclein amyloid, such as pd and other α-synucleinopathies.

[0010] It is a further object of the present invention to identify and provide peptide-based inhibitors of α-synuclein fibril formation that have the potential to cross the blood-brain barrier. [Means for solving the problem]

[0011] Thus, the present invention provides peptides for treating α-synuclein amyloid-based disorders, more particularly, the present invention relates to cell-permeable peptides of formula 1 that inhibit fibril formation of intrinsically disordered proteins and therefore can be used as potential lead molecules for the treatment of α-synuclein amyloid-based disorders such as PD and other α-synucleinopathies.

[0012] Thus, in one embodiment, the present invention provides a compound of formula 1X1[RQI] for the inhibition of α-synuclein protein aggregation involved in amyloid-related disorders. mKIWFX2NRRMK[WKK] m X3, wherein X1 and X3 may be absent or are cysteine, X2 may be either Gln or Ala, and m may have a value of 0 or 1.

[0013] In a particularly preferred embodiment, the peptide according to formula 1, wherein X1 and X3 can be either cysteine ​​or cystine.

[0014] In a particularly preferred embodiment, the peptide according to formula 1, wherein m=0 or 1.

[0015] In a particularly preferred embodiment, the peptide according to formula 1, wherein m=0 or 1 and X2 can be Gln or Ala.

[0016] In another embodiment, the peptide according to formula 1 further comprising a group selected from an acetyl group at the N-terminus and an amide group at the C-terminus.

[0017] In another embodiment, a peptide according to formula 1 for use as a medicament.

[0018] In another embodiment, a pharmaceutical composition comprising a peptide of formula 1, optionally together with a pharma- ceutically acceptable excipient.

[0019] In another embodiment, a pharmaceutical composition comprising a peptide of formula 1, optionally together with a pharma- ceutically acceptable excipient, for treating PD.

[0020] In another embodiment, a pharmaceutical composition comprising a peptide of formula 1 having inhibitory activity against beta-sheet polymerization of amyloidogenic proteins.

[0021] In another embodiment, a pharmaceutical composition comprising a peptide of formula 1 having inhibitory activity against β-sheet polymerization of an amyloidogenic protein, wherein the protein is α-synuclein.

[0022] Another embodiment provides a method for inhibiting α-synuclein amyloid fibril formation with the amino acid sequence of Formula 1 and salts thereof.

[0023] Another embodiment relates to the use of a peptide having the amino acid sequence of formula 1 and salts thereof for the inhibition of alpha-synuclein protein aggregation.

[0024] Another embodiment relates to the use of a peptide having the amino acid sequence of Formula 1 and salts thereof for the preparation of a medicament for the treatment of disorders and diseases associated with α-synuclein amyloid, such as Parkinson's disease and other α-synucleinopathies. [Brief description of the drawings]

[0025] [Figure 1] SEQ ID NO: 4 inhibits α-synuclein fibril formation. α-synuclein fibril formation was monitored using Thioflavin T (ThT), a dye that specifically binds to amyloid. Binding of ThT results in an increase in its fluorescence intensity at 482 nm. Purified α-synuclein (400 μM) was incubated at 37° C. with shaking in the presence and absence of equimolar concentrations of the indicated peptides. Amyloid formation was determined by monitoring ThT fluorescence at various time intervals. [Diagram 2]SEQ ID NO:4 promotes α-synuclein solubilization. (A) α-synuclein fibril formation assays were performed in the presence and absence of SEQ ID NO:4 as described above. Aliquots were collected before (0 h) or after (7 h) fibril formation and fractionated into supernatant and pellet by centrifugation at 4000 rpm for 1 min. Fractions were incubated with SDS loading buffer at 99° C. for 15 min and further separated by 15% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Shown are supernatant and pellet fractions from α-synuclein without (lanes 1, 2) and with (lanes 3, 4) SEQ ID NO:4 at 0 h and without (lanes 5, 6) and with (lanes 7, 8) SEQ ID NO:4 after 7 h of incubation at 37° C. (B) The secondary structure content of α-synuclein (12.5 μM) in the presence and absence of equimolar concentrations of SEQ ID NO:4 was analyzed by CD. In each case, the average of three spectra is reported. As expected, the CD spectrum corresponding to α-synuclein alone shows the characteristics of an intrinsically disordered protein. An α-synuclein aliquot collected after 7 hours of incubation at 37° C. shows the presence of β-sheet content. As can be seen, a similar aliquot from a reaction containing α-synuclein in the presence of SEQ ID NO:4 shows a CD spectrum similar to that observed with α-synuclein alone. [Diagram 3] SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 inhibit α-synuclein fibril formation. Thioflavin T (ThT) dye, which specifically binds to amyloid, was used to monitor α-synuclein fibril formation by the increase in fluorescence intensity recorded at 482 nm of the dye. Purified α-synuclein (400 μM) was incubated at 37° C. with shaking in the presence and absence of equimolar concentrations of the indicated peptides. Amyloid formation was determined by monitoring ThT fluorescence at various time intervals. The reduction in ThT fluorescence observed for up to 6 hours suggests that the peptides inhibit the formation of α-syn fibrils. [Figure 4A]Inhibition of α-synuclein fibril formation in the presence of various peptides as shown by SEQ ID NO: Data are the same as in FIG. 3A, except for clarity, only the expanded (magnified) Y-axis for α-synuclein fibril formation in the presence of peptides is shown. As shown, compared to penetratin, SEQ ID NO: 7 and SEQ ID NO: 8 did not increase α-synuclein fibril formation as evidenced by a slight increase in fluorescence intensity. [Figure 4B] The amplitudes of the fluorescence intensity at 0 and 5 hours were taken from the data shown in Figure 3A. The ratio of the amplitude at 5 hours versus 0 hours for each curve was calculated and normalized. As shown, peptide derivatives of SEQ ID NO:7 and SEQ ID NO:8 are significantly better inhibitors of fibril formation than SEQ ID NO:4, since less change in fluorescence intensity was observed when α-synuclein fibril formation was carried out in the presence of these derivatives, relative to the change in fluorescence intensity at the start of the fibril formation reaction. [Figure 5A] Sequence alignment showed approximately 83% homology (identity) between SEQ ID NO:4 and SEQ ID NO:7. [Figure 5B] Sequence alignment showed approximately 75% homology (identity) between SEQ ID NO:4 and SEQ ID NO:8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Many of the amyloid-based disorders are neurodegenerative in nature. Therefore, one of the main challenges in designing therapeutics against amyloid diseases has been to develop inhibitors that can not only prevent the formation of amyloid fibrils but also cross the blood-brain barrier. Therefore, the main objective of this study is the identification of blood-brain barrier-permeable peptides that can inhibit the formation of fibrils by the intrinsically disordered protein α-synuclein. Based on previous reports that there are a few CPPs that can cross the BBB, we investigated their potential for inhibiting the formation of fibrils.

[0027] The intrinsically disordered protein α-synuclein has been purified and an assay for monitoring its fibril formation has been established. We have shown that α-synuclein starts to form fibrils when incubated at 37° C. under shaking conditions. We then investigated the formation of α-synuclein fibrils in the presence and absence of various CPPs, such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. As shown in FIG. 1, SEQ ID NO:4 significantly inhibited α-synuclein fibril formation (approximately 90%), whereas in the presence of other CPPs, fibril formation was less affected. To further confirm that the decrease in Thioflavin T fluorescence in the presence of SEQ ID NO:4 is due to its ability to inhibit α-synuclein fibril formation, the solubility of fibril-forming proteins was examined by SDS-PAGE. Samples containing α-synuclein incubated at 37° C. in the presence or absence of equimolar concentrations of SEQ ID NO:4 were collected at the beginning of incubation (0 h) or after 7 h. The samples were fractionated into supernatant and pellet, and the fractions were separated using 15% SDS-PAGE. As shown in Figure 2a, α-synuclein was found to be primarily in the supernatant at the start of the incubation, but after 7 hours, more than 50% of the protein was found to be precipitated in the pellet fraction. Interestingly, the reaction containing co-incubation with SEQ ID NO:4 contained nearly all of the α-synuclein in the supernatant at both time points, suggesting that the addition of SEQ ID NO:4 prevents α-synuclein from aggregating into amyloid fibrils and helps the intrinsically disordered protein remain in the soluble fraction.

[0028] It is known that the intrinsically disordered protein α-synuclein, which lacks ordered secondary structural features, becomes converted into β-sheet-rich amyloid fibrils. Thus, changes in β-sheet content are used as a strategy to monitor the presence of amyloid fibrils. To further investigate that SEQ ID NO: 4 inhibits α-synuclein fibril formation in vitro, the secondary structure of α-synuclein with and without SEQ ID NO: 4 was analyzed using CD. α-synuclein was incubated at 37° C. in the presence and absence of SEQ ID NO: 4, and CD spectra were recorded from aliquots collected before and after fibril formation. As is evident in FIG. 2B, aliquots collected after fibril formation show β-sheet or disordered secondary structural features depending on whether SEQ ID NO: 4 was absent or present during the reaction, clearly suggesting that the peptide inhibits α-synuclein fibril formation, thereby promoting its native disordered structural content.

[0029] In general, the main drawback in the use of peptides as drugs is their poor pharmacokinetics. To increase the stability of SEQ ID NO:4, N- and C-terminal capping was performed by acetylation and amidation, respectively (SEQ ID NO:5). A cyclic derivative (SEQ ID NO:6) was also synthesized by introducing Cys residues at the N- and C-termini of the SEQ ID NO:4 sequence. SEQ ID NO:4-like peptides were tested for defibrillation activity by incubating with α-syn and performing a Thioflavin T fibrillation assay. To increase cost-effectiveness and activity, several shorter truncated forms of SEQ ID NO:4 were identified and further cyclized to obtain SEQ ID NO:7 and SEQ ID NO:8. SEQ ID NO:8 also has further mutations in addition to being a shorter cyclized derivative. The activity of all derivatives to inhibit α-synuclein fibril formation was confirmed using a ThT-based fluorescence assay. Figure 3 shows that SEQ ID NO:4-like peptides are also capable of inhibiting α-synuclein fibrils. Taken together, these results suggest that SEQ ID NO: 4 and its derivatives have high potential as lead therapeutic molecules for the treatment of various disorders caused by the accumulation of α-synuclein amyloid fibrils. Furthermore, the ability of all derivatives to inhibit α-synuclein fibril formation was compared by measuring the fluorescence change before and after α-synuclein fibril formation (Figure 4). The fluorescence intensity at 5 hours was normalized with respect to the fluorescence intensity at the beginning of the reaction. Figure 4B compares the normalized fluorescence intensity of the fibril formation reaction in the presence of SEQ ID NO: 4 with the fluorescence intensity obtained in the presence of the other derivatives. The analysis showed that peptides corresponding to SEQ ID NO: 7 and SEQ ID NO: 8 are better inhibitors of α-synuclein fibril formation than SEQ ID NO: 4.

[0030] SEQ ID NO:4 is a well-known CPP (Turner, JJ et al., Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Research 33, 6837-6849, 2005 (doi:10.1093 / nar / gki991)) and has been shown to cross the BBB (Bolton, SJ et al., Cellular uptake and spread of the cell-permeable peptide penetratin in adult rat brain. European Journal of Neuroscience 12, 2847-2855, 2000 (doi:10.1046 / j.1460-9568.2000.00171.x) and Bera, S., Kar, RK, Mondal, S., Pahan, K. & Bhunia, A., Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier., Biochemistry 55, pp. 4982-4996, 2016 (doi:10.1021 / acs.biochem.6b00518), which suggests that SEQ ID NO:4 could be used as a cargo to deliver neuroprotective agents to the brain (Meade, A., Meloni, B., Mastaglia, F. &Knuckey, N., The application of cell penetrating peptides for the delivery of neuroprotective peptides / proteins in experimental cerebral ischaemia studies., JOURNAL OF EXPERIMENTAL STROKE AND TRANSLATIONAL MEDICINE 2, pp. 22-40, 2009 and Joliot, A. & Prochiantz, A., Transduction peptides: from technology to physiology., Nat Cell Biol 6, pp. 189-196, 2004). There is prior art that describes the ability of SEQ ID NO:4 as a carrier to deliver anti-cancer drugs (Fahraeus, R., Paramio, JM, Ball, KL, Lain, S. & Lane, DP. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide from p16CDKN2 / INK4A. Current Biology 6, 84-91, 1996, doi:https: / / doi.org / 10.1016 / S0960-9822(02)00425-6) and anti-amyloid drugs. Regarding use against α-synuclein, there is prior art showing that α-synuclein has the ability to interact with TAT-derived CPPs (Zigoneanu, IG &Pielak, GJ, Interaction of α-Synuclein and a Cell Penetrating Fusion Peptide with Higher Eukaryotic Cell Membranes Assessed by 19F NMR., Molecular Pharmaceutics 9, pp. 1024-1029, 2012, doi:10.1021 / mp200615m). Also, there is a small amount of prior art reporting peptide sequences that act on α-synuclein fibril formation (WO2004009625 and Shaltiel-Karyo, R.et al., Inhibiting α-Synuclein Oligomerization by Stable Cell-Penetrating β-Synuclein Fragments Recovers Phenotype of Parkinson's Disease Model Flies., PLoS ONE 5, e13863, 2010, doi:10.1371 / journal.pone.0013863). Based on these prior art, we can state that CPPs are widely used as cargos to deliver drugs to various mammalian tissues, including the brain, implying that CPPs are not toxic to cells. Also, researchers have reported peptide sequences that act against α-synuclein amyloidogenesis, but they lack the ability to cross the BBB. Therefore, Formula 1(X1[RQI]. m KIWFX2NRRMK[WKK] m The ability of peptides of formula (X3) to act as potent inhibitors of α-synuclein amyloidogenesis makes them extremely useful in drug development for Parkinson's disease. EXAMPLES

[0031] The following examples are given by way of illustration and therefore should not be construed as limiting the scope of the invention. The peptides can be used in pharmaceutical compositions such as lipid formulations, such as liposomes, solid lipid nanoparticles with peptide stability and penetration enhancers.

[0032] Example 1 Expression and purification of α-synuclein. Human wild-type α-synuclein protein was expressed in E. coli Rossetta (DE3) using a pT7-7 based expression vector. After IPTG induction, bacterial cell pellets were collected by centrifugation and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM Pierce protease inhibitor cocktail. Protein purification was performed using a previously described protocol with minor modifications (van Raaij, ME, Segers-Nolten, IMJ &Subramaniam, V. Quantitative Morphological Analysis Reveals Ultrastructural Diversity of Amyloid Fibrils from α-Synuclein Mutants. Biophysical Journal 91, L96-L98, 2006, doi:10.1529 / biophysj.106.090449). Briefly, cell lysis was performed by sonication and the lysate was boiled at 95°C for 30 min, followed by centrifugation at 10,000 x g for 30 min at 4°C. DNA precipitated with streptomycin sulfate and glacial acetic acid was removed by centrifugation at 13,500 x g for 30 min at 4°C. α-synuclein protein was selectively precipitated by addition of 50% ammonium sulfate and incubation on ice for 1 h with periodic shaking every 10 min. The pellet was isolated by centrifugation at 13,500 x g for 30 min at 4°C and washed with an equal volume of 100 mM ammonium acetate followed by an equal volume of ethanol. The pellet was dissolved in 10 mM HEPES, 50 mM NaCl, pH 7.4 and dialyzed extensively to remove ammonium sulfate. Protein purity was confirmed by 15% SDS-PAGE.

[0033] Example 2 Peptide synthesis SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 were synthesized at our peptide synthesis facility at the Institute of Microbial Technology, Chandigarh, India. The peptides were synthesized by solid-phase peptide synthesis strategy using Fmoc (N-(9-fluoroyl)-methoxycarbonyl) chemistry on a PS-3 peptide synthesizer at Protein Technologies Inc, USA on a 0.01 mmol scale as described (Gautam, A. et al., Identification and characterization of novel protein-derived arginine-rich cell-penetrating peptides., European Journal of Pharmaceutics and Biopharmaceutics 89, 93-106, 2015, doi:http: / / dx.doi.org / 10.1016 / j.ejpb.2014.11.020). Other truncated derivatives of SEQ ID NO:4 (SEQ ID NO:7 and SEQ ID NO:8) were synthesized and obtained from Genscript, Inc. (Piscataway, NJ, USA).

[0034] Example 3 In vitro fibril formation assay Thioflavin T (ThT) (4 mM) was added to 400 μM purified α-synuclein with and without cell-penetrating peptide at equimolar concentrations in a 96-microwell plate. The plate was incubated in a multimode plate reader (TECAN infinite M200 PRO) in linear mode at 37° C. with a shaking speed of 900 rpm. Fluorescence kinetics was measured every 15 min with an emission wavelength of 482 nm upon excitation at 442 nm. Each experiment was repeated at least three times.

[0035] Example 4 SDS-PAGE analysis. SDS-PAGE analysis was performed using 15% Tris-bisacrylamide gels operated at a constant current of 30 mA. The running buffer was 25 mM Tris-HCl, 193 mM glycine, and 0.1% SDS (pH 8.3). Samples were taken before (0 h) and after (7 h) fibril formation and fractionated into supernatant and pellet by centrifugation at 4000 rpm for 1 min. The fractions were resuspended in 1×SDS loading dye, respectively, and further separated by 15% SDS-PAGE. Protein bands were visualized by staining SDS-PAGE with 0.1% Coomassie Blue G-250.

[0036] Example 5 CD analysis. Far-UV CD spectra were recorded on a JASCO-J-815 spectropolarimeter. Spectra were recorded from 250 to 195 nm using a 1 mm pathlength cuvette at 25 °C with a scan speed of 10 nm / min and a step size of 1 nm, and averaged over three scans. Raw CD data were reported as mean residue ellipticity (θ ) expressed in degrees squared centimeters per decimol as follows: MRE ) converted to: θ MRE =(100 * θ obs ) / [d * C * (n-1)] where θ is the observed ellipticity (in degrees), d is the optical path length (in centimeters), C is the protein concentration (molar), and n is the total number of amino acids in the protein.

[0037] 1. We report that the cell-penetrating peptide SEQ ID NO:4 and its derivatives have the ability to inhibit α-synuclein fibril formation, whereas other CPPs tested in this study are unable to do so.

[0038] 2. Since it has been reported that SEQ ID NO:4 can cross the BBB, SEQ ID NO:4 and its derivatives are likely to be potent lead therapeutic molecules for the treatment of amyloid-based diseases.

[0039] 3. Although SEQ ID NO:4 has been used as a vehicle for drug delivery in various mammalian tissues, including the brain, the efficacy of SEQ ID NO:4 alone against neurodegenerative disorders has not been studied. The present invention reports the novelty of peptide inhibition of α-synuclein protein aggregation by SEQ ID NO:4.

[0040] 4. SEQ ID NO:4 is already known to cross the BBB and it has been established that SEQ ID NO:4 is not toxic to mammalian cells and therefore its direct application as an anti-amyloid drug is of high therapeutic value.

[0041] 5. The novel derivatives SEQ ID NO:7 and SEQ ID NO:8 were found to be more potent than SEQ ID NO:4 in inhibiting α-synuclein fibril formation and therefore may be better anti-amyloid drugs.

Claims

1. Formula 1 X 3 , 2 , m , m [RQI] m KIWFX 2 NRRMK[WKK] m X 3 (In the formula, X 1 and X 3 is cysteine ​​or cystine, X 2 is Gln or Ala, m = 0 or 1) A peptide having the amino acid sequence of the above and its derivatives.

2. 2. The peptide of claim 1, wherein SEQ ID NO:7 and SEQ ID NO:8 are truncated forms of SEQ ID NO:4 with 83% and 75% homology, respectively.

3. 2. The peptide of claim 1, wherein an acetyl group is added to the N-terminus and an amide group is added to the C-terminus.

4. 2. The peptide of claim 1 for use as a medicament for an amyloid disorder.

5. A method for inhibiting α-synuclein amyloid fibril formation with a peptide having the amino acid sequence of formula 1, comprising: (a) mixing said peptide with alpha-synuclein to obtain a sample; (b) mixing the sample obtained in step (a) with Thioflavin T dye to obtain purified alpha-synuclein; (c) incubating the purified alpha-synuclein obtained in step (b) under shaking conditions at room temperature or at 37°C; (d) measuring the fluorescence intensity at 482 nm to monitor amyloid formation. The method includes:

6. A pharmaceutical composition comprising the peptide of formula 1 according to claim 1 and a pharma- ceutically acceptable excipient thereof.

7. 10. A method for treating an alpha-synuclein amyloid-related disorder as defined in claim 1, comprising administering a therapeutic peptide as defined in claim 1 or a pharmaceutical composition as defined in claim 6.

8. 8. The method of claim 7, wherein the disorder is selected from Parkinson's disease and other alpha-synucleinopathies.

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