WO2019229771A2 - Nonapeptide of formula i, pharmaceutical compositions and methods for preparation thereof - Google Patents

Abstract

The present invention relates to a nonapeptide of Formula (I), designed from the taxol binding cavity of β-tubulin. The nonapeptide has the ability to bind at taxol pocket of β-tubulin with a significant binding affinity and promotes microtubule polymerization. More particularly, the nonapeptide of the present invention binds to the 17-21 region of amyloid beta, inhibits acetylcholinesterase (AChE) induced amyloid beta (Aβ) aggregation by interacting at the peripheral anionic site (PAS) of AChE, enters into the derived neurons, promotes neurite growth, provides microtubule stabilization and shows significant neuroprotection against nerve growth factor (NGF) deprived neurons. Furthermore, the nonapeptide doesn't show any toxicity to both rat pheochromocytoma (PC12) derived neurons as well as primary cortical neurons.

Description

NONAPEPTIDE OF FORMULA I, PHARMACEUTICAL COMPOSITIONS AND METHODS FOR PREPARATION THEREOF

FIELD OF THE INVENTION

The present invention relates to a novel short nonapeptide of Formula I, designed from the taxol binding cavity of b-tubulin. It has the ability to bind at taxol pocket of b-tubulin with a significant binding affinity and promotes microtubule polymerization. In addition, this peptide binds to the 17-21 region of amyloid beta, inhibits acetylcholinesterase (AChE) induced amyloid beta (Ab) aggregation by interacting at the peripheral anionic site (PAS) of AChE, enters into the derived neurons, promotes neurite growth, provides microtubule stabilization and shows significant neuroprotection against nerve growth factor (NGF) deprived neurons. Moreover, it doesn’t show any toxicity to both rat pheochromocytoma (PC12) derived neurons as well as primary cortical neurons.

Formula I

BACKGROUND OF THE INVENTION

In Alzheimer Disease (AD), amyloid plaques deposits in neuron cells that causes rupturing of the neuronal network and neuron cell membrane. [Verdier Y, Zarandi M, Penke B, Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease, J Pept Sci. 2004, 10, 229-48] This major problem arises due to the accumulation and aggregation of Ab in membrane region. [Sasahara K, Morigaki K, Shinya K. Effects of membrane interaction and aggregation of amyloid b-peptide on lipid mobility and membrane domain structure, Phys. Chem. Chem. Phys. 2013, 15, 8929-39 and Hoshino T, Mahmood MI, Mori K, Matsuzaki K, Binding and aggregation mechanism of amyloid b-peptides onto the GM1 ganglioside-containing lipid membrane. J. Phys. Chem. B. 2013, 117, 8085-94] After disruption of cellular membrane, Ab aggregates propagate from one cell to another cell. [Saha A, Mondal G, Biswas A, Chakraborty I, Jana B, Ghosh S, In vitro reconstitution of a cellular like environment using liposome for amyloid beta peptide aggregation and its propagation. Chem. Commun., 2013, 49, 6119-21] In this disease progression, microtubule, one of the key cytoskeleton proteins disrupted severely. [Zempel H, Mandelkow EM, Tau missorting and spastin-induced microtubule disruption in neurodegeneration: Alzheimer Disease and Hereditary Spastic Paraplegia, Molecular Neurodegeneration, 2015, 10, 1-12] Therefore, inhibition of Ab fibrillation as well as microtubule stabilization is the key prerequisite for the development of novel anti-Alzheimer therapeutic agents. [Speck-Planche A, Luan F, Cordeiro MN, Discovery of anti-Alzheimer agents: current ligand-based approaches toward the design of acetylcholinesterase inhibitors. Mini Rev Med Chem., 2012, 12, 583-91]

It was earlier described that microtubule stabilizing agent can be a potent therapeutic for neuroprotection. [Brunden KR, Trojanowski JQ, Smith AB 3rd, Lee VM, Ballatore C, Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease. Bioorg Med Chem. 2014, 22, 5040-9]

Taxol, a microtubule stabilizing anti-cancer drug has been considered as potential anti-AD drug, which was clinically unsuccessful due to its toxicity. [Baas PW, Fridoon J. A, Beyondtaxol: microtubule-based treatment of disease and injury of the nervous system Brain. 2013, 136, 2937- 51] On the other hand, the 17-21 hydrophobic core of amyloid beta is mainly responsible for its fibrillation and aggregation. Any molecule that binds to this hydrophobic core can inhibit amyloid aggregation. [Liihrs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Dobeli H, Schubert D, Riek R, 3D structure of Alzheimer's amyloid-Pfl -42) fibrils, Proc. Natl. Acad. Sci. USA., 2005, 102, 17342-347]

These facts motivate the inventors to develop this novel peptide based therapeutics for AD, which may execute dual role of action. Therefore, the inventors adopted an innovative strategy for the development of peptide based microtubule stabilizer, considering the taxol binding pocket of b-tubulin and hydrophobic region of amyloid beta (Ab). The 17-21 region of amyloid beta contains some non-polar and hydrophobic amino acids whereas the taxol pocket of b-tubulin consists of polar as well as non-polar amino acids. In the present invention, the inventors have designed one nonapeptide“NVRDLTEFQ” (NVR) by the counter interaction of amino acids of those pockets by using relative frequencies of amino acids contacts. [Faure G, Bomot A, de Brevem A G, Protein contacts, inter-residue interactions and side-chain modeling, Biochimie., 2008, 90, 626-39] This approach leads to a potential nonapeptide, which strongly binds with taxol pocket of b-tubulin, serves as an excellent microtubule stabilizer, Ab aggregation inhibitor and neuroprotective agent. Further, results revealed that this peptide is non-toxic against both PC12 derived neurons as well as primary cortical neurons. This novel strategy and discovery of peptide- based microtubule stabilizer will open the door for the development of potential anti-AD therapeutics in near future.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to provide a peptide molecule from the taxol binding pocket of b-tubulin that can inhibit amyloid beta fibrillation for neuro-protective therapeutics.

Another objective of the present invention is to design said peptide molecule.

Yet another objective of the invention is to develop a peptide-based effective AD drug that is bio compatible and less cytotoxic.

Yet another objective of the invention is to develop a peptide based therapeutic molecule which will serve dual purpose such as keeping the microtubule network healthy as well as inhibit the Ab fibrillations. Yet another objective of the present invention is to prepare said“Peptide”.

Yet another objective of the present invention is to prepare pharmaceutical compositions comprising the peptide.

Yet another objective of the present invention is to use the peptide in treatment of Alzheimer Disease.

ABBREVIATIONS:

AD: Alzheimer Disease, Ab: Amyloid beta, PAS: Peripheral anionic site, AChE: Acetylcholinesterase, PC12: Rat pheochromocytoma cells, NGF: Neuronal growth factor, MD: Molecular docking, HPLC: High pressure liquid chromatography, K_b: Binding constant, DAPI: 4',6-diamidino-2-phenylindole, DMEM: Dulbecco’s Modified Eagle’s Medium, ThT: Thioflavin T, TEM: Transmission Electron Microscopy, FBS: Fetal Bovine Serum, DMSO: Dimethyl sulphoxide, MeOH: Methanol, MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, MALDI: Matrix assisted laser desorption/ionisation, PBS: Phosphate Buffer Saline, TFA: Trifluoroacetic acid, EDT: l,2-Ethanedithiol, GTP: Guanosine-5'-triphosphate, DAPI: 2-(4- (Aminoiminomethyl)phenyl)-lH-indole-6-carboximidamide, BSA: Bovine serum albumin, DIC: N, N'-Diisopropylcarbodiimide.

SUMMARY OF THE INVENTION

The present invention provides an improved peptide based molecule that can inhibit amyloid beta fibrillation, stabilize intracellular microtubule and show neuroprotection against Ab infected cells.

The present invention provides a nonapeptide of formula I

Formula I

In an embodiment, the peptide has a molecular weight of 1120.22 Da.

In an embodiment, the nonapeptide is“NVRDLTEFQ”.

In an embodiment, the nonapeptide binds in the taxol pocket of b-tubulin.

In a preferred embodiment, the nonapeptide interacts with taxol binding site of tubulin with significant binding affinity (K_¾-2.5c10⁴ M ¹). In an embodiment, the nonapeptide undergoes self-assembly process and forms nanovesicles (255 nm) through the formation of b-sheet structure.

In an embodiment, the nonapeptide binds to the hydrophobic (17-21) region of amyloid-beta (Ab).

In an embodiment, the nonapeptide binds the peripheral anionic site of acetylcholinesterase (AChE) enzyme.

In an embodiment, the nonapeptide inhibits amyloid aggregation by binding to Ab and AChE enzyme.

In an embodiment, the nonapeptide has significant stability against human blood serum (23.5 % remains after 24 hours of incubation).

In an embodiment, the nonapeptide stabilizes intercellular microtubule of derived PC 12 neurons.

In an embodiment, the nonapeptide displays significant protection in anti-NGF treated PC12 derived neurons.

In an embodiment, the nonapeptide provides stability to the primary cortical neurons.

In an embodiment, the nonapeptide is able to penetrate Blood-Brain Barrier seen in in vivo mice model.

In an embodiment, the nonapeptide is non-toxic to differentiated PC12 neurons.

The present invention provides a method of preparing the nonapeptide, comprising solid phase synthesis of peptide using Rink Amide resin, F-moc deprotection followed by cleaving of peptide from the resin by using 20% piperidine and standard cleavage solution respectively and purification of crude peptide by RP-HPLC.

The present invention provides for a pharmaceutical composition comprising the peptide along with pharmaceutically acceptable excipients.

In an embodiment, the pharmaceutically acceptable excipient is selected from water or saline solution.

The present invention provides for a method of treating Alzheimer’s disease by administering effective amount of nonapeptide.

The present invention discloses that the peptide is administrated with only water or saline solution.

The present invention discloses that the peptide is administered for treating Alzheimer’s disease through intraperitoneal (IP) and intranasal (IN) route.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 provides a schematic representation of the designing concept of NVR peptide from the taxol pocket of b-tubulin using the theory of relative frequencies of amino acids contact. Figure 2 illustrates (a) Molecular docking reveals that NVR peptide binds close to the taxol site of tubulin; (b) Various amino acids of taxol site interact with NVR peptide; (c) Tubulin turbidity assay using various concentrations of NVR peptide; (d) Microtubule assembly assay using various concentrations of NVR peptide; (e) Determination of binding constant (K_b) by measuring the intrinsic tryptophan fluorescence quenching value of tubulin with increasing cone of NVR; (c) Stoichiometric binding of NVR peptide with tubulin, showing (1 : 1) binding in JOB plot; (f) FRET study between Tubulin-colchicine complex (donor) and Fluorescein-NVR (acceptor) to find the binding region of NVR in tubulin.

Figure 3 shows (a) Atomic force microscopy and (b) Scanning electronic microscopy study of NVR peptide shows formation excellent vesicle; (c) Dynamic light scattering (DLS) study reveals the size of the vesicle was 255nm.

Figure 4: illustrates (a) Molecular docking of NVR peptide with Ab (PDB-1IYT); (b) Inhibition of amyloid aggregation by different concentration of NVR peptide monitored by measuring the fluorescence intensity of ThT; (c) Inhibition of preformed amyloid aggregates by different concentration of NVR peptides monitored by ThT assay; (d) Bar diagram shows the percent (%) inhibition of amyloid aggregation by NVR peptide; (e) FT-IR spectrum shows no b sheet character when Ab peptide was incubated with NVR for 7 days at 37°C.

Figure 5: illustrates (a) AChE induced Ab aggregation inhibition by various NVR peptide concentrations. Error bar corresponds to standard deviation of the value (*p<0.00l, performing one way anova); (b) Lineweaver-Burk plot of AChE activity using various concentrations (2mM, ImM, 0.5mM, 0.25mM) of NVR in different substrate concentrations (87.5-700mM); (c) Molecular docking of NVR peptide with AChE (PDB ID-2CKM) showing binding energy of -7.7 kcal/mol; (d) Different binding partner of CAS and PAS site interacting with NVR peptide.

Figure 6: illustrates (Upper panel) Snapshots from a MD simulation study of NVR peptide with two KLVFFAE peptides at different timeframe 0ns, l2ns, l5ns, 50ns (Lower panel) Control simulation of two KLVFFAE sequence at different timeframe 0ns, 2ns, 5ns and l2ns (a-NVR peptide, b,b*-KLVFFAE sequence).

Figure 7: provides (a-d) Microscopic study in different channels (DIC, 405 and 488 nm) and their merged image shows significant cellular uptake of Fluorescein-NVR peptide in differentiated PC12 derived neuron cells. Scale bars correspond to 20 pm; (e) Cytotoxicity effect of NVR peptide in differentiated PC12 derived neurons. Error bar corresponds to standard deviation of the value (*p<0.05, two tailed student’s t-test). Neurite outgrowth of differentiated PC12 derived neurons; (f) control cells; (g) cells are treated with NVR peptide. Scale bars correspond to 100 pm; (h) Average length of neurites for control cell and NVR treated cells, error bar corresponds to standard deviation of the value (**p<0.05, two tailed student’s t-test).

Figure 8: provides (a-d) Microscopic study in different channels (DIC, 405 and 561 nm) and their merged image reveals stable microtubule network of PC12 derived neurons for NVR treated neurons; (e-h) Microscopic study in different channels (DIC, 405 and 561 nm) and their merged image reveals stable microtubule network of PC 12 derived neurons for control study. Scale bars correspond to 20 pm. Figure 9: provides (a) Microscopic images of NGF treated control; (b) Anti-NGF treated; and (c)

Anti-NGF with NVR peptide treated differentiated PC 12 derived neuron cells in DIC mode (scale bar corresponds to 100 pm); (d) Histogram showing anti NGF study with various concentrations of NVR peptide in differentiated PC 12 neuron cell, error bar corresponds to standard deviation of the value (*p<0.05, two tailed student’s t-test); (e) Serum stability of NVR peptide in Human serum up to 24 hours (error bar corresponds to standard deviation of the value).

Figure 10 provides (a) Maldi-TOF mass spectra of mice brain extract after performing Blood- Brain crossing experiment with NVR peptide. Expected mass (M)- 1120 Da, Observed mass- 1165 Da [M+Na⁺], Cleaved peptide (CP) 1051 Da [CPl+2Na⁺], 882 Da [CP2+K⁺], 868 Da [CP2+Na⁺+H⁺], 768 Da [CP3+K⁺], 713 Da [CP4-H⁺]; (b) Microscopic images of cortical neurons (control); and (c) NVR peptide treated, observed in DIC mode shows healthy neuronal network. Scale bar corresponds to 30 pm; Microscopic study of MAP2 staining of cortical neurons seen in (d) DIC mode (e) merged of 405 nm and 561 nm and (f) merged of DIC, 405 and 561 nm channel shows significant staining of healthy neuronal network. Scale bar corresponds to 30 pm.

DETAILED DESCRIPTION OF THE INVENTION

In last three decades, Alzheimer’s disease (AD) has become a major threat for elderly people around the globe and millions of people are suffering in this devastating disease. [Barrow C J, Zagorski, M G, Solution structures of beta peptide and its constituent fragments: relation to amyloid deposition, Science, 1991, 253, 179-182] Amyloid-beta (A/?) peptide plays central role in AD, misfolds into the ?-sheet rich conformation, forms long unbranched fibers and becomes insoluble inside the cellular milieu, which deposits as amyloid plaques followed by disruption of neuronal networks. [Zempel H, Mandelkow EM, Tau missorting and spastin-induced microtubule disruption in neurodegeneration: Alzheimer Disease and Hereditary Spastic Paraplegia, Molecular Neurodegeneration, 2015, 10, 1-12] Neuron cells are rich with tubulin/microtubule and A ffiber is also known to disrupt intracellular microtubule networks. [Zempel H, Mandelkow EM, Tau missorting and spastin-induced microtubule disruption in neurodegeneration: Alzheimer Disease and Hereditary Spastic Paraplegia, Molecular Neurodegeneration, 2015, 10, 1-12 and LaFerla F M, Green K N, Oddo S, Intracellular amyloid-beta in Alzheimer's disease, Nature Rev. Neurosci., 2007, 8, 499-509] Therefore, development of novel molecules, which can simultaneously inhibit Ab fibrillizations and stabilize microtubules, is extremely important for potential therapy of AD. Till date, there are no approved therapies for AD. However, many small molecules including one octapeptide (NP: NAPVSIPQ) derived from activity dependent neuroprotective protein (ADNP) inhibit fibrillizations in vitro. [Biswas A, Kurkute P, Saleem S, Jana B, Mohapatra S, Mondal P, Adak A, Ghosh S, Saha A, Bhunia D, Biswas S. C, Ghosh S, Novel hexapeptide interacts with tubulin and microtubules, inhibits Ab fibrillation, and shows significant neuroprotection, ACS Chem. Neurosci. 2015, 6, 1309-16] Among them, small molecule clioquinol inhibits in vivo Ab fibrillizations and NP inhibits in vivo tau hyperphosphorillation. [Biswas A, Kurkute P, Saleem S, Jana B, Mohapatra S, Mondal P, Adak A, Ghosh S, Saha A, Bhunia D, Biswas S. C, Ghosh S, Novel hexapeptide interacts with tubulin and microtubules, inhibits Ab fibrillation, and shows significant neuroprotection, ACS Chem. Neurosci., 2015, 6, 1309-16] It was described before that Ab fibril formation occurs through antiparallel interaction of one Ab monomer with the adjacent Ab peptide through hydrophobic stretch of residues 17-21 of Ab molecule. [Liihrs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Dobeli H, Schubert D, Riek R, 3D structure of Alzheimer's amyloid-P(l-42) fibrils, Proc. Natl. Acad. Sci. USA., 2005, 102, 17342-347]

Further, it was shown that Ab peptide inhibitors binds to the hydrophobic stretch of residue 17-21, thus Ab fibril formation can be prevented by inhibiting incoming Ab molecule through blocking this site. [Liihrs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Dobeli H, Schubert D, Riek

R, 3D structure of Alzheimer's amyl o -b( 1-42) fibrils, Proc. Natl. Acad. Sci. USA., 2005, 102, 17342-347] Further, it was described before that microtubule stabilizing molecule can behave as a neuroprotective agent. [Brunden KR, Trojanowski JQ, Smith AB 3rd, Lee VM, Ballatore C, Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease. Bioorg Med Chem. 2014, 22, 5040-49] Inventors have find out the polar and non-polar amino acid of taxol pocket and designed a nonapeptide“NVRDLTEFQ” considering the counter amino acid interaction partner (Figure 1). Two non-polar amino acids are inserted in the designed sequence keeping in mind the hydrophobic core of Ab. We envisioned that this peptide will serve dual role such as inhibition of A//fi brill at ions through binding with 17-21 stretch of Ab42 peptide and stabilization of microtubule through binding with taxol binding site of //-tubulin.

Present invention involves synthesis of NVR peptide and its fluorescein conjugation by solid phase peptide synthesis (SPPS) method using Rink amide AM resin. This crude NVRpeptide was purified by HPLC and charaterized by MALDI-TOF mass spectrometry. In AD, intracellular tubulin/microtubule is severely affected and disrupted, therefore the inventors are interested to know whether NVR can provide microtubule stabilization or not.

For that purpose, researchers have checked the binding of this designed peptide in the taxol binding pocket of tubulin (PDB ID-1JFF) [Lowe J, Li H, Downing K. H, Nogales E, Refined structure of alpha beta-tubulin at 3.5 A resolution, J. Mol. Biol., 2001, 313, 1045-57] by molecular docking experiment in AutodockVina l. l.2.[Trott O, Olson A. J, AutoDockVina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, J. Comput. Chem. 2010, 31, 455-61] Inventors found that the binding energy is quite high (-7.1 kcal/mol), which indicate significant binding of NVR around taxol site of tubulin (Figure2a). The interaction partners of that pocket are shown in Figure 2b. Also, the interaction of this designed peptide was further checked by performing tubulin turbidity assay and microtubule assembly assay using purified tubulin with various concentrations of NVR in presence of GTP. Inventors found enhancement of tubulin polymerization with increasing concentration of NVR indicating its binding with intracellular tubulin, which gives substantial stability to microtubule (Figure 2c and 2d). [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh

S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32] The binding affinity of NVR with tubulin was calculated by measuring the intrinsic tryptophan fluorescence quenching value at various concentrations of NVR using the modified Stern- Volmerequation. [Gupta K, Panda D, Perturbation of Microtubule Polymerization by Quercetin through Tubulin Binding: A Novel Mechanism of Its Antiproliferative Activity, Biochemistry, 2002, 41, 13029-038. and Chakraborti S, Das L, Kapoor N, Das A, Dwivedi V, Poddar A, Chakraborti G, Janik M, Basu G, Panda D, Chakrabarti P, Surolia A, Bhattacharyya B, Curcumin Recognizes a Unique Binding Site of Tubulin, J Med. Chem. 2011, 54, 6183-96] Inventors found that the fluorescence intensity of intrinsic tryptophan of tubulin is reduced upon addition of NVR. The calculated binding constant of NVR with tubulin is 2.5>< l0⁴ kcal mol ¹ (Figure 2e). Then inventors performed Forster Resonance Energy Transfer (FRET) experiment [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32 and Chakraborti S, Das L, Kapoor N, Das

A, Dwivedi V, Poddar A, Chakraborti G, Janik M, Basu G, Panda D, Chakrabarti P, Surolia A, Bhattacharyya B, Curcumin Recognizes a Unique Binding Site of Tubulin, J Med. Chem. 2011, 54, 6183-96] to measure the possible binding site of NVR at tubulin using tubulin bound colchicine (Tub-col complex) as a donor and Fluorescein-NVR as an acceptor. From Figure 2f, inventors observed FRET between the Tubulin-colchicine complex and fluorescein-NVR. Forster distance (Ro) between this donor-acceptor pair was 29.5 ± 1 A. [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32] So, the calculated distance (R_DA) between the tubulin-colchicine complex and fluorescein-NVR peptide is 30.8 ± 1 A, which indicates that NVR binds to tubulin in a site, which is~30 A apart from colchicine binding site in tubulin. Beside this, inventors have seen from tubulin turbidity assay and microtubule assembly assay that NVR promotes tubulin polymerization, which indicates that NVR binds close to the taxol binding pocket of b-tubulin. To further analyses the characteristic nature of these nonapeptide, the inventors have prepared a fresh peptide solution and incubated at 37°C for 3-7 days. After 7 days FT-IR, SEM, AFM and DLS studies were performed. Inventors found that this peptide was forming b-sheet structure and forms nice vesicles of size 255nm (Figure 3). As this peptide was designed for neuroprotection which related to the inhibition of amyloid aggregation, inventors was further understand the binding of NVR with amyloid beta (Ab) (PDB ID- llYT) [Crescenzi O, Tomaselli S, Guerrini R, Salvadori S, D'Ursi A. M, Temussi P. A, Solution structure of the Alzheimer amyloid beta-peptide (1-42) in an apolar microenvironment. Similarity with a virus fusion domain, Eur J Biochem., 2002, 269, 5642-48] through molecular docking study. Inventors found that NVR has very good binding affinity with Ab peptide, having binding energy of - 4.8kcal/mol. The amino acids L17 and A21 of Ab peptide showing H-bonding interaction with NVR peptide (Figure 4a). This result clearly shows that NVR is binding at the 17-21 hydrophobic stretch of Ab peptide, which is known as key amino acids sequences for amyloid aggregation. [Liihrs T, Ritter C, Adrian M, Riek-Loher D, Bohrmann B, Dobeli H, Schubert D, Riek R, 3D structure of Alzheimer's amyloid^(l-42) fibrils, Proc. Natl. Acad. Sci. USA., 2005, 102, 17342- 347] To experimentally prove this hypothesis, inventors have incubated Ab solution (10 mM) alone, with different concentration of NVR (10-50 mM) peptide at 37°C for 10 days and monitoring the aggregation of Ab by taking fluorescence of ThT after mixing it with different incubated solution. Also, in another case inventors have mixed peptide solution with preformed amyloid fibril to check its activity on fibril Ab. [Paul A, Nadimpally K. C, Mondal T, Thalluri K, Mandal B, Inhibition of Alzheimer's amyloid-b peptide aggregation and its disruption by a conformationally restricted a/b hybrid peptide, Chem. Commun., 2015, 51, 2245-48] Inventors found that this peptide has significant ability to inhibit oligomeric as well as preformed fibril amyloid aggregation (Figures 3b and 3c). After 7 days, this peptide inhibits almost 45 % of the aggregation (Figure 3d). This inhibition of Ab aggregation was further supported by FT-IR analysis (Figure 3e). [Biswas A, Kurkute P, Saleem S, Jana B, Mohapatra S, Mondal P, Adak A, Ghosh S, Saha A,Bhunia D, Biswas S. C, Ghosh S, Novel hexapeptide interacts with tubulin and microtubules, inhibits Ab fibrillation, and shows significant neuroprotection, ACS Chem. Neurosci., 2015, 6, 1309-16] Then, inventors further studied the inhibition activity of this peptide in extreme amyloid aggregation condition. Inventors mixed AChE enzyme with amyloid solution that result in rapid aggregation of Ab. Inventors found that this peptide has the ability to inhibit aggregation on that condition also. It was documented before that amyloid beta peptide binds at the hydrophobic catalytic binding pocket of the AChE and induces its aggregation. This event can be inhibited by a suitable inhibitor, which has ability to interact with either CAS or PAS binding site of AChE. Thus, inventors evaluated whether NVR has any inhibitory effect on AChE induced amyloid aggregation or not. Co-incubation of NVR and amyloid beta with acetyl cholinesterase enzyme for 24 hours resulted significant decrease of amyloid aggregation with increasing concentration of NVR (5 mM, 10 mM) (Figure 5a). [Chen X, Wehle S, Kuzmanovic N, Merget B, Holzgrabe U, Ko nig B, Sotriffer C. A, Decker M, Acetylcholinesterase inhibitors with photoswitchable inhibition of b-amyloid aggregation, ACS Chem. Neurosci., 2014, 5, 377-389] This result clearly indicates that NVR not only inhibits AChE activity but also it has substantial inhibitory effect on AChE induced amyloid aggregation. Therefore, inventors were further investigated the mechanistic aspects of inhibition of AChE activity by NVR. Inventors have calculated the substrate- velocity curve with varying concentration of NVR (0.25 - 5 mM) with different substrate (acetylthiocholine) concentration (87.5 to 700 mM) to analyze the binding site of this peptide and understand the mechanism of AChE inhibition. From the Lineweaver-Burk plot (Figure 5b), inventors found changes of K_m value (negative reciprocal of X-intercept) and V_max value (reciprocal of Y-intercept) with increasing concentration of NVR, indicating its binding with AChE at CAS site in a competitive manner. [Chen X, Wehle S, Kuzmanovic N, Merget B, Holzgrabe U, Ko nig B, Sotriffer C. A, Decker M, Acetylcholinesterase inhibitors with photoswitchable inhibition of b-amyloid aggregation, ACS Chem. Neurosci., 2014, 5, 377-389] This result was further supported by molecular docking experiment using PDB structure of AChE (PDB ID-2CKM) [Rydberg E. H, Brumshtein B, Greenblatt H. M, Wong D. M, Shaya D, Williams L. D, Carrier P. R, Pang Y. P, Silman I, Sussman J. L, Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of Bis5-tacrine produces a dramatic rearrangement in the active-site gorge, J. Med. Chem. 2006, 49, 5491-5500] where NVR has very good binding with AChE (Figure 5c and 5d). Afterthat, inventors were keen to understand the inhibition of fibril formation through MD simulation study. For that purpose, two short peptides sequence“KLVFFAE” with NVRwas placed in a cubic simulation box having volume of 4.5x4.5x4.5. Then simulation for 20 ns was performed and found that upto 15 ns those two short peptides can’t form any stable b-sheet structure (also seen from the conformational profile diagram (Figure 6, upper panel). In the control experiment, they form stable beta-sheet structure only after 2 ns of simulation (Figure 6, lower panel). These findings clearly reveal that NVR binds to the hydrophobic core of Ab peptide and inhibits its aggregation process.

In vitro assay revealed that NVR significantly inhibits Ab aggregation. Therefore, inventors thought of continuing further studies of NVR on neuronal cells. First, the entry of NVR in PC12 (Rat pheochromocytoma cells) derived neurons was studied where inventors found substantial uptake (Figures 7a-7d). Next, toxicity of NVR in differentiated PC 12 cells was evaluated, which shows that NVR has no toxicity upto 100 mM concentration (Figure 7e). Interestingly, inventors also found from microscopic DIC images that neurons are healthy and neurites are protruding upon treatment with 10 mM of NVR (Figures 7f and 7g). The average length of neurites in NVR treated cells compare to control is quite high, which indicates neuroprotective potential of NVR (Figure 7h). In AD, tau (a microtubule associated protein) hyperphosphorylated and forms neurofibrillary triangle which results in disruption of intracellular microtubule network. Therefore, inventors assessed the microtubule stabilization ability of NVR in neurons. PC 12 derived neurons were treated with 20 mM of NVR and intracellular microtubules were immunostained with tubulin specific antibody followed by fluorescence microscopic imaging. Fluorescence microscopic images reveal that the intracellular microtubules were found to be stable (Figures 8a-8d) as compared to the control cell (Figures 8e-8h). Next, neuroprotective potential of NVR peptide was investigated by using nerve growth factor (NGF) deprived model where differentiated PC 12 cells were treated with anti-NGF. [Adak A, Das G, Barman S, Mohapatra S, Bhunia D, Ghosh S, Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection, and Delivers Anti -Alzheimer Drug, ACS Appl Mater Interfaces. 2017, 9, 5067-76] This assay was performed by measuring the cell viability of neurons through MTT with various concentrations (1-50 mM) of NVR. We found that NVR provides significant neuroprotection and helps to maintain neuronal morphology of the NGF deprived neurons (Figures 9a-9c). This neuroprotection ability increases with increasing concentration of NVR peptide (Figure 9d).

The present invention also involves the serum stability study of NVR for evaluation of its suitability in in vivo system. NVR was incubated in human serum solution at 37°C and HPLC was performed in every 2 hours of incubation to monitor the percentage of remaining peptide upto 24 hours. HPLC data reveals that NVR was quite stable in human serum and 28 % of peptide was still remaining after 24 hours of incubation (Figure 9e). Since NVR has been designed for neuroprotection and potential therapeutic application in AD, we were curious to know whether NVR has ability to cross the blood-brain barrier (BBB) or not. For this purpose, inventors have performed in-vivo mice model experiment with NVR to check its BBB crossing ability. [Prades R, Oller-Salvia B, Schwarzmaier S. M, Selva J, Moros M, Balbi M, Grazii V, de La Fuente J. M, Egea G, Plesnila N, Teixido M, Giralt E, Applying the retro-enantio approach to obtain a peptide capable of overcoming the blood-brain barrier, Angew Chem Int Ed Engl. 2015, 54, 3967-3972] Intra-cardiac injections of NVR (2.5 mM, 100 pL) solution into the healthy mice was performed. Animals were sacrificed after 6 hours, brains were separated out and blood vessel and meninges were removed carefully under stereo microscope. Brain extract was prepared in acetonitrile solution. MALDI mass spectrum of NVR treated brain extract revealed the presence of NVR mass with Na⁺ and K⁺ ions (Figure lOa) with some cleaved peptide, whereas in case of untreated (control) brain extract no such mass peak was observed (Figure lOa, inset). This result indicates that NVR has ability to cross the BBB. Finally, inventors checked whether NVR has any toxicity in primary cortical neurons or not. Primary cortical neuron culture has been performed following previously optimized protocol. [Beaudoin G M J, Lee S H, Singh D, Yuan Y, Ng Y G, Reichardt L F, Arikkath J, Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex, Nature Protocols, 2012, 7, 1741] The primary cortical neurons were further validated for their neuronal character by staining them with anti-MAP2 antibody (MAP2 is a known marker for staining the dendrites of neurons) (Figure lOb and lOc). [Beaudoin G M J, Lee S H, Singh D, Yuan Y, Ng Y G, Reichardt L F, Arikkath J, Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex, Nature Protocols, 2012, 7, 1741] Microscopic images of treated and untreated cortical neurons indicates that there are no significant morphological changes (Figure lOd-lOf) confirming that NVR does not have any toxic effect on primary neurons.

It is within the confines of the present invention that the formulations of the nonapeptide of formula I may be further associated with a pharmaceutically-acceptable carrier, thereby comprising a pharmaceutical composition. The pharmaceutically-acceptable carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. The formulations of the present invention may be prepared by the methods well known in the pharmaceutical art. EXAMPLES

Example 1: Synthesis and method of preparing Nonapeptide

'NVR'(NH₂-NVRDLTEFQ-NH₂) and ‘Fluorescein-NVR’ (FITC-NVRDLTEF Q-NH₂) was synthesized by solid phase peptide synthesis method using Rink Amide AM resin in CEM Liberty 1 automated peptide synthesizer. F-moc deprotection was performed using 20% piperidine in DMF solution. DIPEA and HBTU was used as a base and coupling reagent respectively. Peptide was cleaved from the resin by using standard cleavage cocktail solution (TFA 91%, EDT 3%, Phenol 3% and Milli-Q 3%) and crude peptides were purified by reverse phase HPLC (shows almost -100% purity) and characterized by MALDI TOF mass (exactly matched). This purified peptide was dissolved in Milli-Q water and used for various experiments without further modification.

Example 2:Protein biochemistry

Isolation and purification of tubulin from goat brain was performed in our laboratory following previously described method. [Hyman A, Drechsel D, Kellogg D, Salser S, Sawin K, Steffen P, Wordeman L, Mitchison T, Preparation of Modified Tubulins, Methods in Enzymology, 1991, 196, 478] The concentration of this purified tubulin was maintained to 200 mM and stored with glycerol in liquid N₂ cryo chamber.

Example 3: Tubulin turbidity assay

Tubulin turbidity assay was performed for monitoring the effect of our peptide on the polymerization rate of tubulin in presence of GTP. [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32] Absorbance of tubulin increases when turbidity of the solution increases. Therefore, the amount of microtubule formation was quantified by addition of various concentrations of NVR peptide into the tubulin. 20 pM tubulin, 4 mM GTP, 10% dimethyl sulfoxide and 3.125 and 6.25 pM of NVR were mixed in Brinkley Reassembly Buffer 80 (BRB80) in ice and injected into 37°C heated quartz cuvettes of path length 10 mm. The turbidity was measured by measuring absorbance of the solution at 350 nm for 40 min in G6860A Cary 60 UV-Vis Spectrophotometer, Agilent Technologies.

Example 4: Microtubule assembly assay

Fluorescence intensity of DAPI increases as it binds with microtubules due to its restricted degree of freedom. Therefore, the amount of microtubule formation was quantified by addition of various concentrations of NVR peptide into the tubulin and DAPI mixture. [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32] A mixture of 10 pM DAPI in BRB80 buffer containing 100 pM tubulin, 10 mM GTP and 3.125 and 6.25 pM of NVR was prepared. The solution was excited at 355 nm wavelength at 37°C and the emission spectra of the solution was recorded in region from 400 nm to 600 nm wavelength for 60 min in five min time interval using PTI QM 40 spectrofluorometer. Example 5: Binding affinity (¾) of peptide with tubulin was monitored by quenching of intrinsic tryptophan fluorescence of tubulin

Intrinsic fluorescence of the tryptophan residues is quenched when a small molecule or drug binds to the tubulin. [Gupta K, Panda D, Perturbation of Microtubule Polymerization by Quercetin through Tubulin Binding: A Novel Mechanism of Its Antiproliferative Activity, Biochemistry, 2002, 41, 13029-038 and Chakraborti S, Das L, Kapoor N, Das A, Dwivedi V, Poddar A, Chakraborti G, Janik M, Basu G, Panda D, Chakrabarti P, Surolia A, Bhattacharyya B, Curcumin Recognizes a Unique Binding Site of Tubulin, J Med. Chem. 2011, 54, 6183-96] Therefore, the binding of small molecule or peptide towards tubulin is monitored by recording the fluorescence value of intrinsic tryptophan residue. The excitation wavelength was 295 nm and the emission wavelength was ranging from 310-450 nm.

Example 6: Identification of binding pocket of NVR into the tubulin using Fluorescence Resonance Energy Transfer (FRET)

FRET was performed to identify the binding region of NVR in tubulin. Recently, tubulin- colchicine complex was used to find out the distance of fluorescein attached small molecule/peptide binds to tubulin as colchicine has a specific binding site in tubulin. [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32 and andChakraborti S, Das L, Kapoor N, Das A, Dwivedi V, Poddar A, Chakraborti G, Janik M, Basu G, Panda D, Chakrabarti P, Surolia A, Bhattacharyya B, Curcumin Recognizes a Unique Binding Site of Tubulin, J Med. Chem. 2011, 54, 6183-96] We have incubated colchicine-tubulin complex at 37°C as with binding to tubulin colchicine weak fluorescence increased. Then FRET was performed using fluorescein-NVR and this tubulin-colchicine complex. The excitation wavelength was 355 nm and the emission was ranging from 450-650 nm. We have recorded the emission of only tubulin-colchicine complex, only fluorescein-NVR, complex (mixed in 1 : 1 molar ratio) and calculated the distance.

Example 7: Preparation of Amyloid-Beta (1-42) peptide stock solution

10 pL aliquots of Amyloid Beta (1-42) (Ab42) peptide solution were prepared by mixing 1.0 mg of Ab42 peptide in 400 pL of l,l,l,3,3,3-Hexafluoro-2-propanol and stored at -20 C. [Saha A, Mondal G, Biswas A, Chakraborty I, Jana B, Ghosh S, In vitro reconstitution of a cellular like environment using liposome for amyloid beta peptide aggregation and its propagation. Chem. Commun., 2013, 49, 6119-21] Then at the time of experiment 4 pL of this stock solution was dried under nitrogen gas, 1 pL of 1% NH₄OH added into it and made the volume to 30 pL by using phosphate buffer saline (PBS buffer). The final concentration of the Ab42 peptide solution will be 80 pM. Then, we further diluted this solution with PBS to adjust the concentration as per our experiment.

Example 8: Thioflavin T (ThT) assay for monitoring inhibition of Amyloid b (Ab) peptide aggregation

Inhibition of Ab peptide aggregation by NVR peptide was monitored using ThT fluorescence intensity. [Paul A, Nadimpally K. C, Mondal T, Thalluri K, Mandal B, Inhibition of Alzheimer's amyloid-b peptide aggregation and its disruption by a conformationally restricted a/b hybrid peptide, Chem. Commun., 2015, 51, 2245-48] We have mixed 10 mM of Ab with different concentration of NVR peptide and agitating the solution for 48 hours at room temparature. ThT was added into the 48 hours incubated solution at room temperature and fluorescence intensity of ThT was recorded in PTI QM-40 spectrofluorimeter. The excitation wavelength was 435 nm and emission wavelength was ranging between 460-650 nm.

Example 9: Molecular docking of NVR peptide

Autodock- Vina version 1.1.2 [Trott O, Olson A. J, AutoDockVina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, J. Comput. Chem. 2010, 31, 455-61] was used to perform the docking study between the receptor tubulin (PDB 1JFF), [Lowe J, Li H, Downing K. H, Nogales E, Refined structure of alpha beta- tubulin at 3.5 A resolution, J. Mol. Biol., 2001, 313, 1045-57] amyloid beta (PDB- 1IYT) [Crescenzi O, Tomaselli S, Guerrini R, Salvadori S, D'Ursi A. M, Temussi P. A, Solution structure of the Alzheimer amyloid beta-peptide (1-42) in an apolar microenvironment. Similarity with a virus fusion domain, Eur J Biochem., 2002, 269, 5642-48] and AChE (PDB -2CKM) [Rydberg E. H, Brumshtein B, Greenblatt H. M, Wong D. M, Shaya D, Williams L. D, Carrier P. R, Pang Y. P, Silman I, Sussman J. L, Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of Bis5-tacrine produces a dramatic rearrangement in the active-site gorge, J. Med. Chem. 2006, 49, 5491-5500] with NVR to find out the most potent peptide from the small nonapeptide library, interaction with amyloid beta and interaction with taxol pocket of beta tubulin respectively. Affinity grids of volume 20x20x 16, 40x26x54 and 28x30x32 were centred on the receptor tubulin, amyloid beta and acetylcholine esterase for docking with NVR peptide. All the images were seen and produced in PyMOL ( The PyMOL Molecular Graphics System, Version 1.7.4 Schrodinger, LLC).

ExamplelO: MD Simulation

MD simulation study was performed to check the secondary structure of this peptide using GROMACS version 4.5.5. Gromos 96 53a6 force field was applied for peptides simulation. Periodic boundary conditions were applied in all three directions. 0.9 nm cut-off radii were set for electrostatic interactions and 1.4 nm for Lennard-Jones interactions. Long-range electrostatics interactions were tested using Particle-Mesh Ewald (PME) method. Simulation was performed at a time step of 2 fs. The first phase involved the simulating for 500 ps under a constant volume (NVT) ensemble. Using V-rescale coupling method Protein and non-protein atoms were coupled to separate coupling baths and temperature maintained to 310 K. Following NVT equilibration, 1 ns of constant-pressure (NPT) equilibration was performed using Parrinello-Rahman coupling method. Relaxation time of 1 ps and 0.1 ps were used for NPT and NVT respectively. Then the production run was started for lOOns.

Example 11: Inhibition of Acetylcholinesterase induced Ab peptide aggregation by NVR peptide

Inhibition of Ab peptide aggregation by NVR peptide was monitored in prescence of AChE enzyme as it accelerates the aggregation process. [Chen X, Wehle S, Kuzmanovic N, Merget B, Holzgrabe U, Ko nig B, Sotriffer C. A, Decker M, Acetylcholinesterase inhibitors with photoswitchable inhibition of b-amyloid aggregation, ACS Chem. Neurosci., 2014, 5, 377-389]

To check the neuroprotective behavior of NVR in extreme condition, we have mixed AChE enzyme (10 mM) with 1 OmM of Ab solution with different concentration of NVR (10 and 5 mM). Fluorescence intensity of Thioflavin T (ThT) was recorded (l_ec-435 nm and k_cm-460-650 nm) for monitoring the AChE induced aggregation of Ab peptide in PTI QM 40 spectrofluorometer for 24 hours.

Example 12: Transmission Electron Microscopy (TEM) study to monitor the Inhibition of Ab fibrillation by NVR peptide

Ab peptide alone (having concentration of 1 mM) and with NVR peptide (final concentration of 1 mM) was incubated at 37 °C for 7 days to check its effect on amyloid beta aggregation. After 7 days, both the solutions were deposited in a 300 mesh copper grid from ProSciTech. After 1 minute, excess solution was removed and the grid was washed with water followed by staining with 2% ETranyl acetate in water. The morphology was studied in a TECNAI G2 SPIRIT BIOTWIN CZECH REPUBLIC 120 KV electron microscope operating at 80 kV electron microscope.

Example 13: Fourier Transform Infrared Spectroscopy

The freshly prepared solid NVR peptide’s FT-IR spectroscopic analysis was carried out in a Perkin-Elmer Spectrum 100 FT-IR spectrometer using KBr pellets. Spectra of these pellets were recorded and accumulated of 5 times scan with speed 0.2 cm/s at a resolution of 1.6 cm^-1 in a Perkin-Elmer Spectrum 100 series Spectrometer. The LiTa03 detector was used for data plotting. Each time background correction was performed to eliminate interference from air (or any other parameters).

Example 14: Cell culture:

PC12 cells are cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) that contains 5% fetal bovine serum (FBS) and 10% horse serum at 37 °C temparature and 5% C0₂ atmosphere in our lab.

Example 15: Understanding the cellular entry of NVR peptide into the PC12 cells using fluorescein conjugated NVR peptide

Cellular uptake of NVR peptide was performed in PC12 cells. PC12 derived neurons were treated with 10 mM of fluorescein conjugated NVR for 4 hours in serum free media and fixed with 4% formaldehyde for 1 hour. Then the images were captured in confocal microscope having a 40X objective (Olympus) and an Andor iXon3 897 EMCCD camera in 405 and 488 nm wavelengths laser lights.

Example 16: Cell viability of NVR was performed by MTT assay using differentiated PC12 cells:

We have used 96 well plate to culture the cells for 24 hours. Then, serum free DMEM in 1% horse serum and 100 ng/mL NGF was used to differentiate the cells for 5 days. Then differentiated PC12 cells were treated with various concentrations of NVR peptide (100, 50, 25, 12.5, 6.25 pg/mL) and incubated for 24 hours at 37 °C under 5 % C0₂ environment. After treatment, for all cases MTT solution (5 mg/mL) in 1 X PBS was added to each well except the background wells and incubated for further 4 hours at 37 °C. The culture media was then replaced with DMSO- MeOH (1 : 1) solution to solubilize the yellow formazan before recording the absorbance. Microplate ELISA reader (Thermo; MultiscanTM GO Microplate Spectrophotometer) was used to read the absorbance value from the 96 well plate at wavelength of 550 nm. The effect of peptide on differentiated PC 12 cells was captured using fluorescence microscope (1X83, Olympus) in DIC mode.

Example 17: Effect of NVR peptide on neuronal microtubule monitored by fluorescence microscope

PC12 cells were cultured in a confocal dish with cell density of 3000-5000 and harvested overnight. The culture media was replaced with 10 mM of NVR peptide containing treatment solution. After 16 hours, the complete media was removed and the confocal dishes were washed with serum free media. 4% paraformaldehyde was used to fix the cells and incubated with 0.2% triton-X and 5% BSA in PBS for 1 hour. After a single wash with IX PBS, cells were incubated with polyclonal anti-a-tubulin IgG antibody with dilution 1 :300 for 2 hours. Cells were then washed with PBS and secondary antibody (Cy3.5 pre-absorbed goat anti-rabbit IgG) was mixed with it for 2 hours having dilution 1 :600. Then, cells were washed with IX PBS and incubated with Hoechst 33258 (1 pg/mL) for 30 min before imaging. The microtubule morphology of differentiated PC 12 cells was captured at various areas of culture dish using confocal microscope having a 40X objective (Olympus) and an Andor iXon3 897 EMCCD camera in 405 and 561 nm wavelengths laser lights.

Example 18: Monitoring neuroprotective effect of NVR peptide in NGF deprived PC12 derived neurons:

The neuronal differentiation of PC12 cells was achieved by treating the cells with 100 ng/mL of NGF in serum free DMEM media containing 1% horse serum and incubated at 37 °C and 5 % C0₂ environment for 5 days. [Adak A, Das G, Barman S, Mohapatra S, Bhunia D, Ghosh S, Biodegradable Neuro-Compatible Peptide Hydrogel Promotes Neurite Outgrowth, Shows Significant Neuroprotection, and Delivers Anti-Alzheimer Drug, ACS Appl Mater Interfaces. 2017, 9, 5067- 76] The cells were treated with anti -NGF (2 pg/mL) alone and with different concentrations of NVR peptide (1-20 pM) after differentiation of cells into neurons for 20 hours. Following incubation, MTT solution (5 mg/mL) was added to cells containing serum free media and incubated for 4 hours at 37 °C. Then, we removed the incubation media and 1 : 1 MeOH:DMSO solution was added to each well. The plate was scanned using a microplate ELISA reader (Thermo; MultiscanTM GO Microplate Spectrophotometer) at 550 nm.

Example 19: Serum stability of NVR peptide:

Serum stability of the NVR peptide was monitored in human serum for 24 hours. [Jana B, Mondal P, Saha A, Adak A, Das G, Mohapatra S, Kurkute P, Ghosh S, Designed Tetrapeptide Interacts with Tubulin and Microtubule, Langmuir, 2018, 34, 1123-32] Serum stability of NVR was performed using 50 pL of 200 pM NVR, 800 pL of human serum and 150 pL of milli-Q followed by incubation at 37 °C. After every 2 hours, 100 pL of incubated solution was taken out and mixed with 100 pL acetonitrile. Mixture was centrifuged and filtrate was used for HPLC. We have plotted the change in intensity profile of NVR’ s molecular peak with time.

Example 20: Blood-Brain Barrier crossing experiment:

Healthy C57BL/6J female mice were used in this experiment. Mice were then divided in two groups (3 mice/group). Blood brain barrier crossing experiment was performed by giving intercardiac injection of 100 pL NVR solution (dissolved in saline) to mice at the concentration of 10 mg/kg body weight of the mice. [Prades R, Oller-Salvia B, Schwarzmaier S. M, Selva J, Moros M, Balbi M, Grazii V, de La Fuente J. M, Egea G, Plesnila N, Teixido M, Giralt E, Applying the retro-enantio approach to obtain a peptide capable of overcoming the blood-brain barrier, AngewChemlnt Ed Engl. 2015, 54, 3967-72] For control experiment, mice were treated with 100 pL of saline solution. Animals were sacrificed after 6 hours, mice brains were separated out and blood vessel and meninges were removed carefully under stereo microscope. Then, cleaned brain was directly transferred into liquid nitrogen and crushed with a mortar and pestle. Acetonitrile and water mixture (1 : 1) was added into the crushed brain for dissolving the BBB crossed NVR. Mixture was centrifuged to separate insoluble part and soluble part. Then the soluble part was collected. HPLC and mass analysis of soluble part was performed for both NVR treated mice as well as control mice.

Example 21: Effect of NVR peptide on primary cortical neuron culture

Primary cortical neurons were cultured to find the effect of this peptide. [Beaudoin G M J, Lee S H, Singh D, Yuan Y, Ng Y G, Reichardt L F, Arikkath J, Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex, Nature Protocols, 2012, 7, 1741] Briefly, El 8 embryos of timed-pregnant spraguedawley rat was taken for this experiment, their brain cortices were isolated, micro-dissected, digested, dissolved, filtered and then suspended in MEM medium containing glucose (0.6% wt/vol) and 10% horse serum. The suspended cells were then seeded on confocal dishes (3-5 x l0⁵/mL) coated with poly-D-lysine and cultured at 37 °C with 5% C0_2. After 4 hours of incubation, medium was changed with neurobasal media supplemented with B27, GlutaMAX and pen/strep. The cells were culture for another 4 days and then we have treated the cortical neuron cells with 10 mM of NVR peptide.

Example 22: MAP2 staining of the primary cortical neurons

Immunostaining of the Primary cortical neurons treated with 10 mM NVR were performed using anti-MAP2 following a previously described protocol. [Beaudoin G M J, Lee S H, Singh D, Yuan Y, Ng Y G, Reichardt L F, Arikkath J, Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex, Nature Protocols, 2012, 7, 1741] In brief, the primary cortical neurons were fixed with 4% formaldehyde, and then permeabilized using 0.3 % Triton-X. Thereafter the fixed cells were incubated with primary Mouse anti-MAP2 overnight at 2-8 °C. The cells were rinsed thrice with PBS the very next day and then incubated with secondary antibody Alexa Fluor 594 goat anti-Mouse IgG for 2 hours at 37 °C. After that the cells were again rinsed with PBS and the nucleus was stained using Hoechst 33258. The stained cells were then observed under confocal microscope having a 60X objective (Olympus).

Example 23: Pharmaceutical Composition: This peptide has very good water solubility as it is negatively charged at pH 7. So, this peptide can be administrated with only water or saline solution. Generally, the mode of administration of peptides for treating Alzheimer’s disease is through intraperitoneal (IP) and intranasal (IN) route. So, this peptide can also be administered by these routes. It was documented before that the NAP peptide can be administered by intranasal route [http://dx.doi.org/l0. l039/978l84973 l072-00l08] by giving 25-150 pg of peptide per Kg body weight of transgenic mice.

Also, the peptoid have been (total concentration of 4.8 mg/mL) intranasally administered [Ross T M, Zuckermann R N, Reinhard C, Frey II W H, Intranasal administration delivers peptoids to the rat central nervous system, Neuroscience Letters, 2008, 439, 30] with a pipette over the alternating nares every 2-3 min in 4-10 drops to promote passive inhalation of the peptoid. Over 25 min, a total volume of 68 mL was administered through IN route.

The nonapeptide of the present invention can be administered for treating Alzheimer’s disease is through intraperitoneal (IP) and intranasal (IN) route. Example 24: Data Analysis:

Microscopic images were analysed using Image J software and spectroscopic data was calculated by Origin pro 8.5. Two tailed student’s t-test and one way annova was performed to calculate the statistical analysis and their values varies between *p<0.05 and **p<0.00l for different experiment. In general, most of the neurodegenerative disorders are characterized by a yloid-// fibrillation/aggregation and deposition of neurofibrillary tangles in the brain or central nervous system (CNS), including Alzheimer's disease and Parkinson's disease. Additionally, neuron cells of brain are rich in tubulin/microtubule and severe disruption of this intracellular microtubule networks occur by Ab fiber deposition. Therefore, inhibition of A// fibrillation as well as microtubule stabilization is the key prerequisite for the development of novel anti-Alzheimer therapeutic agents. In the development process of AD therapeutic agent, an innovative strategy has been adopted by considering the taxol binding pocket of b-tubulin and hydrophobic region of Ab. The 17-21 region of A// contains some non-polar and hydrophobic amino acids whereas the taxol pocket of b-tubulin consists of polar as well as non-polar amino acids. The claimed nonapeptide has been designed by the counter interaction of amino acids of those pockets by using relative frequencies of amino acids contacts. Thus, the claimed peptide based amyloid aggregation inhibitor having microtubule stabilization activity is inventive. Its’ not obvious to design a peptide based molecule from the taxol binding cavity of //-tubulin, which will show microtubule stabilization due to the following reason- · When taxol binds to //-tubulin it enforces a structural change in //-tubulin through interaction with the key amino acids of that pocket, causing a long range structural stability of microtubule, that results in highly toxic nature of taxol towards microtubule. Therefore, the design molecule should have moderate/weak binding without interacting with all the active amino acids of that pocket. This contributed to the technical advancement of the claimed invention. The observed association constant of the claimed peptide (2.5 ^c 10⁴ M ¹ at 4 °C) is quite low compare to taxol (3.6 ^c 10⁶ M ¹ at 37 °C and 7.6 ^c 10⁸ M ¹ at 4°C), which is almost 144 times weaker in 37°C and 30400 times weaker in 4°C. This weaker binding affinity of the claimed nonapeptide resulted non-toxicity and this is the crucial point in this invention. Tubulin is a heterodimeric protein that comprises of a and b tubulin monomer with a high degree of homology. Active research is going on to develop a potent anti-cancer or neuroprotective molecule from the interface or from various binding sites of tubulin. As this tubulin is a small 110 kD protein having 900 amino acids and consisting of very less active amino acids. Therefore, the chance of getting similarity of the claimed sequence with the prior art is common but the desired activity by constructing a peptide sequence in a logical way is inventive.

Further in the claimed sequence some non-polar/hydrophobic amino acids are deliberately incorporated and the position of the specific amino acids is important to be a potent amyloid inhibitor.

Advantages:

The present invention provides a novel nonapeptide which acts as a microtubule stabilizer and excellent neuroprotector, having better neuroprotection than any other earlier reported peptide in this field.

The peptide based therapeutic of the present invention has excellent bioavailability.

The process for the synthesis of said peptide is simple and inexpensive.

^ The peptide sequence of the present invention is easy to modify according to the pharmacophore requirement.

The novel nonapeptide of the present invention exhibits dual property like stabilizes microtubule as well as provides excellent protection to neuron cells against Ab infection.

The nonapeptide of the present invention does not cause toxicity.

· The novel peptide based formulation(s) is used as a potential therapy for Alzheimer’s disease (AD).

Claims

We claim:

1) A nonapeptide of formula I.

2) The nonapeptide as claimed in claim 1, having a molecular weight of 1120.22 Da.

3) The nonapeptide as claimed in claim 1, wherein the nonapeptide binds in the taxol pocket of b-tubulin.

4) The nonapeptide as claimed in claim 1, wherein the nonapeptide binds to the hydrophobic (17-21) region of amyloid-beta (Ab).

5) The nonapeptide as claimed in claim 1, wherein the nonapeptide binds the peripheral anionic site of acetylcholinesterase (AChE) enzyme. 6) The nonapeptide as claimed in claim 1, wherein the nonapeptide inhibits amyloid aggregation by binding to Ab and AChE enzyme.

7) A method of preparing the nonapeptide as claimed in claim 1, comprising solid phase synthesis of peptide using Rink Amide resin, F-moc deprotection followed by cleaving of peptide from the resin by using 20% piperidine and standard cleavage solution respectively and purification of crude peptide by RP-HPLC.

8) A pharmaceutical composition comprising the nonapeptide as claimed in claim 1 along with pharmaceutically acceptable excipient(s).

9) The pharmaceutical composition as claimed in claim 8, wherein the pharmaceutically acceptable excipient(s) is selected from water or saline solution. 10) A method of treating Alzheimer’s disease by administering effective amount of nonapeptide as claimed in claim 1.